334 results on '"David E. Cane"'
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2. Prelog Lecture: Mechanism and Structure of Biosynthetic Enzymes. The Biosynthesis of Vitamin B6
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David E. Cane
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Biosynthesis ,Crystal structure ,Oxidative decarboxylation ,Pyridoxine synthase ,Vitamin b6 ,Chemistry ,QD1-999 - Abstract
On Monday, November 11, 2002, the vice president, Prof. Dr. Gerhard Schmitt, presented the Prelog Medal 2002 to Prof. Dr. David E. Cane, Brown University, Providence, Rhode Island, USA. The title of the lecture that followed was 'Mechanism and Structure of Biosynthetic Enzymes'.
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- 2003
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3. Stereospecific Formation of Z-Trisubstituted Double Bonds by the Successive Action of Ketoreductase and Dehydratase Domains from trans-AT Polyketide Synthases
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Xinqiang Xie and David E. Cane
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chemistry.chemical_classification ,Double bond ,010405 organic chemistry ,Stereochemistry ,Stereoisomerism ,010402 general chemistry ,01 natural sciences ,Biochemistry ,Article ,Substrate Specificity ,0104 chemical sciences ,Polyketide ,Stereospecificity ,Bacterial Proteins ,chemistry ,Biocatalysis ,Dehydratase ,Methacrylates ,Bongkrekic Acid ,Polyketide Synthases ,Oxazolomycin ,Hydro-Lyases ,NADP - Abstract
Incubation of (±)-2-methyl-3-ketobutyryl-SNAC (3) and (±)-2-methyl-3-ketopentanoyl-SNAC (4) with BonKR2 or OxaKR5, ketoreductase domains from the bongkrekic acid (1) and oxazolomycin (2) polyketide synthases, in the presence of NADPH gave in each case the corresponding (2 R,3 S)-2-methyl-3-hydroxybutyryl-SNAC (5) or (2 R,3 S)-2-methyl-3-hydroxypentanoyl-SNAC (6) products, as established by chiral gas chromatography-mass spectrometry analysis of the derived methyl esters. Identical results were obtained by BonKR2- and OxaKR5-catalyzed reduction of chemoenzymatically prepared (2 R)-2-methyl-3-ketopentanoyl-EryACP6, (2 R)-2-methyl-3-ketobutyryl-BonACP2 (12), and (2 R)-2-methyl-3-ketopentanoyl-BonACP2 (13). The paired dehydratase domains, BonDH2 and OxaDH5, were then shown to catalyze the reversible syn dehydration of (2 R,3 S)-2-methyl-3-hydroxybutyryl-BonACP2 (14) to give the corresponding trisubstituted ( Z)-2-methylbutenoyl-BonACP2 (16).
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- 2018
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4. Exploring the Influence of Domain Architecture on the Catalytic Function of Diterpene Synthases
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Golda G. Harris, Lian Duan, Travis A. Pemberton, Alex S. Genshaft, Mustafa Köksal, David W. Christianson, Wayne K. W. Chou, David E. Cane, and Mengbin Chen
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Models, Molecular ,0301 basic medicine ,Sesterterpenes ,Architecture domain ,Stereochemistry ,Mutant Chimeric Proteins ,Gene Expression ,Protein Engineering ,Biochemistry ,Article ,Protein Structure, Secondary ,03 medical and health sciences ,chemistry.chemical_compound ,Protein Domains ,Isomerases ,chemistry.chemical_classification ,Alkyl and Aryl Transferases ,ATP synthase ,biology ,biology.organism_classification ,Terpenoid ,Kinetics ,Aspergillus ,030104 developmental biology ,Enzyme ,chemistry ,Cyclization ,Taxadiene synthase ,Saccharomycetales ,Domain (ring theory) ,biology.protein ,Diterpenes ,Taxus ,Diterpene ,Aspergillus clavatus - Abstract
Terpenoid synthases catalyze isoprenoid cyclization reactions underlying the generation of more than 80,000 natural products. Such dramatic chemodiversity belies the fact that these enzymes generally consist of only three domain folds designated α, β, and γ. Catalysis by class I terpenoid synthases occurs exclusively in the α domain, which is found with α, αα, αβ, and αβγ domain architectures. Here, we explore the influence of domain architecture on catalysis by taxadiene synthase from Taxus brevifolia (TbTS, αβγ), fusicoccadiene synthase from Phomopsis amygdali (PaFS, (αα)6), and ophiobolin F synthase from Aspergillus clavatus (AcOS, αα). We show that the cyclization fidelity and catalytic efficiency of the α domain of TbTS are severely compromised by deletion of the βγ domains; however, retention of the β domain preserves significant cyclization fidelity. In PaFS, we previously demonstrated that one α domain similarly influences catalysis by the other α domain [Chen, M., Chou, W. K. W., Toyomasu, T., Cane, D. E., Christianson, D. W. (2016) ACS Chem. Biol. 11, 889–899]. Here, we show that the hexameric quaternary structure of PaFS enables cluster channeling. We also show that the α domains of PaFS and AcOS can be swapped so as to make functional chimeric αα synthases. Notably, both cyclization fidelity and catalytic efficiency are altered in all chimeric synthases. Twelve newly formed and uncharacterized C20 diterpene products and three C25 sesterterpene products are generated by these chimeras. Thus, engineered αβγ and αα terpenoid cyclases promise to generate chemodiversity in the greater family of terpenoid natural products.
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- 2017
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5. The T296V Mutant of Amorpha-4,11-diene Synthase Is Defective in Allylic Diphosphate Isomerization but Retains the Ability To Cyclize the Intermediate (3R)-Nerolidyl Diphosphate to Amorpha-4,11-diene
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Ruiping Gao, Zhenqiu Li, Qinggang Hao, Fang He, Huifang Zhao, David E. Cane, Xiuhua Liu, Wayne K. W. Chou, Hua-Jie Zhu, Longbin Cheng, and Li Liu
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0301 basic medicine ,Amorpha-4,11-diene ,Allylic rearrangement ,Stereochemistry ,Mutant ,Mutation, Missense ,Stereoisomerism ,Artemisia annua ,010402 general chemistry ,01 natural sciences ,Biochemistry ,Article ,Gas Chromatography-Mass Spectrometry ,Substrate Specificity ,03 medical and health sciences ,chemistry.chemical_compound ,Polyisoprenyl Phosphates ,Plant Proteins ,Polycyclic Sesquiterpenes ,Alkyl and Aryl Transferases ,Molecular Structure ,Bicyclic molecule ,ATP synthase ,biology ,Chemistry ,0104 chemical sciences ,Diphosphates ,Kinetics ,030104 developmental biology ,Models, Chemical ,Cyclization ,Biocatalysis ,biology.protein ,Sesquiterpenes ,Cyclase activity - Abstract
The T296V mutant of amorpha-4, 11-diene synthase catalyzes the abortive conversion of the natural substrate (E,E)-farnesyl diphosphate mainly into the acyclic product (E)-β-farnesene (88%) instead of the natural bicyclic sesquiterpene amorphadiene (7%). Incubation of the T296V mutant with (3R,6E)-nerolidyl diphosphate resulted in cyclization to amorphadiene. Analysis of additional mutants of amino acid residue 296 and in vitro assays with the intermediate analogue (2Z,6E)-farnesyl diphosphate as well as (3S,6E)-nerolidyl diphosphate demonstrated that the T296V mutant can no longer catalyze the allylic rearrangement of farnesyl diphosphate to the normal intermediate (3R,6E)-nerolidyl diphosphate, while retaining the ability to cyclize (3R,6E)-nerolidyl diphosphate to amorphadiene. The T296A mutant predominantly retained amorphadiene synthase activity, indicating that neither the hydroxyl nor the methyl group of the Thr296 side chain is required for cyclase activity.
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- 2016
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6. The Cytochrome P450-Catalyzed Oxidative Rearrangement in the Final Step of Pentalenolactone Biosynthesis: Substrate Structure Determines Mechanism
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Gerwald Jogl, Lian Duan, and David E. Cane
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Models, Molecular ,0301 basic medicine ,Cytochrome ,Protein Conformation ,Stereochemistry ,Kinetics ,Oxidative phosphorylation ,Hydroxylation ,010402 general chemistry ,01 natural sciences ,Biochemistry ,Article ,Catalysis ,03 medical and health sciences ,chemistry.chemical_compound ,Colloid and Surface Chemistry ,Cytochrome P-450 Enzyme System ,Biosynthesis ,Molecular Structure ,030102 biochemistry & molecular biology ,biology ,Ligand ,Chemistry ,Mutagenesis ,Substrate (chemistry) ,General Chemistry ,Streptomyces arenae ,Streptomyces ,0104 chemical sciences ,Mutagenesis, Site-Directed ,biology.protein ,Oxidation-Reduction ,Sesquiterpenes - Abstract
The final step in the biosynthesis of the sesquiterpenoid antibiotic pentalenolactone (1) is the highly unusual cytochrome P450-catalyzed, oxidative rearrangement of pentalenolactone F (2), involving the transient generation and rearrangement of a neopentyl cation. In Streptomyces arenae this reaction is catalyzed by CYP161C2 (PntM), with highly conserved orthologues being present in at least 10 other Actinomycetes. Crystal structures of substrate-free PntM, as well as PntM with bound substrate 2, product 1, and substrate analogue 6,7-dihydropentalenolactone F (7) revealed interactions of bound ligand with three residues, F232, M77, and M81 that are unique to PntM and its orthologues and absent from essentially all other P450s. Site-directed mutagenesis, ligand-binding measurements, steady-state kinetics, and reaction product profiles established there is no special stabilization of reactive cationic intermediates by these side chains. Reduced substrate analogue 7 did not undergo either oxidative rearrangement or simple hydroxylation, suggesting that the C1 carbocation is not anchimerically stabilized by the 6,7-double bond of 2. The crystal structures also revealed plausible proton relay networks likely involved in the generation of the key characteristic P450 oxidizing species, Compound I, and in mediating stereospecific deprotonation of H-3re of the substrate. We conclude that the unusual carbocation intermediate results from outer shell electron transfer from the transiently generated C1 radical to the tightly paired heme-•Fe3+-OH radical species. The oxidative electron transfer is kinetically dominant as a result of the unusually strong steric barrier to oxygen rebound to the neopentyl center C-1si which is flanked on each neighboring carbon by syn-axial substituents.
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- 2016
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7. Protein-Protein Interactions, Not Substrate Recognition, Dominate the Turnover of Chimeric Assembly Line Polyketide Synthases
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Jonas Austerjost, Chaitan Khosla, Maja Klaus, Thomas Robbins, Matthew P. Ostrowski, David E. Cane, and Brian Lowry
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0301 basic medicine ,biology ,Recombinant Fusion Proteins ,Protein domain ,Heterologous ,Cell Biology ,Protein engineering ,Biochemistry ,Protein–protein interaction ,03 medical and health sciences ,Polyketide ,Acyl carrier protein ,Chimera (genetics) ,030104 developmental biology ,Bacterial Proteins ,Protein Domains ,Polyketide synthase ,Enzymology ,biology.protein ,Polyketide Synthases ,Molecular Biology - Abstract
The potential for recombining intact polyketide synthase (PKS) modules has been extensively explored. Both enzyme-substrate and protein-protein interactions influence chimeric PKS activity, but their relative contributions are unclear. We now address this issue by studying a library of 11 bimodular and 8 trimodular chimeric PKSs harboring modules from the erythromycin, rifamycin, and rapamycin synthases. Although many chimeras yielded detectable products, nearly all had specific activities below 10% of the reference natural PKSs. Analysis of selected bimodular chimeras, each with the same upstream module, revealed that turnover correlated with the efficiency of intermodular chain translocation. Mutation of the acyl carrier protein (ACP) domain of the upstream module in one chimera at a residue predicted to influence ketosynthase-ACP recognition led to improved turnover. In contrast, replacement of the ketoreductase domain of the upstream module by a paralog that produced the enantiomeric ACP-bound diketide caused no changes in processing rates for each of six heterologous downstream modules compared with those of the native diketide. Taken together, these results demonstrate that protein-protein interactions play a larger role than enzyme-substrate recognition in the evolution or design of catalytically efficient chimeric PKSs.
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- 2016
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8. Nature as organic chemist
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David E. Cane
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0301 basic medicine ,Pharmacology ,Antifungal ,030102 biochemistry & molecular biology ,medicine.drug_class ,Stereochemistry ,business.industry ,Biography ,010402 general chemistry ,01 natural sciences ,Organic chemist ,0104 chemical sciences ,Beta-lactam ,03 medical and health sciences ,chemistry.chemical_compound ,chemistry ,Drug Discovery ,medicine ,Fall of man ,business ,Classics - Abstract
By the time I entered Harvard College in the fall of 1962, I knew, or thought I knew, that I wanted to become a scientist. In fact, I had very little idea what that actually meant, nor did I even know what branch of science to choose. My interest in chemistry was quickly kindled, however, by Prof Leonard K Nash’s introductory course in general chemistry. I was not only smitten by the subject itself but also I was inspired by Prof Nash’s informative and entertaining lectures, and his warm and scintillating personality. Indeed for many years thereafter, Prof Nash continued to be a source of encouragement and advice on both science and the art of teaching.
