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A wide variety of antibiotics are targeted to the bacterial membrane due to its unique arrangement and composition relative to the host mammalian membranes. By modification of their membranes, some gram-negative pathogens resist the action of antibiotics. Lipid A phosphoethanolamine transferase (EptA) is an intramembrane enzyme that modifies the lipid A portion of lipopolysaccharide/lipooligosaccharide by the addition of phosphoethanolamine. This modification reduces the overall net-negative charge of the outer membrane of some gram-negative bacteria, conferring resistance to polymyxin. This resistance mechanism has resulted in a global public health issue due to the increased use of polymyxin as last-resort antibiotic treatments against multi-drug-resistant pathogens. Studies show that, without EptA, pathogenic bacteria become more sensitive to polymyxin and to clearance by the host immune system, suggesting the importance of this target enzyme for the development of novel therapeutic agents. In this review, EptA will be discussed comprehensively. Specifically, this review will cover the regulation of eptA expression by the two component systems PmrA/PmrB and PhoP/PhoQ, the site of modification on lipid A, the structure and catalytic mechanism of EptA in comparison to MCR-1 and Escherichia coli alkaline phosphatase, and the host immune system's response to lipid A modification by EptA. The overarching aim of this review is to provide a comprehensive overview of polymyxin resistance mediated by EptA.

Lipopolysaccharides are complex molecules found in the cell envelop of many Gram-negative bacteria. The toxic activity of these molecules has led to the terminology of endotoxins. They provide bacteria with structural integrity and protection from external environmental conditions, and they interact with host signaling receptors to induce host immune responses. Bacteria have evolved enzymes that act to modify lipopolysaccharides, particularly the lipid A region of the molecule, to enable the circumvention of host immune system responses. These modifications include changes to lipopolysaccharide by the addition of positively charged sugars, such as N-Ara4N, and phosphoethanolamine (pEtN). Other modifications include hydroxylation, acylation, and deacylation of fatty acyl chains. We review the two-component regulatory mechanisms for enzymes that carry out these modifications and provide details of the structures of four enzymes (PagP, PagL, pEtN transferases, and ArnT) that modify the lipid A portion of lipopolysaccharides. We focus largely on the three-dimensional structures of these enzymes, which provide an understanding of how their substrate binding and catalytic activities are mediated. A structure-function-based understanding of these enzymes provides a platform for the development of novel therapeutics to treat antibiotic resistance.

Society has benefitted greatly from the use of antibiotics. Unfortunately, the misuse of these valuable molecules has resulted in increased levels of antibiotic resistance, a major global and public health issue. This resistance and the reliance on a small number of biological targets for the development of antibiotics emphasizes the need for new targets. A critical aspect guiding the development of new antimicrobials through a rational structure-guided approach is to understand the molecular structures of specific biological targets of interest. Here we give an overview of the structures of bacterial virulence enzyme targets involved in protein folding, peptidoglycan biosynthesis and cell wall modification. These include enzymes of the thiol-disulphide oxidoreductase pathway (DSB enzymes), peptidyl-proly cis/trans isomerases (Mips), enzymes from the Mur pathway and enzymes involved in lipopolysaccharide modification (EptA and ArnT). We also present progress towards inhibitor design of these targets for the development of novel anti-virulence therapeutic agents.

Cholesterol oxidase (ChOx), a member of the glucose-methanol-choline (GMC) family, catalyzes the oxidation of the substrate via a hydride transfer mechanism and concomitant reduction of the FAD cofactor. Unlike other GMC enzymes, the conserved His447 is not the catalytic base that deprotonates the substrate in ChOx. Our QM/MM MD simulations indicate that the Glu361 residue acts as a catalytic base facilitating the hydride transfer from the substrate to the cofactor. We find that two rationally chosen point mutations (His447Gln and His447Asn) cause notable decreases in the catalytic activity. The binding free energy calculations show that the Glu361 and His447 residues are important in substrate binding. We also performed high-level double-hybrid density functional theory simulations using small model systems, which support the QM/MM MD results. Our work provides a basis for unraveling the substrate oxidation mechanism in GMC enzymes in which the conserved histidine does not act as a base.

Cholesterol oxidase (ChOx) is a flavoenzyme that oxidises and isomerises cholesterol (CHL) to form cholest-4-en-3-one. Molecular docking and molecular dynamics simulations were conducted to predict the binding interactions of CHL in the active site. Several key interactions (E361-CHL, N485-FAD and H447-CHL) were identified and which are likely to determine the correct positioning of CHL relative to flavin-adenine dinucleotide (FAD). Binding of CHL also induced changes in key residues of the active site leading to the closure of the oxygen channel. A group of residues, Y107, F444 and Y446, known as the hydrophobic triad, are believed to affect the binding of CHL in the active site. Computational site-directed mutagenesis of these residues revealed that their mutation affects the conformations of key residues in the active site, leading to non-optimal binding of CHL and to changes in the structure of the oxygen channel, all of which are likely to reduce the catalytic efficiency of ChOx. This article is protected by copyright. All rights reserved.

The heterodimeric transcription elongation factor Spt4/Spt5 (Spt4/5) tightly associates with RNAPII to regulate both transcriptional elongation and co-transcriptional pre-mRNA processing; however, the mechanisms by which Spt4/5 acts are poorly understood. Recent studies of the human and Drosophila Spt4/5 complexes indicate that they can bind nucleic acids in vitro. We demonstrate here that yeast Spt4/5 can bind in a sequence-specific manner to single stranded RNA containing AAN repeats. Furthermore, we show that the major protein determinants for RNA-binding are Spt4 together with the NGN domain of Spt5 and that the KOW domains are not required for RNA recognition. These findings attribute a new function to a domain of Spt4/5 that associates directly with RNAPII, making significant steps towards elucidating the mechanism behind transcriptional control by Spt4/5.

Bacteria cause disease by two general mechanisms: the action of their toxins on host cells and induction of a pro-inflammatory response that can lead to a deleterious cytokine/chemokine response (e.g., the so-called cytokine storm) often seen in bacteremia/septicemia. These major mechanisms may overlap due to the action of surface structures that can have direct and indirect actions on phagocytic or non-phagocytic cells. In this respect, the lipid A (endotoxin) component of lipopolysaccharide (LPS) possessed by Gram-negative bacteria has been the subject of literally thousands of studies over the past century that clearly identified it as a key virulence factor in endotoxic shock. In addition to its capacity to modulate inflammatory responses, endotoxin can also modulate bacterial susceptibility to host antimicrobials, such as the host defense peptides termed cationic antimicrobial peptides. This review concentrates on the phosphoethanolamine (PEA) decoration of lipid A in the pathogenic species of the genus Neisseria [N. gonorrhoeae and N. meningitidis]. PEA decoration of lipid A is mediated by the enzyme EptA (formerly termed LptA) and promotes resistance to innate defense systems, induces the pro-inflammatory response and can influence the in vivo fitness of bacteria during infection. These important biological properties have driven efforts dealing with the biochemistry and structural biology of EptA that will facilitate the development of potential inhibitors that block PEA addition to lipid A.

BackgroundBemisia tabacispecies (whiteflies) are the world’s most devastating insect pests within crops in the tropics. They cause billions of dollars (US) of damage each year and are leaving farmers in the developing world food insecure. Understanding the genetic and transcriptomic composition of these insect pests, the viruses they transmit and the microbiota is crucial to sustainable insect and virus management solutions for farmers. Currently, publically available transcriptome data forB. tabacihas been generated from pooled samples (mainly inbred lab colonies) consisting of several individuals because whiteflies are small (approximately 0.2 mm wide and 0.1 mm in height). Pooling individuals can lead to high heterozygosity and skewed representation of the genetic diversity. The ability to extract enough RNA from a single whitefly has remained elusive due to their small size and technology limitations. Therefore, the understanding of whitefly-microbiotad-viral species composition of an individual field-collected whitefly has also remained unknown. In this study, we developed a single whitefly RNA extraction procedure and subsequently successfully sequenced the transcriptome of four individual adult SubdSaharan Africa (SSA1)B. tabaci.ResultsTranscriptome sequencing on individual whiteflies resulted in between 39-42 million raw reads.De novoassembly of trimmed reads yielded between 65,000-162,000 transcripts across all fourB. tabacitranscriptomes. In addition, Bayesian phylogenetic analysis of mitochondrion cytochrome I oxidase (mtCOI) grouped the four whiteflies within the SSA1 clade. BLAST searches on assembled transcripts within the four individual transcriptomes identified five endosymbionts; the primary endosymbiontPortiera,aleyrodidarumand four secondary endosymbionts:Arsenophonus, Wolbachia, Rickettsia,andCardinium spp.These five endosymbionts were predominant across all four SSA1B. tabacistudy samples with prevalence levels of between 54.1d75%. Nucleotide and amino acid sequence alignments of theNusG gene ofP. aleyrodidarumfor the SSA1B. tabacitranscriptomes of samples WF2 and WF2b revealed an eleven amino acid residue deletion that was absent in samples WF1 and WF2a. Comparison of the protein structure of theNusG protein fromP. aleyrodidarumin SSA1 with knownNusG structures showed the deletion resulted in a shorter D loop. AlthoughNusG is key in regulating of transcription elongation, it is believed that the shortening of the loop region in the N-terminal domain is unlikely to affect transcription termination. Therefore, the effect of variability in this region across species is unknown.ConclusionIn this study, we optimised a single whitefly high quality RNA extraction procedure and successfully carried out individual whitefly transcriptome sequencing on adultB. tabaciwhiteflies. This enabled the detection of unique genetic differences in theNusG genes of the primary endosymbiontP. aleyrodidarumin four field-collected SSA1 whiteflies that may not have been detected using lab-pooledB. tabaciisolines. The use of field-collected specimens means that both time and money will be saved in future studies using single whitefly transcriptomes in monitoring vector and viral interactions. In addition, the methods we have developed here are applicable to any small organism where RNA quantity has limited transcriptome studies.

Multidrug-resistant (MDR) gram-negative bacteria have increased the prevalence of fatal sepsis in modern times. Colistin is a cationic antimicrobial peptide (CAMP) antibiotic that permeabilizes the bacterial outer membrane (OM) and has been used to treat these infections. The OM outer leaflet is comprised of endotoxin containing lipid A, which can be modified to increase resistance to CAMPs and prevent clearance by the innate immune response. One type of lipid A modification involves the addition of phosphoethanolamine to the 1 and 4′ headgroup positions by phosphoethanolamine transferases. Previous structural work on a truncated form of this enzyme suggested that the full-length protein was required for correct lipid substrate binding and catalysis. We now report the crystal structure of a full-length lipid A phosphoethanolamine transferase from Neisseria meningitidis , determined to 2.75-A resolution. The structure reveals a previously uncharacterized helical membrane domain and a periplasmic facing soluble domain. The domains are linked by a helix that runs along the membrane surface interacting with the phospholipid head groups. Two helices located in a periplasmic loop between two transmembrane helices contain conserved charged residues and are implicated in substrate binding. Intrinsic fluorescence, limited proteolysis, and molecular dynamics studies suggest the protein may sample different conformational states to enable the binding of two very different- sized lipid substrates. These results provide insights into the mechanism of endotoxin modification and will aid a structure-guided rational drug design approach to treating multidrug-resistant bacterial infections.