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- 2016
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9. Structure and Function of Fusicoccadiene Synthase, a Hexameric Bifunctional Diterpene Synthase
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Tomonobu Toyomasu, Wayne K. W. Chou, David W. Christianson, Mengbin Chen, and David E. Cane
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0301 basic medicine ,Stereochemistry ,Crystallography, X-Ray ,Biochemistry ,Cyclase ,Article ,Catalysis ,Ligases ,Structure-Activity Relationship ,03 medical and health sciences ,chemistry.chemical_compound ,Transferase ,030102 biochemistry & molecular biology ,biology ,ATP synthase ,Active site ,General Medicine ,Lyase ,Plant disease ,030104 developmental biology ,chemistry ,Fusicoccin ,biology.protein ,Molecular Medicine ,Diterpenes ,Diterpene - Abstract
Fusicoccin A is a diterpene glucoside phytotoxin generated by the fungal pathogen Phomopsis amygdali that causes the plant disease constriction canker, first discovered in New Jersey peach orchards in the 1930’s. Fusicoccin A is also an emerging new lead in cancer chemotherapy. The hydrocarbon precursor of fusicoccin A is the tricyclic diterpene fusicoccadiene, which is generated by a bifunctional terpenoid synthase. Here, we report X-ray crystal structures of the individual catalytic domains of fusicoccadiene synthase: the C-terminal domain is a chain elongation enzyme that generates geranylgeranyl diphosphate, and the N-terminal domain catalyzes the cyclization of geranylgeranyl diphosphate to form fusicoccadiene. Crystal structures of each domain complexed with bisphosphonate substrate analogues suggest that three metal ions and three positively charged amino acid side chains trigger substrate ionization in each active site. While in vitro incubations reveal that the cyclase domain can utilize farnesyl diphosphate and geranyl diphosphate as surrogate substrates, these shorter isoprenoid diphosphates are mainly converted into acyclic alcohol or hydrocarbon products. Gel filtration chromatography and analytical ultracentrifugation experiments indicate that full-length fusicoccadiene synthase adopts hexameric quaternary structure, and small-angle X-ray scattering data yield a well-defined molecular envelope illustrating a plausible model for hexamer assembly.
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- 2016
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10. Structure–Function Analysis of the Extended Conformation of a Polyketide Synthase Module
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Natalia Sevillano, Marc C. Deller, David E. Cane, Yu-Chen Liu, Florencia La Greca, Charles S. Craik, Lindsay Deis, Tsutomu Matsui, Irimpan I. Mathews, Chaitan Khosla, and Xiuyuan Li
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0301 basic medicine ,Models, Molecular ,Stereochemistry ,Protein Conformation ,010402 general chemistry ,Crystallography, X-Ray ,01 natural sciences ,Biochemistry ,Catalysis ,Article ,03 medical and health sciences ,Polyketide ,chemistry.chemical_compound ,Colloid and Surface Chemistry ,Protein structure ,Chain (algebraic topology) ,X-Ray Diffraction ,Polyketide synthase ,Scattering, Small Angle ,Humans ,Tandem ,biology ,Chemistry ,Structure function ,General Chemistry ,0104 chemical sciences ,Kinetics ,030104 developmental biology ,Biocatalysis ,Functional group ,biology.protein ,Polyketide Synthases - Abstract
Catalytic modules of assembly-line polyketide synthases (PKSs) have previously been observed in two very different conformations—an “extended” architecture and an “arch-shaped” architecture—although the catalytic relevance of neither has been directly established. By the use of a fully human naïve antigen-binding fragmenẗ (F(ab)) library, a high-affinity antibody was identified that bound to the extended conformation of a PKS module, as verified by X-ray crystallography and tandem size-exclusion chromatography–small-angle X-ray scattering (SEC–SAXS). Kinetic analysis proved that this antibody-stabilized module conformation was fully competent for catalysis of intermodular polyketide chain translocation as well as intramodular polyketide chain elongation and functional group modification of a growing polyketide chain. Thus, the extended conformation of a PKS module is fully competent for all of its essential catalytic functions.
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- 2018
11. Structural Studies of Geosmin Synthase, a Bifunctional Sesquiterpene Synthase with αα Domain Architecture That Catalyzes a Unique Cyclization–Fragmentation Reaction Sequence
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Golda G. Harris, David W. Christianson, Thomas M. Weiss, Patrick M. Lombardi, Wayne K. W. Chou, Frank V. Murphy, Travis A. Pemberton, Tsutomu Matsui, David E. Cane, Kathryn E. Cole, Mustafa Köksal, L. Sangeetha Vedula, Köksal, Mustafa, and Izmir Institute of Technology. Molecular Biology and Genetics
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Geosmin synthase ,Models, Molecular ,Architecture domain ,Stereochemistry ,Streptomyces coelicolor ,Naphthols ,Crystallography, X-Ray ,Biochemistry ,Cyclase ,Catalysis ,Article ,Acetone ,Protein structure ,Bacterial Proteins ,Polyisoprenyl Phosphates ,Magnesium ,Homology modeling ,Alendronate ,biology ,Chemistry ,Crystal structure ,Active site ,biology.organism_classification ,Lyase ,Protein Structure, Tertiary ,Cyclization ,biology.protein ,Sesquiterpenes - Abstract
Geosmin synthase from Streptomyces coelicolor (ScGS) catalyzes an unusual, metal-dependent terpenoid cyclization and fragmentation reaction sequence. Two distinct active sites are required for catalysis: the N-terminal domain catalyzes the ionization and cyclization of farnesyl diphosphate to form germacradienol and inorganic pyrophosphate (PPi), and the C-terminal domain catalyzes the protonation, cyclization, and fragmentation of germacradienol to form geosmin and acetone through a retro-Prins reaction. A unique αα domain architecture is predicted for ScGS based on amino acid sequence: each domain contains the metal-binding motifs typical of a class I terpenoid cyclase, and each domain requires Mg2+ for catalysis. Here, we report the X-ray crystal structure of the unliganded N-terminal domain of ScGS and the structure of its complex with three Mg2+ ions and alendronate. These structures highlight conformational changes required for active site closure and catalysis. Although neither full-length ScGS nor constructs of the C-terminal domain could be crystallized, homology models of the C-terminal domain were constructed on the basis of 36% sequence identity with the N-terminal domain. Small-angle X-ray scattering experiments yield low-resolution molecular envelopes into which the N-terminal domain crystal structure and the C-terminal domain homology model were fit, suggesting possible αα domain architectures as frameworks for bifunctional catalysis. © 2015 American Chemical Society., National Institutes of Health (NIH) (GM56838/GM30301) NIH Structural Biology and Molecular Biophysics Training Grant; Radcliffe Institute for Advanced Study
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- 2015
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12. Substitution of Aromatic Residues with Polar Residues in the Active Site Pocket of epi-Isozizaene Synthase Leads to the Generation of New Cyclic Sesquiterpenes
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Lian Duan, David W. Christianson, Wayne K. W. Chou, Patrick N. Blank, Golda H. Barrow, and David E. Cane
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0301 basic medicine ,Models, Molecular ,Stereochemistry ,Mutation, Missense ,Streptomyces coelicolor ,Carbocation ,Sesquiterpene ,Crystallography, X-Ray ,Biochemistry ,Article ,03 medical and health sciences ,chemistry.chemical_compound ,Bacterial Proteins ,Catalytic Domain ,Side chain ,Carbon-Carbon Lyases ,chemistry.chemical_classification ,030102 biochemistry & molecular biology ,biology ,ATP synthase ,Active site ,Substrate (chemistry) ,Lyase ,030104 developmental biology ,Enzyme ,chemistry ,Amino Acid Substitution ,biology.protein ,Hydrophobic and Hydrophilic Interactions ,Sesquiterpenes - Abstract
The sesquiterpene cyclase epi-isozizaene synthase (EIZS) catalyzes the cyclization of farnesyl diphosphate to form the tricyclic hydrocarbon precursor of the antibiotic albaflavenone. The hydrophobic active site pocket of EIZS serves as a template as it binds and chaperones the flexible substrate and carbocation intermediates through the conformations required for a multistep reaction sequence. We previously demonstrated that the substitution of hydrophobic residues with other hydrophobic residues remolds the template and expands product chemodiversity [Li, R., Chou, W. K. W., Himmelberger, J. A., Litwin, K. M., Harris, G. G., Cane, D. E., and Christianson, D. W. (2014) Biochemistry 53, 1155-1168]. Here, we show that the substitution of hydrophobic residues-specifically, Y69, F95, F96, and W203-with polar side chains also yields functional enzyme catalysts that expand product chemodiversity. Fourteen new EIZS mutants are reported that generate product arrays in which eight new sesquiterpene products have been identified. Of note, some mutants generate acyclic and cyclic hydroxylated products, suggesting that the introduction of polarity in the hydrophobic pocket facilitates the binding of water capable of quenching carbocation intermediates. Furthermore, the substitution of polar residues for F96 yields high-fidelity sesquisabinene synthases. Crystal structures of selected mutants reveal that residues defining the three-dimensional contour of the hydrophobic pocket can be substituted without triggering significant structural changes elsewhere in the active site. Thus, more radical nonpolar-polar amino acid substitutions should be considered when terpenoid cyclase active sites are remolded by mutagenesis with the goal of exploring and expanding product chemodiversity.
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- 2017
13. Stereospecific Formation of E- and Z-Disubstituted Double Bonds by Dehydratase Domains from Modules 1 and 2 of the Fostriecin Polyketide Synthase
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David E. Cane, Dhara D. Shah, and Young Ok You
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Double bond ,Stereochemistry ,Protein domain ,Stereoisomerism ,Polyenes ,010402 general chemistry ,01 natural sciences ,Biochemistry ,Catalysis ,Article ,Substrate Specificity ,Colloid and Surface Chemistry ,Stereospecificity ,Protein Domains ,Polyketide synthase ,Fostriecin ,chemistry.chemical_classification ,biology ,010405 organic chemistry ,Substrate (chemistry) ,General Chemistry ,Streptomyces ,0104 chemical sciences ,Biosynthetic Pathways ,chemistry ,Pyrones ,Dehydratase ,biology.protein ,Polyketide Synthases - Abstract
The dehydratase domain FosDH1 from module 1 of the fostriecin polyketide synthase (PKS) catalyzed the stereospecific interconversion of (3R)-3-hydroxybutyryl-FosACP1 (5) and (E)-2-butenoyl-FosACP1 (11), as established by a combination of direct LC-MS/MS and chiral GC-MS. FosDH1 did not act on either (3S)-3-hydroxybutyryl-FosACP1 (6) or (Z)-2-butenoyl-FosACP1 (12). FosKR2, the ketoreductase from module 2 of the fostriecin PKS that normally provides the natural substrate for FosDH2, was shown to catalyze the NADPH-dependent stereospecific reduction of 3-ketobutyryl-FosACP2 (23) to (3S)-3-hydroxybutyryl-FosACP2 (8). Consistent with this finding, FosDH2 catalyzed the interconversion of the corresponding triketide substrates (3R,4E)-3-hydroxy-4-hexenoyl-FosACP2 (18) and (2Z,4E)-2,4-hexadienoyl-FosACP2 (21). FosDH2 also catalyzed the stereospecific hydration of (Z)-2-butenoyl-FosACP2 (14) to (3S)-3-hydroxybutyryl-FosACP2 (8). Although incubation of FosDH2 with (3S)-3-hydroxybutyryl-FosACP2 (8) did not result in detectable accumulation of (Z)-2-butenoyl-FosACP2 (14), FosDH2 catalyzed the slow exchange of the 3-hydroxy group of 8 with [18O]-water. FosDH2 unexpectedly could also support the stereospecific interconversion of (3R)-3-hydroxybutyryl-FosACP2 (7) and (E)-2-butenoyl-FosACP2 (13).
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- 2017
14. Elucidation of the Cryptic Methyl Group Epimerase Activity of Dehydratase Domains from Modular Polyketide Synthases Using a Tandem Modules Epimerase Assay
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Xinqiang Xie, Chaitan Khosla, Ashish Garg, and David E. Cane
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0301 basic medicine ,Nigericin ,Stereochemistry ,Protein domain ,Racemases and Epimerases ,010402 general chemistry ,01 natural sciences ,Biochemistry ,Catalysis ,Article ,03 medical and health sciences ,chemistry.chemical_compound ,Polyketide ,Colloid and Surface Chemistry ,Protein Domains ,Polyketide synthase ,Hydro-Lyases ,Enzyme Assays ,030102 biochemistry & molecular biology ,ATP synthase ,biology ,Molecular Structure ,General Chemistry ,0104 chemical sciences ,Acyl carrier protein ,chemistry ,Dehydratase ,biology.protein ,Polyketide Synthases ,Function (biology) - Abstract
Dehydratase (DH) domains of cryptic function are often found in polyketide synthase (PKS) modules that produce epimerized (2S)-2-methyl-3-ketoacyl-ACP (acyl carrier protein) intermediates. A combination of tandem equilibrium isotope exchange (EIX) and a newly developed Tandem Modules Epimerase assay revealed the intrinsic epimerase activity of NanDH1 and NanDH5, from modules 1 and 5, respectively, of the nanchangmycin (1) PKS as well of NigDH1, from module 1 of the nigericin (3) PKS. Unexpectedly, all three epimerase-active DH domains were also found to possess intrinsic dehydratase activity, whereas the conventional DH domains, EryDH4, from module 4 of the erythromycin synthase, and NanDH2 from module 2 of the nanchangmycin synthase, were shown to have cryptic epimerase activity.