Made available in DSpace on 2022-04-28T19:11:00Z (GMT). No. of bitstreams: 0 Previous issue date: 2017-01-01 Bill and Melinda Gates Foundation Department for International Development Background: Bemisia tabaci species (B. tabaci), or whiteflies, are the world’s most devastating insect pests. They cause billions of dollars (US) of damage each year, and are leaving farmers in the developing world food insecure. Currently, all publically available transcriptome data for B. tabaci are generated from pooled samples, which can lead to high heterozygosity and skewed representation of the genetic diversity. The ability to extract enough RNA from a single whitefly has remained elusive due to their small size and technological limitations. Methods: In this study, we optimised a single whitefly RNA extraction procedure, and sequenced the transcriptome of four individual adult Sub-Saharan Africa 1 (SSA1) B. tabaci. Transcriptome sequencing resulted in 39-42 million raw reads. De novo assembly of trimmed reads yielded between 65,000-162,000 Contigs across B. tabaci transcriptomes. Results: Bayesian phylogenetic analysis of mitochondrion cytochrome I oxidase (mtCOI) grouped the four whiteflies within the SSA1 clade. BLASTn searches on the four transcriptomes identified five endosymbionts; the primary endosymbiont Portiera aleyrodidarum and four secondary endosymbionts: Arsenophonus, Wolbachia, Rickettsia, and Cardinium spp. that were predominant across all four SSA1 B. tabaci samples with prevalence levels of between 54.1 to 75%. Amino acid alignments of the NusG gene of P. aleyrodidarum for the SSA1 B. tabaci transcriptomes of samples WF2 and WF2b revealed an eleven amino acid residue deletion that was absent in samples WF1 and WF2a. Comparison of the protein structure of the NusG protein from P. aleyrodidarum in SSA1 with known NusG structures showed the deletion resulted in a shorter D loop. Conclusions: The use of field-collected specimens means time and money will be saved in future studies using single whitefly transcriptomes in monitoring vector and viral interactions. Our method is applicable to any small organism where RNA quantity has limited transcriptome studies. Mikocheni Agriculture Research Institute (MARI), P.O. Box 6226 School of Molecular Sciences and Australian Research Council Centre of Excellence in Plant Energy Biology University of Western Australia National Agricultural Research Laboratories, P.O. Box 7065, Kampala Kawanda-Senge Rd Ministry of Agriculture Key Laboratory of Agricultural Entomology Institute of Insect Sciences Zhejiang University State Key Laboratory for the Biology of Plant Diseases and Insect Pests Institute of Plant Protection Chinese Academy of Agricultural Sciences Faculdade de Ciências Agronômicas Universidade Estadual Paulista Department of Primary Industries and Regional Development DPIRD Diagnostic Laboratory Services Faculdade de Ciências Agronômicas Universidade Estadual Paulista

Cholesterol oxidase (CO) is a flavoenzyme that catalyzes the oxidation and isomerization of cholesterol to cholest-4-en-3-one. The reductive half reaction occursviaa hydride transfer from the substrate to the FAD cofactor. The structures of CO reduced with dithionite under aerobic conditions and in the presence of the substrate 2-propanol under both aerobic and anaerobic conditions are presented. The 1.32 Å resolution structure of the dithionite-reduced enzyme reveals a sulfite molecule covalently bound to the FAD cofactor. The isoalloxazine ring system displays a bent structure relative to that of the oxidized enzyme, and alternate conformations of a triad of aromatic residues near to the cofactor are evident. A 1.12 Å resolution anaerobically trapped reduced enzyme structure in the presence of 2-propanol does not show a similar bending of the flavin ring system, but does show alternate conformations of the aromatic triad. Additionally, a significant difference electron-density peak is observed within a covalent-bond distance of N5 of the flavin moiety, suggesting that a hydride-transfer event has occurred as a result of substrate oxidation trapping the flavin in the electron-rich reduced state. The hydride transfer generates a tetrahedral geometry about the flavin N5 atom. High-level density-functional theory calculations were performed to correlate the crystallographic findings with the energetics of this unusual arrangement of the flavin moiety. These calculations suggest that strong hydrogen-bond interactions between Gly120 and the flavin N5 centre may play an important role in these structural features.

Spt4/5 is a hetero-dimeric transcription elongation factor that can both inhibit and promote transcription elongation by RNA polymerase II (RNAPII). However, Spt4/5's mechanism of action remains elusive. Spt5 is an essential protein and the only universally-conserved RNAP-associated transcription elongation factor. The protein contains multiple Kyrpides, Ouzounis and Woese (KOW) domains. These domains, in other proteins, are thought to bind RNA although there is little direct evidence in the literature to support such a function in Spt5. This could be due, at least in part, to difficulties in expressing and purifying recombinant Spt5. When expressed in Escherichia coli (E. coli), Spt5 is innately insoluble. Here we report a new approach for the successful expression and purification of milligram quantities of three different multi-KOW domain complexes of Saccharomyces cerevisiae Spt4/5 for use in future functional studies. Using the E. coli strain Rosetta2 (DE3) we have developed strategies for co-expression of Spt4 and multi-KOW domain Spt5 complexes from the bi-cistronic pET-Duet vector. In a second strategy, Spt4/5 was expressed via co-transformation of Spt4 in the vector pET-M11 with Spt5 ubiquitin fusion constructs in the vector pHUE. We characterized the multi-KOW domain Spt4/5 complexes by Western blot, limited proteolysis, circular dichroism, SDS-PAGE and size exclusion chromatography-multiangle light scattering and found that the proteins are folded with a Spt4:Spt5 hetero-dimeric stoichiometry of 1:1. These expression constructs encompass a larger region of Spt5 than has previously been reported, and will provide the opportunity to elucidate the biological function of the multi-KOW containing Spt5.

The study aims to gain insight into the mode of ligand recognition by tetratricopeptide repeat (TPR) domains of chloroplast translocon at the outer envelope of chloroplast (Toc64) and mitochondrial Om64, two paralogous proteins that mediate import of proteins into chloroplast and mitochondria, respectively. Chaperone proteins associate with precursor proteins in the cytosol to maintain them in a translocation competent conformation and are recognized by Toc64 and Om64 that are located on the outer membrane of the target organelle. Heat shock proteins (Hsp70) and Hsp90 are two chaperones, which are known to play import roles in protein import. The C-termini of these chaperones are known to interact with the TPR domain of chloroplast Toc64 and mitochondrial Om64 in Arabidopsis thaliana (At). Using a molecular dynamics approach and binding energy calculations, we identify important residues involved in the interactions. Our findings suggest that the TPR domain from AtToc64 has higher affinity towards C-terminal residues of Hsp70. The interaction occurs as the terminal helices move towards each other enclosing the cradle on interaction of AtHsp70 with the TPR domain. In contrast, the TPR domain from AtOm64 does not discriminate between the C-termini of Hsp70 and Hsp90. These binding affinities are discussed with respect to our knowledge of protein targeting and specificity of protein import into endosymbiotic organelles in plant cells. Copyright © 2014 John Wiley & Sons, Ltd.

The crystal structure of the wild-type form of glutaryl-7-ACA (7-aminocephalosporanic acid) acylase from Pseudomonas N176 and a double mutant of the protein (H57βS/H70βS) that displays enhanced catalytic efficiency on cephalosporin C over glutaryl-7-aminocephalosporanic acid has been determined. The structures show a heterodimer made up of an α-chain (229 residues) and a β-chain (543 residues) with a deep cavity, which constitutes the active site. Comparison of the wild-type and mutant structures provides insights into the molecular reasons for the observed enhanced specificity on cephalosporin C over glutaryl-7-aminocephalosporanic acid and offers the basis to evolve a further improved enzyme variant. The nucleophilic catalytic serine residue, Ser1β, is situated at the base of the active site cavity. The electron density reveals a ligand covalently bound to the catalytic serine residue, such that a tetrahedral adduct is formed. This is proposed to mimic the transition state of the enzyme for both the maturation step and the catalysis of the substrates. A view of the transition state configuration of the enzyme provides important insights into the mechanism of substrate binding and catalysis.

Pathogenic meningococci have acquired a 24 kb capsule synthesis island (cps) by horizontal gene transfer which consists of a synthetic locus and associated capsule transport genes flanked by repetitive Regions D and D’. Regions D and D’ contain an intact gene encoding a UDP-galactose epimerase (galE1) and a truncated remnant (galE2), respectively. In this study, GalE protein alleles were shown to be either mono-functional, synthesising UDP-galactose (UDP-Gal), or bi-functional, synthesising UDP-Gal and UDP-galactosamine (UDP-GalNAc). Meningococci possessing a capsule null locus (cnl) typically possessed a single bi-functional galE. Separation of functionality between galE1 and galE2 alleles in meningococcal isolates was retained for all serogroups except serogroup E which has a synthetic requirement for UDP-GalNAc. The truncated galE2 remnant in Region D’ was also phylogenetically related to the bi-functional galE of the cnl locus suggesting common ancestry. A model is proposed in which the illegitimate recombination of the cps island into the galE allele of the cnl locus results in the formation of Region D’ containing the truncated galE2 locus and the capture of the cps island en bloc. The retention of the duplicated Regions D and D’ enables inversion of the synthetic locus within the cps island during bacterial growth.

Snake venoms are cocktails of enzymes and non-enzymatic proteins used for both the immobilization and digestion of prey. The most common snake venom enzymes include acetylcholinesterases, l-amino acid oxidases, serine proteinases, metalloproteinases and phospholipases A(2) . Higher catalytic efficiency, thermal stability and resistance to proteolysis make these enzymes attractive models for biochemists, enzymologists and structural biologists. Here, we review the structures of these enzymes and describe their structure-based mechanisms of catalysis and inhibition. Some of the enzymes exist as protein complexes in the venom. Thus we also discuss the functional role of non-enzymatic subunits and the pharmacological effects of such protein complexes. The structures of inhibitor-enzyme complexes provide ideal platforms for the design of potent inhibitors which are useful in the development of prototypes and lead compounds with potential therapeutic applications.