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- 2017
15. Elucidation of the Stereospecificity of C-Methyltransferases from trans-AT Polyketide Synthases
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David E. Cane, Chaitan Khosla, and Xinqiang Xie
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Methyltransferase ,Stereochemistry ,Stereoisomerism ,010402 general chemistry ,01 natural sciences ,Biochemistry ,Catalysis ,Article ,Polyketide ,chemistry.chemical_compound ,Colloid and Surface Chemistry ,Stereospecificity ,Polyketide synthase ,Methionine ,biology ,Molecular Structure ,010405 organic chemistry ,Chemistry ,General Chemistry ,Methylation ,Methyltransferases ,0104 chemical sciences ,Acyltransferases ,biology.protein ,lipids (amino acids, peptides, and proteins) ,Polyketide Synthases - Abstract
S-Adenosyl methionine (SAM)-dependent C-methyltransferases are responsible for the C2-methylation of 3-ketoacyl-acyl carrier protein (ACP) intermediates to give the corresponding 2-methy-3-ketoacyl-ACP products during bacterial polyketide biosynthesis mediated by trans-AT polyketide synthases that lack integrated acyl transferase (AT) domains. A coupled ketoreductase (KR) assay was used to assign the stereochemistry of the C-methyltransferase-catalyzed reaction. Samples of chemoenzymatically-generated 3-ketopentanoyl-ACP (9) were incubated with SAM and BonMT2 from module 2 of the bongkrekic acid polyketide synthase. The resulting 2-methyl-3-ketopentanoyl-ACP (10) was incubated separately with five (2R)- or (2S)-methyl specific KR domains. Analysis of the derived 2-methyl-3-hydroxypentanoate methyl esters (8) by chiral GC-MS established that the BonMT2-catalyzed methylation generated exclusively (2R)-2-methyl-3-ketopentanoyl-ACP ((2R)-10). Identical results were also obtained with three additional C-methyltransferases – BaeMT9, DifMT1, and MupMT1 – from the bacillaene, difficidin, and mupirocin trans-AT polyketide synthases
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- 2017
16. Mechanism and Stereochemistry of Polyketide Chain Elongation and Methyl Group Epimerization in Polyether Biosynthesis
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Ashish Garg, Xinqiang Xie, Chaitan Khosla, and David E. Cane
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Stereochemistry ,Polymers ,Molecular Conformation ,Stereoisomerism ,010402 general chemistry ,01 natural sciences ,Biochemistry ,Catalysis ,Article ,Polyketide ,chemistry.chemical_compound ,Colloid and Surface Chemistry ,Biosynthesis ,Salinomycin ,ATP synthase ,biology ,010405 organic chemistry ,Substrate (chemistry) ,General Chemistry ,0104 chemical sciences ,chemistry ,Polyketides ,biology.protein ,lipids (amino acids, peptides, and proteins) ,Epimer ,Polyketide Synthases ,Methyl group ,Ethers - Abstract
The polyketide synthases responsible for the biosynthesis of the polyether antibiotics nanchangmycin (1) and salinomycin (4) harbor a number of redox-inactive ketoreductase (KR0) domains that are implicated in the generation of C2-epimerized (2S)-2-methyl-3-ketoacyl-ACP intermediates. Evidence that the natural substrate for the polyether KR0 domains is, as predicted, a (2R)-2-methyl-3-ketoacyl-ACP intermediate, came from a newly developed coupled ketosynthase (KS)-ketoreductase (KR) assay that established that the decarboxylative condensation of methylmalonyl-CoA with S-propionyl-N-acetylcysteamine catalyzed by the Nan[KS1][AT1] didomain from module 1 of the nanchangmycin synthase generates exclusively the corresponding (2R)-2-methyl-3-ketopentanoyl-ACP (7a) product. In tandem equilibrium isotope exchange experiments, incubation of [2-2H]-(2R,3S)-2-methyl-3-hydroxypentanoyl-ACP (6a) with redox-active, epimerase-inactive EryKR6 from module 6 of the 6-deoxyerythronolide B synthase and catalytic quantities of NADP+ in the presence of redox-inactive, recombinant NanKR10 or NanKR50, from modules 1 and 5 of the nanchangmycin synthase, or recombinant SalKR70 from module 7 of the salinomycin synthase, resulted in first-order, time-dependent washout of deuterium from 6a. Control experiments confirmed that this washout was due to KR0-catalyzed isotope exchange of the reversibly-generated, transiently-formed oxidation product [2-2H]-(2R)-2-methyl-3-ketopentanoyl-ACP (7a), consistent with the proposed epimerase activity of each of the KR0 domains. Although they belong to the superfamily of short chain dehydrogenase-reductases, the epimerase-active KR0 domains from polyether synthases lack one or both residues of the conserved Tyr-Ser dyad that has previously been implicated in KR-catalyzed epimerizations.
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- 2017
17. Elucidation of the Cryptic Epimerase Activity of Redox-Inactive Ketoreductase Domains from Modular Polyketide Synthases by Tandem Equilibrium Isotope Exchange
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David E. Cane, Chaitan Khosla, Xinqiang Xie, Ashish Garg, and Adrian T. Keatinge-Clay
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Stereochemistry ,Racemases and Epimerases ,Biochemistry ,Catalysis ,Cofactor ,Substrate Specificity ,Polyketide ,Colloid and Surface Chemistry ,Protein structure ,Bacterial Proteins ,Binding site ,chemistry.chemical_classification ,Binding Sites ,ATP synthase ,biology ,Chemistry ,Communication ,Mutagenesis ,Stereoisomerism ,General Chemistry ,Deuterium ,Protein Structure, Tertiary ,Alcohol Oxidoreductases ,Enzyme ,Biocatalysis ,biology.protein ,NADPH binding ,Macrolides ,Oxidation-Reduction ,Polyketide Synthases ,NADP - Abstract
Many modular polyketide synthases harbor one or more redox-inactive domains of unknown function that are highly homologous to ketoreductase (KR) domains. A newly developed tandem equilibrium isotope exchange (EIX) assay has now established that such "KR(0)" domains catalyze the biosynthetically essential epimerization of transient (2R)-2-methyl-3-ketoacyl-ACP intermediates to the corresponding (2S)-2-methyl-3-ketoacyl-ACP diastereomers. Incubation of [2-(2)H]-(2R,3S)-2-methyl-3-hydroxypentanoyl-SACP ([2-(2)H]-3b) with the EryKR3(0) domain from module 3 of the 6-deoxyerythronolide B synthase, and the redox-active, nonepimerizing EryKR6 domain and NADP(+) resulted in time- and cofactor-dependent washout of deuterium from 3b, as a result of EryKR3(0)-catalyzed epimerization of transiently generated [2-(2)H]-2-methyl-3-ketopentanoyl-ACP (4). Similar results were obtained with redox-inactive PicKR3(0) from module 3 of the picromycin synthase. Four redox-inactive mutants of epimerase-active EryKR1 were engineered by mutagenesis of the NADPH binding site of this enzyme. Tandem EIX established that these EryKR1(0) mutants retained the intrinsic epimerase activity of the parent EryKR1 domain. These results establish the intrinsic epimerase activity of redox-inactive KR(0) domains, rule out any role for the NADPH cofactor in epimerization, and provide a general experimental basis for decoupling the epimerase and reductase activities of a large class of PKS domains.
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- 2014
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18. Comparative Analysis of the Substrate Specificity of trans- versus cis-Acyltransferases of Assembly Line Polyketide Synthases
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Chaitan Khosla, Briana J. Dunn, Thomas Robbins, Katharine R. Watts, and David E. Cane
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Models, Molecular ,Pyridones ,Stereochemistry ,Recombinant Fusion Proteins ,Context (language use) ,Biology ,Protein Engineering ,01 natural sciences ,Biochemistry ,Article ,Substrate Specificity ,03 medical and health sciences ,Polyketide ,Transacylation ,Protein structure ,Bacterial Proteins ,Catalytic Domain ,Protein Interaction Domains and Motifs ,Protein Structure, Quaternary ,Oxazoles ,030304 developmental biology ,0303 health sciences ,010405 organic chemistry ,Stereoisomerism ,Protein engineering ,Peptide Fragments ,Anti-Bacterial Agents ,0104 chemical sciences ,Malonyl Coenzyme A ,Protein Subunits ,Acyl carrier protein ,Acyltransferases ,Polyketides ,Acyltransferase ,Biocatalysis ,biology.protein ,Mutant Proteins ,Acyl Coenzyme A ,Macrolides ,Polyketide Synthases - Abstract
Due to their pivotal role in extender unit selection during polyketide biosynthesis, acyltransferase (AT) domains are important engineering targets. A subset of assembly line polyketide synthases (PKSs) are serviced by discrete, trans-acting ATs. Theoretically, these trans-ATs can complement an inactivated cis-AT, promoting introduction of a noncognate extender unit. This approach requires a better understanding of the substrate specificity and catalytic mechanism of naturally occurring trans-ATs. We kinetically analyzed trans-ATs from the disorazole and kirromycin synthases and compared them to a representative cis-AT from the 6-deoxyerythronolide B synthase (DEBS). During transacylation, the disorazole AT favored malonyl-CoA over methylmalonyl-CoA by >40000-fold, whereas the kirromycin AT favored ethylmalonyl-CoA over methylmalonyl-CoA by 20-fold. Conversely, the disorazole AT had broader specificity than its kirromycin counterpart for acyl carrier protein (ACP) substrates. The presence of the ACP had little effect on the specificity (k(cat)/K(M)) of the cis-AT domain for carboxyacyl-CoA substrates but had a marked influence on the corresponding specificity parameters for the trans-ATs, suggesting that these enzymes do not act strictly by a canonical ping-pong mechanism. To investigate the relevance of the kinetic analysis of isolated ATs in the context of intact PKSs, we complemented an in vitro AT-null DEBS assembly line with either trans-AT. Whereas the disorazole AT efficiently complemented the mutant PKS at substoichiometric protein ratios, the kirromycin AT was considerably less effective. Our findings suggest that knowledge of both carboxyacyl-CoA and ACP specificity is critical to the choice of a trans-AT in combination with a mutant PKS to generate novel polyketides.
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- 2014
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19. Assembly Line Polyketide Synthases: Mechanistic Insights and Unsolved Problems
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Chaitan Khosla, Daniel Herschlag, Christopher T. Walsh, and David E. Cane
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Models, Molecular ,biology ,Stereochemistry ,Extramural ,Polyketide biosynthesis ,Stereoisomerism ,Multienzyme complexes ,Biochemistry ,Protein Structure, Tertiary ,Stereocenter ,Polyketide ,Protein structure ,Models, Chemical ,Multienzyme Complexes ,Polyketide synthase ,Current Topic ,biology.protein ,Assembly line ,Polyketide Synthases - Abstract
Two hallmarks of assembly line polyketide synthases have motivated an interest in these unusual multienzyme systems, their stereospecificity and their capacity for directional biosynthesis. In this review, we summarize the state of knowledge regarding the mechanistic origins of these two remarkable features, using the 6-deoxyerythronolide B synthase as a prototype. Of the 10 stereocenters in 6-deoxyerythronolide B, the stereochemistry of nine carbon atoms is directly set by ketoreductase domains, which catalyze epimerization and/or diastereospecific reduction reactions. The 10th stereocenter is established by the sequential action of three enzymatic domains. Thus, the problem has been reduced to a challenge in mainstream enzymology, where fundamental gaps remain in our understanding of the structural basis for this exquisite stereochemical control by relatively well-defined active sites. In contrast, testable mechanistic hypotheses for the phenomenon of vectorial biosynthesis are only just beginning to emerge. Starting from an elegant theoretical framework for understanding coupled vectorial processes in biology [Jencks, W. P. (1980) Adv. Enzymol. Relat. Areas Mol. Biol. 51, 75–106], we present a simple model that can explain assembly line polyketide biosynthesis as a coupled vectorial process. Our model, which highlights the important role of domain–domain interactions, not only is consistent with recent observations but also is amenable to further experimental verification and refinement. Ultimately, a definitive view of the coordinated motions within and between polyketide synthase modules will require a combination of structural, kinetic, spectroscopic, and computational tools and could be one of the most exciting frontiers in 21st Century enzymology.