The disulfide relay system of the mitochondrial intermembrane space has been extensively characterized in Saccharomyces cerevisiae. It contains two essential components, Mia40 and Erv1. The genome of Arabidopsis thaliana contains a single gene for each of these components. Although insertional inactivation of Erv1 leads to a lethal phenotype, inactivation of Mia40 results in no detectable deleterious phenotype. A. thaliana Mia40 is targeted to and accumulates in mitochondria and peroxisomes. Inactivation of Mia40 results in an alteration of several proteins in mitochondria, an absence of copper/zinc superoxide dismutase (CSD1), the chaperone for superoxide dismutase (Ccs1) that inserts copper into CSD1, and a decrease in capacity and amount of complex I. In peroxisomes the absence of Mia40 leads to an absence of CSD3 and a decrease in abnormal inflorescence meristem 1 (Aim1), a β-oxidation pathway enzyme. Inactivation of Mia40 leads to an alteration of the transcriptome of A. thaliana, with genes encoding peroxisomal proteins, redox functions, and biotic stress significantly changing in abundance. Thus, the mechanistic operation of the mitochondrial disulfide relay system is different in A. thaliana compared with other systems, and Mia40 has taken on new roles in peroxisomes and mitochondria.

Background: Bemisia tabaci species (B. tabaci), or whiteflies, are the world’s most devastating insect pests. They cause billions of dollars (US) of damage each year, and are leaving farmers in the developing world food insecure. Currently, all publically available transcriptome data for B. tabaci are generated from pooled samples, which can lead to high heterozygosity and skewed representation of the genetic diversity. The ability to extract enough RNA from a single whitefly has remained elusive due to their small size and technological limitations. Methods: In this study, we optimised a single whitefly RNA extraction procedure, and sequenced the transcriptome of four individual adult Sub-Saharan Africa 1 (SSA1) B. tabaci. Transcriptome sequencing resulted in 39-42 million raw reads. De novo assembly of trimmed reads yielded between 65,000-162,000 Contigs across B. tabaci transcriptomes. Results: Bayesian phylogenetic analysis of mitochondrion cytochrome I oxidase (mtCOI) grouped the four whiteflies within the SSA1 clade. BLASTn searches on the four transcriptomes identified five endosymbionts; the primary endosymbiont Portiera aleyrodidarum and four secondary endosymbionts: Arsenophonus, Wolbachia, Rickettsia, and Cardinium spp. that were predominant across all four SSA1 B. tabaci samples with prevalence levels of between 54.1 to 75%. Amino acid alignments of the NusG gene of P. aleyrodidarum for the SSA1 B. tabaci transcriptomes of samples WF2 and WF2b revealed an eleven amino acid residue deletion that was absent in samples WF1 and WF2a. Comparison of the protein structure of the NusG protein from P. aleyrodidarum in SSA1 with known NusG structures showed the deletion resulted in a shorter D loop. Conclusions: The use of field-collected specimens means time and money will be saved in future studies using single whitefly transcriptomes in monitoring vector and viral interactions. Our method is applicable to any small organism where RNA quantity has limited transcriptome studies.

Cholesterol oxidases are bifunctional flavoenzymes that catalyze the oxidation of steroid substrates which have a hydroxyl group at the 3beta position of the steroid ring system. The enzyme is found, in a wide range of bacterial species, in two forms: one with the FAD cofactor bound noncovalently to the enzyme; and one with the cofactor linked covalently to the protein. Here we discuss, compare and contrast the salient biochemical properties of the two forms of the enzyme. Specifically, the structural features are discussed that affect the redox potentials of the flavin cofactor, the chemical mechanism of substrate dehydrogenation by active-center amino acid residues, the kinetic parameters of both types of enzymes and the reactivity of reduced enzymes with molecular dioxygen. The presence of a molecular tunnel that is proposed to serve in the access of dioxygen to the active site and mechanisms of its control by a 'gate' formed by amino acid residues are highlighted.

The crystal structure of cholesterol oxidase, a 56kDa flavoenzyme was anisotropically refined to 0.95A resolution. The final crystallographic R-factor and R(free) value is 11.0% and 13.2%, respectively. The quality of the electron density maps has enabled modeling of alternate conformations for 83 residues in the enzyme, many of which are located in the active site. The additional observed structural features were not apparent in the previous high-resolution structure (1.5A resolution) and have enabled the identification of a narrow tunnel leading directly to the isoalloxazine portion of the FAD prosthetic group. The hydrophobic nature of this narrow tunnel suggests it is the pathway for molecular oxygen to access the isoalloxazine group for the oxidative half reaction. Resolving the alternate conformations in the active site residues provides a model for the dynamics of substrate binding and a potential oxidation triggered gating mechanism involving access to the hydrophobic tunnel. This structure reveals that the NE2 atom of the active site histidine residue, H447, critical to the redox activity of this flavin oxidase, acts as a hydrogen bond donor rather than as hydrogen acceptor. The atomic resolution structure of cholesterol oxidase has revealed the presence of hydrogen atoms, dynamic aspects of the protein and how side-chain conformations are correlated with novel structural features such as the oxygen tunnel. This new structural information has provided us with the opportunity to re-analyze the roles played by specific residues in the mechanism of the enzyme.

Summary Whether the highly dynamic structure of the vimentin intermediate filament (IF) cytoskeleton responds to cues from cellular organelles, and what proteins might participate in such events is largely unknown. We have shown previously that the Golgi protein formiminotransferase cyclodeaminase (FTCD) binds to vimentin filaments in vivo and in vitro, and that overexpression of FTCD causes dramatic rearrangements of the vimentin IF cytoskeleton (Gao and Sztul, J. Cell Biol. 152, 877 – 894, 2001). Using real-time imaging, we now show that FTCD causes bundling of individual thinner vimentin filaments into fibers and that the bundling always originates at the Golgi. FTCD appears to be the molecular “glue” since FTCD cross-links vimentin filaments in vitro. To initiate the analysis of structural determinants required for FTCD function in vimentin dynamics, we used structure-based design to generate individual formiminotransferase (FT) and cyclodeaminase (CD) domains, and to produce an enzymatically inactive FTCD. We show that the intact octameric structure is required for FTCD binding to vimentin filaments and for promoting filament assembly, but that eliminating enzymatic activity does not affect FTCD effects on the vimentin cytoskeleton. Our findings indicate that the Golgi protein FTCD is a potent modulator of the vimentin IF cytoskeleton, and suggest that the Golgi might act as a reservoir for proteins that regulate cytoskeletal dynamics.

The decoration of the lipid A headgroups of the lipooligosaccharide (LOS) by the LOS phosphoethanolamine (PEA) transferase (LptA) in Neisseria spp. is central for resistance to polymyxin. The structure of the globular domain of LptA shows that the protein has five disulphide bonds, indicating that it is a potential substrate of the protein oxidation pathway in the bacterial periplasm. When neisserial LptA was expressed in Escherichia coli in the presence of the oxidoreductase, EcDsbA, polymyxin resistance increased 30-fold. LptA decorated one position of the E. coli lipid A headgroups with PEA. In the absence of the EcDsbA, LptA was degraded in E. coli. Neisseria spp. express three oxidoreductases, DsbA1, DsbA2 and DsbA3, each of which appear to donate disulphide bonds to different targets. Inactivation of each oxidoreductase in N. meningitidis enhanced sensitivity to polymyxin with combinatorial mutants displaying an additive increase in sensitivity to polymyxin, indicating that the oxidoreductases were required for multiple pathways leading to polymyxin resistance. Correlates were sought between polymyxin sensitivity, LptA stability or activity and the presence of each of the neisserial oxidoreductases. Only meningococcal mutants lacking DsbA3 had a measurable decrease in the amount of PEA decoration on lipid A headgroups implying that LptA stability was supported by the presence of DsbA3 but did not require DsbA1/2 even though these oxidoreductases could oxidise the protein. This is the first indication that DsbA3 acts as an oxidoreductase in vivo and that multiple oxidoreductases may be involved in oxidising the one target in N. meningitidis. In conclusion, LptA is stabilised by disulphide bonds within the protein. This effect was more pronounced when neisserial LptA was expressed in E. coli than in N. meningitidis and may reflect that other factors in the neisserial periplasm have a role in LptA stability.

The fatty aldehyde dehydrogenase (Vh-ALDH) isolated from the luminescent bacterium, Vibrio harveyi, differs from other aldehyde dehydrogenases in its high affinity for NADP(+). The binding of NADP(+) appears to arise from the interaction of the 2'-phosphate of the adenosine moiety of NADP(+) with a threonine (T175) in the nucleotide recognition site just after the beta(B) strand as well as with an arginine (R210) that pi stacks over the adenosine moiety. The active site of Vh-ALDH contains the usual suspects of a cysteine (C289), two glutamates (E253 and E377) and an asparagine (N147) involved in the aldehyde dehydrogenase mechanism. However, Vh-ALDH has one polar residue in the active site that distinguishes it from other ALDHs; a histidine (H450) is in close contact with the cysteine nucleophile. As a glutamate has been implicated in promoting the nucleophilicity of the active site cysteine residue in ALDHs, the close contact of a histidine with the cysteine nucleophile in Vh-ALDH raises the possibility of alternate routes to increase the reactivity of the cysteine nucleophile. The effects of mutation of these residues on the different functions catalyzed by Vh-ALDH including acylation, (thio)esterase, reductase and dehydrogenase activities should help define the specific roles of the residues in the active site of ALDHs.

The specific targeting of protein to organelles is achieved by targeting signals being recognised by their cognate receptors. Cytosolic chaperones, bound to precursor proteins, are recognized by specific receptors of the import machinery enabling transport into the specific organelle. The aim of this study was to gain greater insight into the mode of recognition of the C-termini of Hsp70 and Hsp90 chaperones by the Tetratricopeptide Repeat (TPR) domain of the chloroplast import receptor Toc64 from Arabidopsis thaliana (At). The monomeric TPR domain binds with 1∶1 stoichiometry in similar micromolar affinity to both Hsp70 and Hsp90 as determined by isothermal titration calorimetry (ITC). Mutations of the terminal EEVD motif caused a profound decrease in affinity. Additionally, this study considered the contributions of residues upstream as alanine scanning experiments of these residues showed reduced binding affinity. Molecular dynamics simulations of the TPR domain helices upon peptide binding predicted that two helices within the TPR domain move backwards, exposing the cradle surface for interaction with the peptide. Our findings from ITC and molecular dynamics studies suggest that AtToc64_TPR does not discriminate between C-termini peptides of Hsp70 and Hsp90.