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- 2014
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20. Generation of Complexity in Fungal Terpene Biosynthesis: Discovery of a Multifunctional Cytochrome P450 in the Fumagillin Pathway
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Sourabh Dhingra, Yi Tang, Kenji Watanabe, Manami Fukutomi, Yit-Heng Chooi, Hsiao-Ching Lin, Yuta Tsunematsu, David E. Cane, Wei Xu, and Ana M. Calvo
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Stereochemistry ,Biochemistry ,Catalysis ,Article ,Hydroxylation ,chemistry.chemical_compound ,Polyketide ,Colloid and Surface Chemistry ,Cytochrome P-450 Enzyme System ,Dioxygenase ,Cyclohexanes ,Polyketide synthase ,medicine ,Fumagillin ,biology ,Molecular Structure ,Chemistry ,Terpenes ,Aspergillus fumigatus ,Cytochrome P450 ,General Chemistry ,Monooxygenase ,METAP2 ,biology.protein ,Fatty Acids, Unsaturated ,Sesquiterpenes ,medicine.drug - Abstract
Fumagillin (1), a meroterpenoid from Aspergillus fumigatus, is known for its antiangiogenic activity due to binding to human methionine aminopeptidase 2. 1 has a highly oxygenated structure containing a penta-substituted cyclohexane that is generated by oxidative cleavage of the bicyclic sesquiterpene β-trans-bergamotene. The chemical nature, order, and biochemical mechanism of all the oxygenative tailoring reactions has remained enigmatic despite the identification of the biosynthetic gene cluster and the use of targeted-gene deletion experiments. Here, we report the identification and characterization of three oxygenases from the fumagillin biosynthetic pathway, including a multifunctional cytochrome P450 monooxygenase, a hydroxylating nonheme-iron-dependent dioxygenase, and an ABM family monooxygenase for oxidative cleavage of the polyketide moiety. Most significantly, the P450 monooxygenase is shown to catalyze successive hydroxylation, bicyclic ring-opening, and two epoxidations that generate the sesquiterpenoid core skeleton of 1. We also characterized a truncated polyketide synthase with a ketoreductase function that controls the configuration at C-5 of hydroxylated intermediates.
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- 2014
21. Reprogramming the Chemodiversity of Terpenoid Cyclization by Remolding the Active Site Contour of epi-Isozizaene Synthase
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Ruiqiong Li, Julie A. Himmelberger, Wayne K. W. Chou, David E. Cane, Kevin M. Litwin, Golda G. Harris, and David W. Christianson
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Models, Molecular ,biology ,ATP synthase ,Molecular Structure ,Stereochemistry ,Terpenes ,Active site ,Substrate (chemistry) ,Streptomyces coelicolor ,Carbocation ,Sesquiterpene ,Lyase ,Crystallography, X-Ray ,Biochemistry ,Cyclase ,Terpenoid ,Article ,chemistry.chemical_compound ,Kinetics ,chemistry ,Bacterial Proteins ,Cyclization ,Catalytic Domain ,biology.protein ,Mutagenesis, Site-Directed - Abstract
The class I terpenoid cyclase epi-isozizaene synthase (EIZS) utilizes the universal achiral isoprenoid substrate, farnesyl diphosphate, to generate epi-isozizaene as the predominant sesquiterpene cyclization product and at least five minor sesquiterpene products, making EIZS an ideal platform for the exploration of fidelity and promiscuity in a terpenoid cyclization reaction. The hydrophobic active site contour of EIZS serves as a template that enforces a single substrate conformation, and chaperones subsequently formed carbocation intermediates through a well-defined mechanistic sequence. Here, we have used the crystal structure of EIZS as a guide to systematically remold the hydrophobic active site contour in a library of 26 site-specific mutants. Remolded cyclization templates reprogram the reaction cascade not only by reproportioning products generated by the wild-type enzyme but also by generating completely new products of diverse structure. Specifically, we have tripled the overall number of characterized products generated by EIZS. Moreover, we have converted EIZS into six different sesquiterpene synthases: F96A EIZS is an (E)-β-farnesene synthase, F96W EIZS is a zizaene synthase, F95H EIZS is a β-curcumene synthase, F95M EIZS is a β-acoradiene synthase, F198L EIZS is a β-cedrene synthase, and F96V EIZS and W203F EIZS are (Z)-γ-bisabolene synthases. Active site aromatic residues appear to be hot spots for reprogramming the cyclization cascade by manipulating the stability and conformation of critical carbocation intermediates. A majority of mutant enzymes exhibit only relatively modest 2-100-fold losses of catalytic activity, suggesting that residues responsible for triggering substrate ionization readily tolerate mutations deeper in the active site cavity.
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- 2014
22. Coupled Methyl Group Epimerization and Reduction by Polyketide Synthase Ketoreductase Domains. Ketoreductase-Catalyzed Equilibrium Isotope Exchange
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Chaitan Khosla, David E. Cane, and Ashish Garg
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Time Factors ,Molecular Structure ,Stereochemistry ,Alcohol oxidoreductase ,General Chemistry ,Deuterium ,Biochemistry ,Article ,Catalysis ,Alcohol Oxidoreductases ,chemistry.chemical_compound ,Polyketide ,Colloid and Surface Chemistry ,Bacterial Proteins ,chemistry ,Biocatalysis ,Molecule ,Epimer ,Oxidation-Reduction ,Methyl group - Abstract
Incubation of [2-2H]-(2S,3R)-2-methyl-3-hydroxypentanoyl-SACP ([2-2H]-1a) with the epimerizing ketoreductase domain EryKR1 in the presence of a catalytic amount NADP+ (0.05 equiv) resulted in time-and cofactor-dependent washout of deuterium from 1a, as a result of equilibrium isotope exchange of transiently generated [2-2H]-2-methyl-3-ketopentanoyl-ACP (2). Incubations of [2-2H]-(2S,3S)-2-methyl-3-hydroxypentanoyl-SACP ([2-2H]-1b) with RifKR7 and with NysKR1 also resulted in time-dependent loss of deuterium. By contrast, incubations of [2-2H]-(2R,3S)-2-methyl-3-hydroxypentanoyl-SACP ([2-2H]-1c) and [2-2H]-(2R,3R)-2-methyl-3-hydroxypentanoyl-SACP ([2-2H]-1d) with the non-epimerizing ketoreductase domains EryKR6 and TylKR1, respectively, did not result in any significant washout of deuterium. The isotope exchange assay directly establishes that specific polyketide synthase ketoreductase domains also have an intrinsic epimerase activity, thus enabling mechanistic analysis of a key determinant of polyketide stereocomplexity.
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- 2013
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23. Mechanistic Insights from the Binding of Substrate and Carbocation Intermediate Analogues to Aristolochene Synthase
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Rudolf Konrad Allemann, Edward L. D'Antonio, Juan A. Faraldos, David W. Christianson, Marine Janvier, David E. Cane, Naeemah Al-Lami, and Mengbin Chen
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Models, Molecular ,Cations, Divalent ,Stereochemistry ,Isomerase ,Carbocation ,Biochemistry ,Article ,Catalysis ,chemistry.chemical_compound ,Polyisoprenyl Phosphates ,Catalytic Domain ,Magnesium ,Isomerases ,Binding Sites ,biology ,Bicyclic molecule ,Hydrogen bond ,Iminium ,Active site ,Hydrogen Bonding ,Stereoisomerism ,Diphosphates ,Quaternary Ammonium Compounds ,Aristolochene synthase ,Aspergillus ,chemistry ,biology.protein ,Aristolochene ,Sesquiterpenes - Abstract
Aristolochene synthase, a metal-dependent sesquiterpene cyclase from Aspergillus terreus, catalyzes the ionization-dependent cyclization of farnesyl diphosphate (FPP) to form the bicyclic eremophilane (+)-aristolochene with perfect structural and stereochemical precision. Here, we report the X-ray crystal structure of aristolochene synthase complexed with three Mg(2+) ions and the unreactive substrate analogue farnesyl-S-thiolodiphosphate (FSPP), showing that the substrate diphosphate group is anchored by metal coordination and hydrogen bond interactions identical to those previously observed in the complex with three Mg(2+) ions and inorganic pyrophosphate (PPi). Moreover, the binding conformation of FSPP directly mimics that expected for productively bound FPP, with the exception of the precise alignment of the C-S bond with regard to the C10-C11 π system that would be required for C1-C10 bond formation in the first step of catalysis. We also report crystal structures of aristolochene synthase complexed with Mg(2+)3-PPi and ammonium or iminium analogues of bicyclic carbocation intermediates proposed for the natural cyclization cascade. Various binding orientations are observed for these bicyclic analogues, and these orientations appear to be driven by favorable electrostatic interactions between the positively charged ammonium group of the analogue and the negatively charged PPi anion. Surprisingly, the active site is sufficiently flexible to accommodate analogues with partially or completely incorrect stereochemistry. Although this permissiveness in binding is unanticipated, based on the stereochemical precision of catalysis that leads exclusively to the (+)-aristolochene stereoisomer, it suggests the ability of the active site to enable controlled reorientation of intermediates during the cyclization cascade. Taken together, these structures illuminate important aspects of the catalytic mechanism.
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- 2013
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24. Substitution of a Single Amino Acid Reverses the Regiospecificity of the Baeyer-Villiger Monooxygenase PntE in the Biosynthesis of the Antibiotic Pentalenolactone
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Chengde Zhang, Zixin Deng, Lu Zhu, Wensheng Xiang, Haruo Ikeda, Dongqing Zhu, David E. Cane, Shiwen Wu, and Ke Chen
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Stereochemistry ,Mutant ,Mutation, Missense ,Stereoisomerism ,Biology ,010402 general chemistry ,Arginine ,01 natural sciences ,Biochemistry ,Article ,Mixed Function Oxygenases ,chemistry.chemical_compound ,Biosynthesis ,Bacterial Proteins ,Amino Acid Sequence ,Peptide sequence ,chemistry.chemical_classification ,Molecular Structure ,Sequence Homology, Amino Acid ,010405 organic chemistry ,Lysine ,Genetic Complementation Test ,Monooxygenase ,Streptomyces ,0104 chemical sciences ,Amino acid ,Anti-Bacterial Agents ,Biosynthetic Pathways ,Enzyme ,chemistry ,Amino Acid Substitution ,Models, Chemical ,Sesquiterpenes ,Lactone - Abstract
In the biosynthesis of pentalenolactone (1), PenE and PntE, orthologous proteins from Streptomyces exfoliatus and S. arenae, respectively, catalyze the flavin-dependent Baeyer-Villiger oxidation of 1-deoxy-11-oxopentalenic acid (4) to the lactone pentalenolactone D (5), in which the less-substituted methylene carbon has migrated. By contrast, the paralogous PtlE enzyme from S. avermitilis catalyzes the oxidation of 4 to neopentalenolactone D (6), in which the more substituted methane substitution has undergone migration. We report the design and analysis of 13 single and multiple mutants of PntE mutants in order to identify the key amino acids that contribute to the regiospecificity of these two classes of Baeyer-Villiger monooxygenases. The L185S mutation in PntE reversed the observed regiospecificity of PntE such that all recombinant PntE mutants harboring this L185S mutation acquired the characteristic regiospecificity of PtlE, catalyzing the conversion of 4 to 6 as the major product. The recombinant PntE mutant harboring R484L exhibited reduced regiospecificity, generating a mixture of lactones containing more than 17% of 6. These in vitro results were corroborated by analysis of the complementation of the S. avermitilis ΔptlED double deletion mutant with pntE mutants, such that pntE mutants harboring L185S produced 6 as the major product, while complemention of the ΔptlED deletion mutant with pntE mutants carrying the R484L mutation gave 6 as more than 33% of the total lactone product mixture.