Aldehyde dehydrogenase from the bioluminescent bacterium, Vibrio harveyi , catalyses the oxidation of long-chain aliphatic aldehydes to acids. The enzyme is unique compared with other forms of aldehyde dehydrogenase in that it exhibits a very high specificity and affinity for the cofactor NADP + . Structural studies of this enzyme and comparisons with other forms of aldehyde dehydrogenase provide the basis for understanding the molecular features that dictate these unique properties and will enhance our understanding of the mechanism of catalysis for this class of enzyme. The X-ray structure of aldehyde dehydrogenase from V. harveyi has been solved to 2.5-A resolution as a partial complex with the cofactor NADP + and to 2.1-A resolution as a fully bound ‘holo’complex. The cofactor preference exhibited by different forms of the enzyme is predominantly determined by the electrostatic environment surrounding the 2´-hydroxy or the 2´-phosphate groups of the adenosine ribose moiety of NAD + or NADP + , respectively. In the NADP + -dependent structures the presence of a threonine and a lysine contribute to the cofactor specificity. In the V. harveyi enzyme an arginine residue (Arg-210) contributes to the high cofactor affinity through a pi stacking interaction with the adenine ring system of the cofactor. Further differences between the V. harveyi enzyme and other aldehyde dehydrogenases are seen in the active site, in particular a histidine residue which is structurally conserved with phosphorylating glyceraldehyde-3-phosphate dehydrogenase. This may suggest an alternative mechanism for activation of the reactive cysteine residue for nucleophilic attack.

Cholesterol oxidase is a monomeric flavoenzyme which catalyzes the oxidation and isomerization of cholesterol to cholest-4-en-3-one. The enzyme interacts with lipid bilayers in order to bind its steroid substrate. The X-ray structure of the enzyme from Brevibacterium sterolicum revealed two loops, comprising residues 78-87 and residues 433-436, which act as a lid over the active site and facilitate binding of the substrate [Vrielink et al. (1991) J. Mol. Biol. 219, 533-554; Li et al. (1993) Biochemistry 32, 11507-11515]. It was postulated that these loops must open, forming a hydrophobic channel between the membrane and the active site of the protein and thus sequestering the cholesterol substrate from the aqueous environment. Here we describe the three-dimensional structure of the homologous enzyme from Streptomyces refined to 1.5 A resolution. Structural comparisons to the enzyme from B. sterolicum reveal significant conformational differences in these loop regions; in particular, a region of the loop comprising residues 78-87 adopts a small amphipathic helical turn with hydrophobic residues directed toward the active site cavity and hydrophilic residues directed toward the external surface of the molecule. It seems reasonable that this increased rigidity reduces the entropy loss that occurs upon binding substrate. Consequently, the Streptomyces enzyme is a more efficient catalyst. In addition, we have determined the structures of three active site mutants which have significantly reduced activity for either the oxidation (His447Asn and His447Gln) or the isomerization (Glu361Gln). Our structural and kinetic data indicate that His447 and Glu361 act as general base catalysts in association with conserved water H2O541 and Asn485. The His447, Glu361, H2O541, and Asn485 hydrogen bond network is conserved among other oxidoreductases. This catalytic tetrad appears to be a structural motif that occurs in flavoenzymes that catalyze the oxidation of unactivated alcohols.

The ubiquitin pathway is a major system for selective proteolysis in eukaryotes. However, the mechanisms underlying substrate selectivity by the ubiquitin system remain unclear. We previously identified isoforms of a rat ubiquitin-conjugating enzyme (E2) homologous to the Saccharomyces cerevisiae class I E2 genes, UBC4/UBC5. Two isoforms, although 93% identical, show distinct features. UBC4-1 is expressed ubiquitously, whereas UBC4-testis is expressed in spermatids. Interestingly, although these isoforms interacted similarly with some ubiquitin-protein ligases (E3s) such as E6-AP and rat p100 and an E3 that conjugates ubiquitin to histone H2A, they also supported conjugation of ubiquitin to distinct subsets of testis proteins. UBC4-1 showed an 11-fold greater ability to support conjugation of ubiquitin to endogenous substrates present in a testis nuclear fraction. Site-directed mutagenesis of the UBC4-testis isoform was undertaken to identify regions of the molecule responsible for the observed difference in substrate specificity. Four residues (Gln-15, Ala-49, Ser-107, and Gln-125) scattered on surfaces away from the active site appeared necessary and sufficient for UBC4-1-like conjugation. These four residues identify a large surface of the E2 core domain that may represent an area of binding to E3s or substrates. These findings demonstrate that a limited number of amino acid substitutions in E2s can dictate conjugation of ubiquitin to different proteins and indicate a mechanism by which small E2 molecules can encode a wide range of substrate specificities.

The involvement of two conserved glycine residues (Gly229 and Gly234) in activity and nucleotide binding in Vibrio harveyi aldehyde dehydrogenase (ALDH) have been investigated. Each of the glycine residues has been mutated to alanine and the mutant ALDHs have been expressed in Escherichia coli and specifically labelled with [ 35 S]methionine. The G229A mutant was inactive with either NADP + or NAD + as coenzyme and did not bind to 2′,5′-ADP Sepharose, indicating a complete loss of nucleotide affinity. In contrast, the G234A mutant showed a high affinity for 2′,5′-ADP Sepharose. Purified G234A mutant showed similar kinetic properties to the native enzyme including a pre-steady-state burst of NADPH; however, the Michaelis constants for NAD + and NADP + were increased by 3- to 9-fold, showing that the mutation had an effect on saturation of the enzyme with NAD(P) + . These data are consistent with the structure for the nucleotide binding domain of Vh.ALDH being similar to that of class 3 or class 2 mammalian ALDHs which differ from the classical nucleotide binding domain found in most dehydrogenases.