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- 2016
25. Incubation of 2-methylisoborneol synthase with the intermediate analog 2-methylneryl diphosphate
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Wayne Kw Chou, David E. Cane, and Colin A. Gould
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0301 basic medicine ,Stereochemistry ,terpene synthase ,Alkenes ,010402 general chemistry ,Crystallography, X-Ray ,01 natural sciences ,Gas Chromatography-Mass Spectrometry ,Article ,03 medical and health sciences ,chemistry.chemical_compound ,Organophosphorus Compounds ,Drug Discovery ,Enzyme kinetics ,Incubation ,Pharmacology ,Camphanes ,030102 biochemistry & molecular biology ,ATP synthase ,biology ,2-methylisoborneol ,0104 chemical sciences ,Monoterpene cyclase ,Kinetics ,2-methylneryl diphosphate ,homo-monoterpene ,chemistry ,Cyclization ,biology.protein ,Monoterpenes ,2-Methylisoborneol - Abstract
Incubation of synthetic 2-methylneryl diphosphate (2-MeNPP, 10) with 2-methylisoborneol synthase (MIBS) gave a mixture of products that differed significantly from that derived from the natural substrate (E)-2-methylgeranyl disphosphate (3, 2-MeGPP. The proportion of (−)-2-methylisoborneol (1) decreased from 89% to 17% while that of 2-methylenebornane (4) increased from 10% to 26%, with the relative yields of the isomeric homo-monoterpenes 2-methyl-2-bornene (5) and 1-methylcamphene (6) remaining essentially unchanged (
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- 2016
26. Roles of Conserved Active Site Residues in the Ketosynthase Domain of an Assembly Line Polyketide Synthase
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David E. Cane, Chaitan Khosla, Thomas Robbins, and Joshuah Kapilivsky
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0301 basic medicine ,Models, Molecular ,Decarboxylation ,Stereochemistry ,Mutant ,Carboxylic Acids ,01 natural sciences ,Biochemistry ,Article ,Conserved sequence ,03 medical and health sciences ,Polyketide ,Polyketide synthase ,Catalytic Domain ,Amino Acid Sequence ,Conserved Sequence ,biology ,ATP synthase ,010405 organic chemistry ,Mutagenesis ,Active site ,0104 chemical sciences ,030104 developmental biology ,Mutation ,biology.protein ,Mutagenesis, Site-Directed ,Polyketide Synthases - Abstract
Ketosynthase (KS) domains of assembly line polyketide synthases (PKSs) catalyze intermodular translocation of the growing polyketide chain as well as chain elongation via decarboxylative Claisen condensation. The mechanistic roles of ten conserved residues in the KS domain of Module 1 of the 6-deoxyerythronolide B synthase were interrogated via site-directed mutagenesis and extensive biochemical analysis. Although the C211A mutant at the KS active site exhibited no turnover activity, it was still a competent methylmalonyl-ACP decarboxylase. The H346A mutant exhibited reduced rates of both chain translocation and chain elongation, with a greater effect on the latter half-reaction. H384 contributed to methylmalonyl-ACP decarboxylation, whereas K379 promoted C-C bond formation. S315 played a role in coupling decarboxylation to C-C bond formation. These findings support a mechanism for the translocation and elongation half-reactions that provides a well-defined starting point for further analysis of the key chain-building domain in assembly line PKSs.
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- 2016
27. Probing the Role of Active Site Water in the Sesquiterpene Cyclization Reaction Catalyzed by Aristolochene Synthase
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David W. Christianson, Wayne K. W. Chou, Naeemah Al-Lami, David E. Cane, Mengbin Chen, Rudolf Konrad Allemann, and Juan A. Faraldos
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0301 basic medicine ,Stereochemistry ,Kinetics ,Mutation, Missense ,Isomerase ,Carbocation ,Crystallography, X-Ray ,Biochemistry ,Article ,Fungal Proteins ,03 medical and health sciences ,chemistry.chemical_compound ,Catalytic Domain ,Isomerases ,030102 biochemistry & molecular biology ,biology ,Bicyclic molecule ,Chemistry ,Hydrogen bond ,Active site ,Water ,Aristolochene synthase ,030104 developmental biology ,Aspergillus ,Amino Acid Substitution ,biology.protein ,Aristolochene ,Sesquiterpenes - Abstract
Aristolochene synthase (ATAS) is a high-fidelity terpenoid cyclase that converts farnesyl diphosphate exclusively into the bicyclic hydrocarbon aristolochene. Previously determined crystal structures of ATAS complexes revealed trapped active site water molecules that could potentially interact with catalytic intermediates: water "w" hydrogen bonds with S303 and N299, water molecules "w1" and "w2" hydrogen bond with Q151, and a fourth water molecule coordinates to the Mg(2+)C ion. There is no obvious role for water in the ATAS mechanism because the enzyme exclusively generates a hydrocarbon product. Thus, these water molecules are tightly controlled so that they cannot react with carbocation intermediates. Steady-state kinetics and product distribution analyses of eight ATAS mutants designed to perturb interactions with active site water molecules (S303A, S303H, S303D, N299A, N299L, N299A/S303A, Q151H, and Q151E) indicate relatively modest effects on catalysis but significant effects on sesquiterpene product distributions. X-ray crystal structures of S303A, N299A, N299A/S303A, and Q151H mutants reveal minimal perturbation of active site solvent structure. Seven of the eight mutants generate farnesol and nerolidol, possibly resulting from addition of the Mg(2+)C-bound water molecule to the initially formed farnesyl cation, but no products are generated that would suggest enhanced reactivity of other active site water molecules. However, intermediate germacrene A tends to accumulate in these mutants. Thus, apart from the possible reactivity of Mg(2+)C-bound water, active site water molecules in ATAS are not directly involved in the chemistry of catalysis but instead contribute to the template that governs the conformation of the flexible substrate and carbocation intermediates.
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- 2016
28. The Epimerase and Reductase Activities of Polyketide Synthase Ketoreductase Domains Utilize the Same Conserved Tyrosine and Serine Residues
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Chaitan Khosla, Xinqiang Xie, Adrian T. Keatinge-Clay, David E. Cane, and Ashish Garg
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0301 basic medicine ,Models, Molecular ,Stereochemistry ,Mutant ,Molecular Sequence Data ,Racemases and Epimerases ,Gene Expression ,Sequence alignment ,010402 general chemistry ,01 natural sciences ,Biochemistry ,Article ,Conserved sequence ,Substrate Specificity ,Serine ,03 medical and health sciences ,Protein structure ,Bacterial Proteins ,Catalytic Domain ,Escherichia coli ,Amino Acid Sequence ,Tyrosine ,Peptide sequence ,Conserved Sequence ,biology ,Bacteria ,Chemistry ,Active site ,0104 chemical sciences ,Protein Structure, Tertiary ,Alcohol Oxidoreductases ,030104 developmental biology ,biology.protein ,Sequence Alignment - Abstract
The role of the conserved active site tyrosine and serine residues in epimerization catalyzed by polyketide synthase ketoreductase (PKS KR) domains has been investigated. Both mutant and wild-type forms of epimerase-active KR domains, including the intrinsically redox-inactive EryKR3° and PicKR3° as well as redox-inactive mutants of EryKR1, were incubated with [2-(2)H]-(2R,3S)-2-methyl-3-hydroxypentanoyl-SACP ([2-(2)H]-2) and 0.05 equiv of NADP(+) in the presence of the redox-active, epimerase-inactive EryKR6 domain. The residual epimerase activity of each mutant was determined by tandem equilibrium isotope exchange, in which the first-order, time-dependent washout of isotope from 2 was monitored by liquid chromatography-tandem mass spectrometry with quantitation of the deuterium content of the diagnostic pantetheinate ejection fragment (4). Replacement of the active site Tyr or Ser residues, alone or together, significantly reduced the observed epimerase activity of each KR domain with minimal effect on substrate binding. Our results demonstrate that the epimerase and reductase activities of PKS KR domains share a common active site, with both reactions utilizing the same pair of Tyr and Ser residues.
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- 2016
29. Recognition of acyl carrier proteins by ketoreductases in assembly line polyketide synthases
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Chaitan Khosla, Matthew P. Ostrowski, and David E. Cane
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0301 basic medicine ,animal structures ,Stereochemistry ,Alcohol oxidoreductase ,010402 general chemistry ,01 natural sciences ,Article ,Stereocenter ,Substrate Specificity ,03 medical and health sciences ,Polyketide ,chemistry.chemical_compound ,Biosynthesis ,Bacterial Proteins ,Catalytic Domain ,Drug Discovery ,Acyl Carrier Protein ,Pharmacology ,chemistry.chemical_classification ,ATP synthase ,biology ,0104 chemical sciences ,Acyl carrier protein ,Alcohol Oxidoreductases ,030104 developmental biology ,Enzyme ,Biochemistry ,chemistry ,biology.protein ,lipids (amino acids, peptides, and proteins) ,Assembly line ,Polyketide Synthases - Abstract
Ketoreductases (KRs) are the most widespread tailoring domains found in individual modules of assembly line polyketide synthases (PKSs), and are responsible for controlling the configurations of both the α-methyl and β-hydroxyl stereogenic centers in the growing polyketide chain. Because they recognize substrates that are covalently bound to acyl carrier proteins (ACPs) within the same PKS module, we sought to quantify the extent to which protein-protein recognition contributes to the turnover of these oxidoreductive enzymes using stand-alone domains from the 6-deoxyerythronolide B synthase (DEBS). Reduced 2-methyl-3-hydroxyacyl-ACP substrates derived from two enantiomeric acyl chains and four distinct ACP domains were synthesized and presented to four distinct KR domains. Two KRs, from DEBS modules 2 and 5, displayed little preference for oxidation of substrates tethered to their cognate ACP domains over those attached to the other ACP domains tested. In contrast, the KR from DEBS module 1 showed a ca. 10-50-fold preference for substrate attached to its native ACP domain, whereas the KR from DEBS module 6 actually displayed a ca. 10-fold preference for the ACP from DEBS module 5. Our findings suggest that recognition of the ACP by a KR domain is unlikely to affect the rate of native assembly line polyketide biosynthesis. In some cases, however, unfavorable KR-ACP interactions may suppress the rate of substrate processing when KR domains are swapped to construct hybrid PKS modules.
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- 2016
30. Precursor Directed Biosynthesis of an Orthogonally Functional Erythromycin Analogue: Selectivity in the Ribosome Macrolide Binding Pocket
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Chaitan Khosla, David E. Cane, Joseph D. Puglisi, Colin J. B. Harvey, and Vijay S. Pande
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Models, Molecular ,medicine.drug_class ,Stereochemistry ,Antibiotics ,Erythromycin ,Biochemistry ,Ribosome ,Article ,Catalysis ,Industrial Microbiology ,chemistry.chemical_compound ,Colloid and Surface Chemistry ,Biosynthesis ,Escherichia coli ,medicine ,Protein biosynthesis ,Binding site ,Binding Sites ,Natural product ,General Chemistry ,Macrolide binding ,Anti-Bacterial Agents ,chemistry ,Ribosomes ,Bacillus subtilis ,medicine.drug - Abstract
The macrolide antibiotic erythromycin A and its semisynthetic analogues have been among the most useful antibacterial agents for the treatment of infectious diseases. Using a recently developed chemical genetic strategy for precursor-directed biosynthesis and colony bioassay of 6-deoxyerythromycin D analogues, we identified a new class of alkynyl- and alkenyl-substituted macrolides with activities comparable to that of the natural product. Further analysis revealed a marked and unexpected dependence of antibiotic activity on the size and degree of unsaturation of the precursor. Based on these leads, we also report the precursor-directed biosynthesis of 15-propargyl erythromycin A, a novel antibiotic that not only is as potent as erythromycin A with respect to its ability to inhibit bacterial growth and cell-free ribosomal protein biosynthesis but also harbors an orthogonal functional group that is capable of facile chemical modification.
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- 2012
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31. Structure of Geranyl Diphosphate C-Methyltransferase from Streptomyces coelicolor and Implications for the Mechanism of Isoprenoid Modification
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David E. Cane, Mustafa Köksal, David W. Christianson, and Wayne K. W. Chou
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S-Adenosylmethionine ,Protein Conformation ,Stereochemistry ,Streptomyces coelicolor ,Crystallography, X-Ray ,Methylation ,Biochemistry ,Cyclase ,Article ,Cofactor ,Bacterial Proteins ,Transferase ,Demethylation ,Cofactor binding ,Binding Sites ,biology ,ATP synthase ,Chemistry ,organic chemicals ,Active site ,Methyltransferases ,biology.organism_classification ,Diphosphates ,biology.protein ,Diterpenes - Abstract
Geranyl diphosphate C-methyltransferase (GPPMT) from Streptomyces coelicolor A3(2) is the first methyltransferase discovered that modifies an acyclic isoprenoid diphosphate, geranyl diphosphate (GPP), to yield a non-canonical acyclic allylic diphosphate product, 2-methylgeranyl diphosphate, which serves as the substrate for a subsequent cyclization reaction catalyzed by a terpenoid cyclase, methylisoborneol synthase. Here, we report the crystal structures of GPPMT in complex with GPP or the substrate analogue geranyl-S-thiolodiphosphate (GSPP) along with S-adenosyl-l-homocysteine in the cofactor binding site, resulting from in situ demethylation of S-adenosyl-l-methionine, at 2.05 Å and 1.82 Å resolution, respectively. These structures suggest that both GPP and GSPP can undergo catalytic methylation in crystalline GPPMT, followed by dissociation of the isoprenoid product. S-adenosyl-l-homocysteine remains bound in the active site, however, and does not exchange with a fresh molecule of cofactor S-adenosyl-l-methionine. These structures provide important clues regarding the molecular mechanism of the reaction, especially with regard to the face of the 2,3 double bond of GPP that is methylated as well as the stabilization of the resulting carbocation intermediate through cation-π interactions.
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- 2012
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32. Structure of 2-Methylisoborneol Synthase from Streptomyces coelicolor and Implications for the Cyclization of a Noncanonical C-Methylated Monoterpenoid Substrate
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David W. Christianson, Wayne K. W. Chou, Mustafa Köksal, and David E. Cane
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Models, Molecular ,Protein Conformation ,Stereochemistry ,Streptomyces coelicolor ,Crystallography, X-Ray ,Biochemistry ,Cyclase ,Article ,Farnesyl diphosphate synthase ,Bacterial Proteins ,Organic chemistry ,neoplasms ,Binding Sites ,Camphanes ,ATP synthase ,biology ,Chemistry ,fungi ,biology.organism_classification ,Lyase ,digestive system diseases ,Terpenoid ,Protein tertiary structure ,Cyclization ,Monoterpenes ,biology.protein ,Protein quaternary structure - Abstract
The crystal structure of 2-methylisoborneol synthase (MIBS) from Streptomyces coelicolor A3(2) has been determined in complex with substrate analogues geranyl-S-thiolodiphosphate and 2-fluorogeranyl diphosphate at 1.80 Å and 1.95 Å resolution, respectively. This terpenoid cyclase catalyzes the cyclization of the naturally-occuring, non-canonical C-methylated isoprenoid substrate, 2-methylgeranyl diphosphate, to form the bicyclic product 2-methylisoborneol, a volatile C11 homoterpene alcohol with an earthy, musty odor. While MIBS adopts the tertiary structure of a class I terpenoid cyclase, its dimeric quaternary structure differs from that previously observed in dimeric terpenoid cyclases from plants and fungi. The quaternary structure of MIBS is nonetheless similar in some respects to that of dimeric farnesyl diphosphate synthase, which is not a cyclase. The structures of MIBS complexed with substrate analogues provide insights regarding differences in the catalytic mechanism of MIBS and the mechanisms of (+)-bornyl diphosphate synthase and endo-fenchol synthase, plant cyclases that convert geranyl diphosphate into products with closely related bicyclic bornyl skeletons, but distinct structures and stereochemistries.
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- 2012
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33. Reprogramming a module of the 6-deoxyerythronolide B synthase for iterative chain elongation
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Chaitan Khosla, Shiven Kapur, Alice Y. Chen, Satoshi Yuzawa, David E. Cane, Sanketha Kenthirapalan, and Brian Lowry
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Models, Molecular ,Multidisciplinary ,ATP synthase ,biology ,Stereochemistry ,Molecular Sequence Data ,Protein domain ,Protein engineering ,Biological Sciences ,Protein Engineering ,Substrate Specificity ,Cell biology ,Protein Transport ,Polyketide ,Acyl carrier protein ,Chain (algebraic topology) ,Polyketides ,6-Deoxyerythronolide B synthase ,Acyl Carrier Protein ,Biocatalysis ,biology.protein ,Amino Acid Sequence ,Polyketide Synthases ,Peptide sequence - Abstract
Multimodular polyketide synthases (PKSs) have an assembly line architecture in which a set of protein domains, known as a module, participates in one round of polyketide chain elongation and associated chemical modifications, after which the growing chain is translocated to the next PKS module. The ability to rationally reprogram these assembly lines to enable efficient synthesis of new polyketide antibiotics has been a long-standing goal in natural products biosynthesis. We have identified a ratchet mechanism that can explain the observed unidirectional translocation of the growing polyketide chain along the 6-deoxyerythronolide B synthase. As a test of this model, module 3 of the 6-deoxyerythronolide B synthase has been reengineered to catalyze two successive rounds of chain elongation. Our results suggest that high selectivity has been evolutionarily programmed at three types of protein–protein interfaces that are present repetitively along naturally occurring PKS assembly lines.
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- 2012
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34. Exploration and Mining of the Bacterial Terpenome
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Haruo Ikeda and David E. Cane
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Alkyl and Aryl Transferases ,Molecular Structure ,biology ,Terpenes ,Extramural ,Gram-positive bacteria ,General Medicine ,General Chemistry ,Gram-Positive Bacteria ,biology.organism_classification ,Article ,Terpenoid ,Terpene ,Biochemistry - Abstract
Tens of thousands of terpenoids are present in both terrestrial and marine plants, as well as fungi. In the last 5-10 years, however, it has become evident that terpenes are also produced by numerous bacteria, especially soil-dwelling Gram-positive organisms such as Streptomyces and other Actinomycetes. Although some microbial terpenes, such as geosmin, the degraded sesquiterpene responsible for the smell of moist soil, the characteristic odor of the earth itself, have been known for over 100 years, few terpenoids have been identified by classical structure- or activity-guided screening of bacterial culture extracts. In fact, the majority of cyclic terpenes from bacterial species have only recently been uncovered by the newly developed techniques of "genome mining". In this new paradigm for biochemical discovery, bacterial genome sequences are first analyzed with powerful bioinformatic tools, such as the BLASTP program or Profile Hidden Markov models, to screen for and identify conserved protein sequences harboring a characteristic set of universally conserved functional domains typical of all terpene synthases. Of particular importance is the presence of variants of two universally conserved domains, the aspartate-rich DDXX(D/E) motif and the NSE/DTE triad, (N/D)DXX(S/T)XX(K/R)(D/E). Both domains have been implicated in the binding of the essential divalent cation, typically Mg(2+), that is required for cyclization of the universal acyclic terpene precursors, such as farnesyl and geranyl diphosphate. The low level of overall sequence similarity among terpene synthases, however, has so far precluded any simple correlation of protein sequence with the structure of the cyclized terpene product. The actual biochemical function of a cryptic bacterial (or indeed any) terpene synthase must therefore be determined by direct experiment. Two common approaches are (i) incubation of the expressed recombinant protein with acyclic allylic diphosphate substrates and identification of the resultant terpene hydrocarbon or alcohol and (ii) in vivo expression in engineered bacterial hosts that can support the production of terpene metabolites. One of the most attractive features of the coordinated application of genome mining and biochemical characterization is that the discovery of natural products is directly coupled to the simultaneous discovery and exploitation of the responsible biosynthetic genes and enzymes. Bacterial genome mining has proved highly rewarding scientifically, already uncovering more than a dozen newly identified cyclic terpenes (many of them unique to bacteria), as well as several novel cyclization mechanisms. Moreover, bioinformatic analysis has identified more than 120 presumptive genes for bacterial terpene synthases that are now ripe for exploration. In this Account, we review a particularly rich vein we have mined in the genomes of two model Actinomycetes, Streptomyces coelicolor and Streptomyces avermitilis, from which the entire set of terpenoid biosynthetic genes and pathways have now been elucidated. In addition, studies of terpenoid biosynthetic gene clusters have revealed a wealth of previously unknown oxidative enzymes, including cytochromes P450, non-heme iron-dependent dioxygenases, and flavin monooxygenases. We have shown that these enzymes catalyze a variety of unusual biochemical reactions, including two-step ketonization of methylene groups, desaturation-epoxidation of secondary methyl groups, and pathway-specific Baeyer-Villiger oxidations of cyclic ketones.
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- 2011
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35. Structure and Mechanism of the trans-Acting Acyltransferase from the Disorazole Synthase
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Chaitan Khosla, Xi Jin, Fong Tian Wong, David E. Cane, and Irimpan I. Mathews
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Stereochemistry ,Molecular Sequence Data ,Streptomyces coelicolor ,Crystallography, X-Ray ,Biochemistry ,Article ,Substrate Specificity ,Structure-Activity Relationship ,Protein structure ,Acyl Carrier Protein ,Transferase ,Structure–activity relationship ,Amino Acid Sequence ,Oxazoles ,Alanine ,ATP synthase ,biology ,Escherichia coli Proteins ,biology.organism_classification ,Protein Structure, Tertiary ,Acyl carrier protein ,Acyltransferase ,Mutagenesis, Site-Directed ,biology.protein ,Macrolides ,Trans-acting ,Fatty Acid Synthases ,Crystallization ,Acyltransferases - Abstract
The 1.51 Å resolution X-ray crystal structure of the trans-acyltransferase (AT) from the "AT-less" disorazole synthase (DSZS) and that of its acetate complex at 1.35 Å resolution are reported. Separately, comprehensive alanine-scanning mutagenesis of one of its acyl carrier protein substrates (ACP1 from DSZS) led to the identification of a conserved Asp45 residue on the ACP, which contributes to the substrate specificity of this unusual enzyme. Together, these experimental findings were used to derive a model for the selective association of the DSZS AT and its ACP substrate. With a goal of structurally characterizing the AT-ACP interface, a strategy was developed for covalently cross-linking the active site Ser → Cys mutant of the DSZS AT to its ACP substrate and for purifying the resulting AT-ACP complex to homogeneity. The S86C DSZS AT mutant was found to be functional, albeit with a transacylation efficiency 200-fold lower than that of its wild-type counterpart. Our findings provide new insights as well as new opportunities for high-resolution analysis of an important protein-protein interface in polyketide synthases.
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- 2011
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36. Probing the interactions of an acyl carrier protein domain from the 6-deoxyerythronolide B synthase
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David E. Cane, Shiven Kapur, Chaitan Khosla, Dieter Seebach, Louise K. Charkoudian, Stefania Capone, Antonio Togni, and Corey W. Liu
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Models, Molecular ,biology ,Stereochemistry ,Protein subunit ,Biochemistry ,Article ,Protein Structure, Tertiary ,Protein–protein interaction ,Acyl carrier protein ,Polyketide ,Protein structure ,Catalytic cycle ,6-Deoxyerythronolide B synthase ,Polyketide synthase ,Protein Interaction Mapping ,Acyl Carrier Protein ,biology.protein ,Nuclear Magnetic Resonance, Biomolecular ,Polyketide Synthases ,Molecular Biology ,Acyltransferases ,Saccharopolyspora - Abstract
The assembly-line architecture of polyketide synthases (PKSs) provides an opportunity to rationally reprogram polyketide biosynthetic pathways to produce novel antibiotics. A fundamental challenge toward this goal is to identify the factors that control the unidirectional channeling of reactive biosynthetic intermediates through these enzymatic assembly lines. Within the catalytic cycle of every PKS module, the acyl carrier protein (ACP) first collaborates with the ketosynthase (KS) domain of the paired subunit in its own homodimeric module so as to elongate the growing polyketide chain and then with the KS domain of the next module to translocate the newly elongated polyketide chain. Using NMR spectroscopy, we investigated the features of a structurally characterized ACP domain of the 6-deoxyerythronolide B synthase that contribute to its association with its KS translocation partner. Not only were we able to visualize selective protein-protein interactions between the two partners, but also we detected a significant influence of the acyl chain substrate on this interaction. A novel reagent, CF₃-S-ACP, was developed as a ¹⁹F NMR spectroscopic probe of protein-protein interactions. The implications of our findings for understanding intermodular chain translocation are discussed.
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- 2011
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37. Genome Mining in Streptomyces. Discovery of an Unprecedented P450-Catalyzed Oxidative Rearrangement That Is the Final Step in the Biosynthesis of Pentalenolactone
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Dongqing Zhu, Haruo Ikeda, David E. Cane, and Myung-Ji Seo
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Reductase ,medicine.disease_cause ,Biochemistry ,Streptomyces ,Article ,Catalysis ,chemistry.chemical_compound ,Colloid and Surface Chemistry ,Cytochrome P-450 Enzyme System ,Biosynthesis ,medicine ,Cloning, Molecular ,Escherichia coli ,Ferredoxin ,Sequence Deletion ,biology ,General Chemistry ,Streptomyces arenae ,biology.organism_classification ,Anti-Bacterial Agents ,Complementation ,chemistry ,Sesquiterpenes ,Streptomyces avermitilis - Abstract
The penM and pntM genes from the pentalenolactone biosynthetic gene clusters of Streptomyces exfoliatus UC5319 and Streptomyces arenae TÜ469 were predicted to encode orthologous cytochrome P450s, CYP161C3 and CYP161C2, responsible for the final step in the biosynthesis of the sesquiterpenoid antibiotic pentalenolactone (1). Synthetic genes optimized for expression in Escherichia coli were used to obtain recombinant PenM and PntM, each carrying an N-terminal His(6)-tag. Both proteins showed typical reduced-CO UV maxima at 450 nm, and each bound the predicted substrate, pentalenolactone F (4), with K(D) values of 153 ± 14 and 126 ± 11 μM for PenM and PntM, respectively, as determined by UV shift titrations. PenM and PntM both catalyzed the oxidative rearrangement of 4 to 1 when incubated in the presence of NADPH, spinach ferredoxin, ferredoxin reductase, and O(2). The steady-state kinetic parameters were k(cat) = 10.5 ± 1.7 min(-1) and K(m) = 340 ± 100 μM 4 for PenM and k(cat) = 8.8 ± 0.9 min(-1) and K(m) = 430 ± 100 μM 4 for PntM. The in vivo function of both gene products was confirmed by the finding that the corresponding deletion mutants S. exfoliatus/ΔpenM ZD22 and S. arenae/ΔpntM ZD23 no longer produced pentalenolactone but accumulated the precursor pentalenolactone F. Complementation of each deletion mutant with either penM or pntM restored production of antibiotic 1. Pentalenolactone was also produced by an engineered strain of Streptomyces avermitilis that had been complemented with pntE, pntD, and either pntM or penM, as well as the S. avermitilis electron-transport genes for ferredoxin and ferrodoxin reductase, fdxD and fprD.
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- 2011
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38. Two Distinct Mechanisms for TIM Barrel Prenyltransferases in Bacteria
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Deborah L. Perlstein, David E. Cane, Suzanne Walker, Emma Doud, and Manuel Wolpert
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chemistry.chemical_classification ,Alkyl and Aryl Transferases ,Bacteria ,Sequence Homology, Amino Acid ,Double bond ,Chemistry ,Stereochemistry ,Molecular Sequence Data ,Prenyltransferase ,General Chemistry ,Dimethylallyltranstransferase ,Biochemistry ,Article ,Catalysis ,Triosephosphate isomerase ,Colloid and Surface Chemistry ,Prenylation ,Nucleophile ,TIM barrel ,Amino Acid Sequence ,Isomerization ,Triose-Phosphate Isomerase - Abstract
The reactions of two bacterial TIM barrel prenyltransferases (PTs), MoeO5 and PcrB, were explored. MoeO5, the enzyme responsible for the first step in moenomycin biosynthesis, catalyzes the transfer of farnesyl to 3-phosphoglyceric acid (3PG) to give a product containing a cis-allylic double bond. We show that this reaction involves isomerization to a nerolidyl pyrophosphate intermediate followed by bond rotation prior to attack by the nucleophile. This mechanism is unprecedented for a prenyltransferase that catalyzes an intermolecular coupling. We also show that PcrB transfers geranyl and geranylgeranyl groups to glycerol-1-phosphate (G1P), making it the first known bacterial enzyme to use G1P as a substrate. Unlike MoeO5, PcrB catalyzes prenyl transfer without isomerization to give products that retain the trans-allylic bond of the prenyl donors. The TIM barrel family of PTs is unique in including enzymes that catalyze prenyl transfer by distinctly different reaction mechanisms.
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- 2011
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39. Improved precursor-directed biosynthesis in E. coli via directed evolution
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David E. Cane, Chaitan Khosla, Colin J. B. Harvey, and Ho Young Lee
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precursor directed biosynthesis ,Mutant ,Microbial Sensitivity Tests ,6-deoxyerythronolide B synthase ,010402 general chemistry ,medicine.disease_cause ,01 natural sciences ,Article ,Mass Spectrometry ,03 medical and health sciences ,Polyketide ,chemistry.chemical_compound ,Plasmid ,polyketide ,Biosynthesis ,Polyketide synthase ,Drug Discovery ,Escherichia coli ,medicine ,directed evolution ,030304 developmental biology ,Pharmacology ,0303 health sciences ,biology ,Directed evolution ,Anti-Bacterial Agents ,Biosynthetic Pathways ,Erythromycin ,0104 chemical sciences ,3. Good health ,Biochemistry ,chemistry ,6-Deoxyerythronolide B synthase ,biology.protein ,Directed Molecular Evolution ,Plasmids - Abstract
Erythromycin and related macrolide antibiotics are widely used polyketide natural products. We have evolved an engineered biosynthetic pathway in Escherichia coli that yields erythromycin analogs from simple synthetic precursors. Multiple rounds of mutagenesis and screening led to the identification of new mutant strains with improved efficiency for precursor-directed biosynthesis. Genetic and biochemical analysis suggested that the phenotypically relevant alterations in these mutant strains were localized exclusively to the host-vector system, and not to the polyketide synthase. We also demonstrate the utility of this improved system through engineered biosynthesis of a novel alkynyl erythromycin derivative with comparable antibacterial activity to its natural counterpart. In addition to reinforcing the power of directed evolution for engineering macrolide biosynthesis, our studies have identified a new lead substance for investigating structure-function relationships in the bacterial ribosome.
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- 2010
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40. Genome-minimized Streptomyces host for the heterologous expression of secondary metabolism
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David E. Cane, Haruo Ikeda, Mamoru Komatsu, Takuma Uchiyama, and Satoshi Omura
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Magnetic Resonance Spectroscopy ,Mutant ,Streptomyces ,Gas Chromatography-Mass Spectrometry ,Industrial Microbiology ,Bacterial Proteins ,Gene cluster ,Cephamycins ,Secondary metabolism ,Gene ,Regulator gene ,Polycyclic Sesquiterpenes ,Genetics ,Genes, Essential ,Multidisciplinary ,biology ,Gene Expression Regulation, Bacterial ,Biological Sciences ,biology.organism_classification ,Multigene Family ,Mutation ,Streptomycin ,Epoxy Compounds ,Macrolides ,Transformation, Bacterial ,Heterologous expression ,Genetic Engineering ,Sesquiterpenes ,Streptomyces avermitilis ,Gene Deletion ,Genome, Bacterial - Abstract
To construct a versatile model host for heterologous expression of genes encoding secondary metabolite biosynthesis, the genome of the industrial microorganism Streptomyces avermitilis was systematically deleted to remove nonessential genes. A region of more than 1.4 Mb was deleted stepwise from the 9.02-Mb S. avermitilis linear chromosome to generate a series of defined deletion mutants, corresponding to 83.12–81.46% of the wild-type chromosome, that did not produce any of the major endogenous secondary metabolites found in the parent strain. The suitability of the mutants as hosts for efficient production of foreign metabolites was shown by heterologous expression of three different exogenous biosynthetic gene clusters encoding the biosynthesis of streptomycin (from S. griseus Institute for Fermentation, Osaka [IFO] 13350), cephamycin C (from S. clavuligerus American type culture collection (ATCC) 27064), and pladienolide (from S. platensis Mer-11107). Both streptomycin and cephamycin C were efficiently produced by individual transformants at levels higher than those of the native-producing species. Although pladienolide was not produced by a deletion mutant transformed with the corresponding intact biosynthetic gene cluster, production of the macrolide was enabled by introduction of an extra copy of the regulatory gene pldR expressed under control of an alternative promoter. Another mutant optimized for terpenoid production efficiently produced the plant terpenoid intermediate, amorpha-4,11-diene, by introduction of a synthetic gene optimized for Streptomyces codon usage. These findings highlight the strength and flexibility of engineered S. avermitilis as a model host for heterologous gene expression, resulting in the production of exogenous natural and unnatural metabolites.
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- 2010
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41. Revisiting the modularity of modular polyketide synthases
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Chaitan Khosla, Shiven Kapur, and David E. Cane
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Flexibility (engineering) ,Modularity (networks) ,business.industry ,Computational biology ,Biology ,Modular design ,Biochemistry ,Article ,Biosynthetic Pathways ,Analytical Chemistry ,Polyketide ,Polyketide synthase ,Biocatalysis ,biology.protein ,business ,Engineering design process ,Polyketide Synthases - Abstract
Modularity is a highly sought after feature in engineering design. A modular catalyst is a multi-component system whose parts can be predictably interchanged for functional flexibility and variety. Nearly two decades after the discovery of the first modular polyketide synthase (PKS), we critically assess PKS modularity in the face of a growing body of atomic structural and in vitro biochemical investigations. Both the architectural modularity and the functional modularity of this family of enzymatic assembly lines are reviewed, and the fundamental challenges that lie ahead for the rational exploitation of their full biosynthetic potential are discussed.
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- 2009
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42. Isolation and Characterization of the Gene Associated with Geosmin Production in Cyanobacteria
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Christopher P. Saint, Jiaoyang Jiang, Steven Giglio, David E. Cane, Paul Monis, Giglio,Steven, Jiang, J, Saint, Christopher Paul, Cane, D E, and Monis, Paul
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DNA, Bacterial ,Cyanobacteria ,Molecular Sequence Data ,Naphthols ,Biology ,Secondary metabolite ,Nucleic Acid Denaturation ,Polymerase Chain Reaction ,Catalysis ,Gas Chromatography-Mass Spectrometry ,Article ,chemistry.chemical_compound ,Bacterial Proteins ,Polyisoprenyl Phosphates ,Environmental Microbiology ,medicine ,Environmental Chemistry ,Amino Acid Sequence ,Geosmin synthase ,Nostoc ,Ecology ,General Chemistry ,biology.organism_classification ,Isolation (microbiology) ,Geosmin ,chemistry ,Odor ,Cyclization ,Genes, Bacterial ,Environmental chemistry ,Water treatment ,Sequence Alignment ,Sesquiterpenes ,Water utility ,medicine.drug - Abstract
Geosmin is a secondary metabolite responsible for earthy tastes and odors in potable water supplies. Geosmin continues to be a challenge to water utility management regimes and remains one of the most common causes of consumer complaints, as the taste of “dirty” water may suggest a failed disinfection regime and that the water may be unsafe to drink. Although cyanobacteria have been reported to be largely responsible for these taste and odor events, the answer as to how or why geosmin is produced has eluded researchers. We describe here for the first time the mechanism by which geosmin is produced in a model cyanobacterium, Nostoc punctiforme PCC 73102 (ATCC 29133), which we demonstrate utilizes a single enzyme to catalyze the cyclization of farnesyl diphosphate to geosmin. Using this information, we have developed a PCR-based assay that allows the rapid detection of geosmin-producing cyanobacteria. This test may be utilized to confirm and track the emergence of taste and odor-producing cyanobacteria in any given water body and thus can be used as an early warning system by managers of water bodies that may suffer from adverse taste and odor episodes.
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- 2008
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43. Mechanism of Thioesterase-Catalyzed Chain Release in the Biosynthesis of the Polyether Antibiotic Nanchangmycin
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Tiangang Liu, Xin Lin, Xiufen Zhou, David E. Cane, and Zixin Deng
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MICROBIO ,Stereochemistry ,Molecular Sequence Data ,Clinical Biochemistry ,Biology ,Thioester ,Biochemistry ,Catalysis ,Article ,Substrate Specificity ,Polyketide ,chemistry.chemical_compound ,Biosynthesis ,Thioesterase ,Polyketide synthase ,Drug Discovery ,Catalytic triad ,Spiro Compounds ,Amino Acid Sequence ,Molecular Biology ,Pharmacology ,chemistry.chemical_classification ,Sequence Homology, Amino Acid ,Mutagenesis ,Esterases ,General Medicine ,Anti-Bacterial Agents ,Kinetics ,CHEMBIO ,Aglycone ,chemistry ,Mutagenesis, Site-Directed ,biology.protein ,Molecular Medicine ,Ethers - Abstract
SummaryThe polyketide backbone of the polyether ionophore antibiotic nanchangmycin (1) is assembled by a modular polyketide synthase in Streptomyces nanchangensis NS3226. The ACP-bound polyketide is thought to undergo a cascade of oxidative cyclizations to generate the characteristic polyether. Deletion of the glycosyl transferase gene nanG5 resulted in accumulation of the corresponding nanchangmycin aglycone (6). The discrete thioesterase NanE exhibited a nearly 17-fold preference for hydrolysis of 4, the N-acetylcysteamine (SNAC) thioester of nanchangmycin, over 7, the corresponding SNAC derivative of the aglycone, consistent with NanE-catalyzed hydrolysis of ACP-bound nanchangmycin being the final step in the biosynthetic pathway. Site-directed mutagenesis established that Ser96, His261, and Asp120, the proposed components of the NanE catalytic triad, were all essential for thioesterase activity, while Trp97 was shown to influence the preference for polyether over polyketide substrates.
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- 2008
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44. X-ray Crystallographic Studies of Substrate Binding to Aristolochene Synthase Suggest a Metal Ion Binding Sequence for Catalysis
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Juan A. Faraldos, E.Y. Shishova, David W. Christianson, Fanglei Yu, Robert M. Coates, David E. Cane, Rudolf Konrad Allemann, David James Miller, and Yuxin X. Zhao
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Stereochemistry ,Sequence (biology) ,Isomerase ,Crystallography, X-Ray ,Biochemistry ,Catalysis ,Fungal Proteins ,chemistry.chemical_compound ,Ion binding ,Polyisoprenyl Phosphates ,Tetramer ,Magnesium ,Isomerases ,Molecular Biology ,Enzyme Catalysis and Regulation ,biology ,Cell Biology ,Lyase ,Protein Structure, Tertiary ,Aristolochene synthase ,Crystallography ,Aspergillus ,Monomer ,chemistry ,biology.protein ,Aristolochene ,Sesquiterpenes - Abstract
The universal sesquiterpene precursor, farnesyl diphosphate (FPP), is cyclized in an Mg(2+)-dependent reaction catalyzed by the tetrameric aristolochene synthase from Aspergillus terreus to form the bicyclic hydrocarbon aristolochene and a pyrophosphate anion (PP(i)) coproduct. The 2.1-A resolution crystal structure determined from crystals soaked with FPP reveals the binding of intact FPP to monomers A-C, and the binding of PP(i) and Mg(2+)(B) to monomer D. The 1.89-A resolution structure of the complex with 2-fluorofarnesyl diphosphate (2F-FPP) reveals 2F-FPP binding to all subunits of the tetramer, with Mg(2+)(B)accompanying the binding of this analogue only in monomer D. All monomers adopt open activesite conformations in these complexes, but slight structural changes in monomers C and D of each complex reflect the very initial stages of a conformational transition to the closed state. Finally, the 2.4-A resolution structure of the complex with 12,13-difluorofarnesyl diphosphate (DF-FPP) reveals the binding of intact DF-FPP to monomers A-C in the open conformation and the binding of PP(i), Mg(2+)(B), and Mg(2+)(C) to monomer D in a predominantly closed conformation. Taken together, these structures provide 12 independent "snapshots" of substrate or product complexes that suggest a possible sequence for metal ion binding and conformational changes required for catalysis.
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- 2008
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45. Structural and mechanistic analysis of trichodiene synthase using site-directed mutagenesis: Probing the catalytic function of tyrosine-295 and the asparagine-225/serine-229/glutamate-233–Mg2+B motif
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Jiaoyang Jiang, David W. Christianson, Tatiana Y. Zakharian, L. Sangeetha Vedula, and David E. Cane
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biology ,Chemistry ,Stereochemistry ,Mutant ,Biophysics ,Trichodiene synthase ,biology.organism_classification ,Lyase ,Biochemistry ,Fusarium sporotrichioides ,Serine ,biology.protein ,Enzyme kinetics ,Asparagine ,Site-directed mutagenesis ,Molecular Biology - Abstract
Trichodiene synthase from Fusarium sporotrichioides contains two metal ion-binding motifs required for the cyclization of farnesyl diphosphate: the “aspartate-rich” motif D100DXX(D/E) that coordinates to Mg2+A and Mg2+C, and the “NSE/DTE” motif N225DXXSXXXE that chelates Mg2+B (boldface indicates metal ion ligands). Here, we report steady-state kinetic parameters, product array analyses, and X-ray crystal structures of trichodiene synthase mutants in which the fungal NSE motif is progressively converted into a plant-like DDXXTXXXE motif, resulting in a degradation in both steady-state kinetic parameters and product specificity. Each catalytically active mutant generates a different distribution of sesquiterpene products, and three newly detected sesquiterpenes are identified. In addition, the kinetic and structural properties of the Y295F mutant of trichodiene synthase were found to be similar to those of the wild-type enzyme, thereby ruling out a proposed role for Y295 in catalysis.
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- 2008
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46. Crystal Structure of the Non-heme Iron Dioxygenase PtlH in Pentalenolactone Biosynthesis
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Haruo Ikeda, Satoshi Omura, Zheng You, David E. Cane, and Gerwald Jogl
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Protein Folding ,Stereochemistry ,Iron ,Coenzymes ,Crystallography, X-Ray ,Biochemistry ,Article ,Catalysis ,Protein Structure, Secondary ,Cofactor ,Dioxygenases ,Substrate Specificity ,Dioxygenase ,Oxidoreductase ,Binding site ,Molecular Biology ,Protein secondary structure ,chemistry.chemical_classification ,Binding Sites ,biology ,Chemistry ,Active site ,Cell Biology ,biology.organism_classification ,Streptomyces ,Protein Structure, Tertiary ,Crystallography ,Mutagenesis, Site-Directed ,biology.protein ,Ketoglutaric Acids ,Protein folding ,Sesquiterpenes ,Streptomyces avermitilis - Abstract
The non-heme iron dioxygenase PtlH from the soil organism Streptomyces avermitilis is a member of the iron(II)/alpha-ketoglutarate-dependent dioxygenase superfamily and catalyzes an essential reaction in the biosynthesis of the sesquiterpenoid antibiotic pentalenolactone. To investigate the structural basis for substrate recognition and catalysis, we have determined the x-ray crystal structure of PtlH in several complexes with the cofactors iron, alpha-ketoglutarate, and the non-reactive enantiomer of the substrate, ent-1-deoxypentalenic acid, in four different crystal forms to up to 1.31 A resolution. The overall structure of PtlH forms a double-stranded barrel helix fold, and the cofactor-binding site for iron and alpha-ketoglutarate is similar to other double-stranded barrel helix fold enzymes. Additional secondary structure elements that contribute to the substrate-binding site in PtlH are not conserved in other double-stranded barrel helix fold enzymes. Binding of the substrate enantiomer induces a reorganization of the monoclinic crystal lattice leading to a disorder-order transition of a C-terminal alpha-helix. The newly formed helix blocks the major access to the active site and effectively traps the bound substrate. Kinetic analysis of wild type and site-directed mutant proteins confirms a critical function of two arginine residues in substrate binding, while simulated docking of the enzymatic reaction product reveals the likely orientation of bound substrate.
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- 2007
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47. Stereospecificity of Ketoreductase Domains of the 6-Deoxyerythronolide B Synthase
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Roselyne Castonguay, Weiguo He, David E. Cane, Chaitan Khosla, and Alice Y. Chen
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Stereochemistry ,Molecular Conformation ,Stereoisomerism ,Thioester ,Biochemistry ,Mass Spectrometry ,Article ,Catalysis ,Substrate Specificity ,Lactones ,Polyketide ,Colloid and Surface Chemistry ,Stereospecificity ,Thioesterase ,Polyketide synthase ,Transferase ,chemistry.chemical_classification ,Binding Sites ,biology ,Chemistry ,General Chemistry ,Protein Structure, Tertiary ,6-Deoxyerythronolide B synthase ,biology.protein ,Oxidoreductases ,Polyketide Synthases - Abstract
6-Deoxyerythronolide B synthase (DEBS) is a modular polyketide synthase (PKS) responsible for the biosynthesis of 6-dEB (1), the parent aglycone of the broad spectrum macrolide antibiotic erythromycin. Individual DEBS modules, which contain the catalytic domains necessary for each step of polyketide chain elongation and chemical modification, can be deconstructed into constituent domains. To better understand the intrinsic stereospecificity of the ketoreductase (KR) domains, an in vitro reconstituted system has been developed involving combinations of ketosynthase (KS) – acyl transferase (AT) didomains with acyl-carrier protein (ACP) and KR domains from different DEBS modules. Incubations with (2S,3R)-2-methyl-3-hydroxypentanoic acid N-acetylcysteamine thioester (2) and methylmalonyl-CoA plus NADPH result in formation of a reduced, ACP-bound triketide that is converted to the corresponding triketide lactone 4 by either base- or enzyme-catalyzed hydrolysis/cyclization. A sensitive and robust GC-mass spectrometry technique has been developed to assign the stereochemistry of the resulting triketide lactones, on the basis of direct comparison with synthetic standards of each of the four possible diasteromers 4a–4d. Using the [KS][AT] didomains from either DEBS module 3 or module 6 in combination with KR domains from modules 2 or 6 gave in all cases exclusively (2R,3S,4R,5R)-3,5-dihydroxy-2,4-dimethyl-n-heptanoic acid-δ-lactone (4a). The same product was also generated by a chimeric module in which [KS3][AT3] was fused to [KR5][ACP5] and the DEBS thioesterase [TE] domain. Reductive quenching of the ACP-bound 2-methyl-3-ketoacyl triketide intermediate with sodium borohydride confirmed that in each case the triketide intermediate carried only an unepimerized D-2-methyl group. The results confirm the predicted stereospecificity of the individual KR domains, while revealing an unexpected configurational stability of the ACP-bound 2-methyl-3-ketoacyl thioester intermediate. The methodology should be applicable to the study of any combination of heterologous [KS][AT] and [KR] domains.
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- 2007
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48. Solution structure and proposed domain-domain recognition interface of an acyl carrier protein domain from a modular polyketide synthase
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Chaitan Khosla, Viktor Yuryevich Alekseyev, Corey W. Liu, Joseph D. Puglisi, and David E. Cane
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Models, Molecular ,Steric effects ,Stereochemistry ,Molecular Sequence Data ,Static Electricity ,Biochemistry ,Article ,Protein Structure, Secondary ,Mixed Function Oxygenases ,Lactones ,Polyketide ,Bacterial Proteins ,Cytochrome P-450 Enzyme System ,Polyketide synthase ,Static electricity ,Acyl Carrier Protein ,Point Mutation ,Amino Acid Sequence ,Homology modeling ,Nuclear Magnetic Resonance, Biomolecular ,Molecular Biology ,Peptide sequence ,Phylogeny ,Binding Sites ,biology ,Protein Structure, Tertiary ,Solutions ,Acyl carrier protein ,Structural Homology, Protein ,Helix ,biology.protein - Abstract
Polyketides are a medicinally important class of natural products. The architecture of modular polyketide synthases (PKSs), composed of multiple covalently linked domains grouped into modules, provides an attractive framework for engineering novel polyketide-producing assemblies. However, impaired domain-domain interactions can compromise the efficiency of engineered polyketide biosynthesis. To facilitate the study of these domain-domain interactions, we have used nuclear magnetic resonance (NMR) spectroscopy to determine the first solution structure of an acyl carrier protein (ACP) domain from a modular PKS, 6-deoxyerythronolide B synthase (DEBS). The tertiary fold of this 10-kD domain is a three-helical bundle; an additional short helix in the second loop also contributes to the core helical packing. Superposition of residues 14-94 of the ensemble on the mean structure yields an average atomic RMSD of 0.64 +/- 0.09 Angstrom for the backbone atoms (1.21 +/- 0.13 Angstrom for all non-hydrogen atoms). The three major helices superimpose with a backbone RMSD of 0.48 +/- 0.10 Angstrom (0.99 +/- 0.11 Angstrom for non-hydrogen atoms). Based on this solution structure, homology models were constructed for five other DEBS ACP domains. Comparison of their steric and electrostatic surfaces at the putative interaction interface (centered on helix II) suggests a model for protein-protein recognition of ACP domains, consistent with the previously observed specificity. Site-directed mutagenesis experiments indicate that two of the identified residues influence the specificity of ACP recognition.
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- 2007
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49. Structure-Based Dissociation of a Type I Polyketide Synthase Module
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Chaitan Khosla, Alice Y. Chen, and David E. Cane
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CHEMBIOL ,MICROBIO ,Stereochemistry ,Protein Conformation ,Acylation ,Clinical Biochemistry ,Biochemistry ,Catalysis ,Article ,Polyketide ,Protein structure ,Polyketide synthase ,Drug Discovery ,Molecular Biology ,DNA Primers ,Pharmacology ,ATP synthase ,biology ,Base Sequence ,Substrate (chemistry) ,General Medicine ,Recombinant Proteins ,Acyl carrier protein ,biology.protein ,Molecular Medicine ,Chromatography, Thin Layer ,Linker ,Polyketide Synthases - Abstract
Individual catalytic modules of modular polyketide synthases (PKSs) such as the 6-deoxyerythronolide B synthase (DEBS) are comprised of highly conserved, covalently linked domains separated by relatively unconserved intervening sequences known as linkers. Recent structural studies have provided the first high-resolution descriptions of the architecture of PKS modules. To better understand the relative significance of protein-protein interactions and enzyme-substrate interactions in controlling module catalysis and specificity, we have exploited these structural insights to prepare stand-alone functional domains of selected DEBS modules. When combined in vitro, the ketosynthase (KS), acyl transferase (AT), and acyl carrier protein (ACP) domains of DEBS module 3 catalyzed methylmalonyl transfer and elongation of a diketide substrate. The 30-residue post-AT linker, which interacts with the KS and AT domains as well as the KS-to-AT linker, was found to be critical for chain elongation but not methylmalonyl transfer. When added to a minimal PKS, ketoreductase (KR) domains from DEBS modules 1, 2, and 6 showed specificity for the polyketide substrate, but were unaffected by the identity of either the ACP domain onto which the β-ketoacylthioester substrate was tethered or the KS domain that synthesized the substrate. By providing hitherto unanticipated insights into factors influencing the catalytic efficiency and specificity of PKS modules, our results provide new guidelines for the optimal construction of hybrid PKS systems.
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- 2007
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50. Pentalenolactone biosynthesis: Molecular cloning and assignment of biochemical function to PtlF, a short-chain dehydrogenase from Streptomyces avermitilis, and identification of a new biosynthetic intermediate
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Haruo Ikeda, David E. Cane, Satoshi Omura, and Zheng You
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chemistry.chemical_classification ,Short-chain dehydrogenase ,biology ,Biophysics ,Dehydrogenase ,Protein Engineering ,biology.organism_classification ,Biochemistry ,Streptomyces ,Recombinant Proteins ,Article ,chemistry.chemical_compound ,chemistry ,Biosynthesis ,Oxidoreductase ,Gene cluster ,NAD+ kinase ,Cloning, Molecular ,Oxidoreductases ,Sesquiterpenes ,Molecular Biology ,Streptomyces avermitilis - Abstract
Pentalenolactone ( 1 ) is an antibiotic that has been isolated from many species of Streptomyces . The putative dehydrogenase encoded by the ptlF gene (SAV2993) found within the Streptomyces avermitilis pentalenolactone gene cluster was cloned and overexpressed in Escherichia coli . PtlF, which belongs to the short-chain dehydrogenase/oxidoreductase superfamily, was shown to catalyze the oxidation of 1-deoxy-11β-hydroxypentalenic acid ( 9 ) to 1-deoxy-11-oxopentalenic acid ( 10 ), a new intermediate of the pentalenolactone biosynthetic pathway. The methyl ester of 10 was characterized by NMR, GC–MS and high resolution mass spectrometry. PtlF exhibited a 150-fold preference for β-NAD + over β-NADP + . PtlF had a pH optimum of 8.0 in the physiological pH range, while a significant activity enhancement was observed from pH 9.0 to 11.3. At pH 8.0, PtlF had a k cat of 0.65 ± 0.03 s −1 , with a K m for 9 of 6.5 ± 1.5 μM and K m for NAD + of 25 ± 3 μM.
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- 2007
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