Gram-negative bacteria possess an outer membrane envelope consisting of an outer leaflet of lipopolysaccharides, also called endotoxins, which protect the pathogen from antimicrobial peptides and have multifaceted roles in virulence. Lipopolysaccharide consists of a glycan moiety attached to lipid A, embedded in the outer membrane. Modification of the lipid A headgroups by phosphoethanolamine (PEA) or 4-amino-arabinose residues increases resistance to the cationic cyclic polypeptide antibiotic, polymyxin. Lipid A PEA transferases are members of the YhjW/YjdB/YijP superfamily and usually consist of a transmembrane domain anchoring the enzyme to the periplasmic face of the cytoplasmic membrane attached to a soluble catalytic domain. The crystal structure of the soluble domain of the protein of the lipid A PEA transferase from Neisseria meningitidis has been determined crystallographically and refined to 1.4 A resolution. The structure reveals a core hydrolase fold similar to that of alkaline phosphatase. Loop regions in the structure differ, presumably to enable interaction with the membrane-localized substrates and to provide substrate specificity. A phosphorylated form of the putative nucleophile, Thr280, is observed. Metal ions present in the active site are coordinated to Thr280 and to residues conserved among the family of transferases. The structure reveals the protein components needed for the transferase chemistry; however, substrate-binding regions are not evident and are likely to reside in the transmembrane domain of the protein.

A PCR-based random mutagenesis procedure was employed to identify several thermosensitive mutants of the MPTP enzyme, the murine homologue of the human T-cell PTPase and rat PTP-S enzymes. Four mutants with varying degrees of thermosensitivity were characterized for their thermostability and refolding properties following incubation at the nonpermissive temperature. Structure analysis of these mutations based on the hPTP1B co-ordinate structure demonstrates a clear relationship between the position of each mutated amino acid relative to the catalytic cysteine residue and their thermostability. Introduction of two of these mutations in the related enzyme hPTP1B suggests that the structural defects and the resulting thermosensitivity of these mutations may represent an intrinsic property of all PTPase catalytic domains.

The enzyme phosphoethanolamine transferase A is involved in the addition of phosphoethanolamine moieties to lipid A in Neisseria meningitidis. The enzyme is composed of an N-terminal transmembrane domain and a C-terminal soluble domain that is present in the periplasm of the bacteria. A membrane-deletion construct of the enzyme was designed and expressed in Escherichia coli. Well ordered crystals that diffracted to 1.7 A resolution were obtained by carrying out a limited trypsin digestion of the protein to remove a predicted N-­terminal disordered portion. The crystals belonged to space group P21, with unit-cell parameters a = 44.3, b = 71.6, c = 49.9 A, β = 109.2°, and contained one molecule in the asymmetric unit.

It has been hypothesized that the bifunctional enzyme DmpFG channels its intermediate, acetaldehyde, from one active site to the next using a buried intermolecular channel identified in the crystal structure. This channel appears to switch between an open and a closed conformation depending on whether the coenzyme NAD + is present or absent. Here, we applied molecular dynamics and metadynamics to investigate channeling within DmpFG in both the presence and absence of NAD + . We found that substrate channeling within this enzyme is energetically feasible in the presence of NAD + but was less likely in its absence. Tyr-291, a proposed control point at the channel's entry, does not appear to function as a molecular gate. Instead, it is thought to orientate the substrate 4-hydroxy-2-ketovalerate in DmpG before reaction occurs, and may function as a proton shuttle for the DmpG reaction. Three hydrophobic residues at the channel's exit appear to have an important role in controlling the entry of acetaldehyde into the DmpF active site.

Various known inhibitors of glycosidases were assessed for their ability to inhibit, both independently as well as with UDP, the cholesterol alpha-glucosyltransferase from Helicobacter pylori. The sub-cloning, expression and purification of the glucosyltransferase is also discussed.

Cholesterol oxidase is a bacterial-specific flavoenzyme that catalyzes the oxidation and isomerisation of steroids containing a 3β hydroxyl group and a double bond at the Δ5–6 of the steroid ring system. The enzyme is a member of a large family of flavin-specific oxidoreductases and is found in two different forms: one where the flavin adenine dinucleotide (FAD) cofactor is covalently linked to the protein and one where the cofactor is non-covalently bound to the protein. These two enzyme forms have been extensively studied in order to gain insight into the mechanism of flavin-mediated oxidation and the relationship between protein structure and enzyme redox potential. More recently the enzyme has been found to play an important role in bacterial pathogenesis and hence further studies are focused on its potential use for future development of novel antibacterial therapeutic agents. In this review the biochemical, structural, kinetic and mechanistic features of the enzyme are discussed.

Cholesterol oxidase is a flavoenzyme that catalyzes the oxidation and isomerization of 3beta-hydroxysteroids. Structural and mutagenesis studies have shown that Asn485 plays a key role in substrate oxidation. The side chain makes an NH...pi interaction with the reduced form of the flavin cofactor. A N485D mutant was constructed to further test the role of the amide group in catalysis. The mutation resulted in a 1800-fold drop in the overall k(cat). Atomic resolution structures were determined for both the N485L and N485D mutants. The structure of the N485D mutant enzyme (at 1.0 A resolution) reveals significant perturbations in the active site. As predicted, Asp485 is oriented away from the flavin moiety, such that any stabilizing interaction with the reduced flavin is abolished. Met122 and Glu361 form unusual hydrogen bonds to the functional group of Asp485 and are displaced from the positions they occupy in the wild-type active site. The overall effect is to disrupt the stabilization of the reduced FAD cofactor during catalysis. Furthermore, a narrow transient channel that is shown to form when the wild-type Asn485 forms the NH...pi interaction with FAD and that has been proposed to function as an access route of molecular oxygen, is not observed in either of the mutant structures, suggesting that the dynamics of the active site are altered.

The usage by enzymes of specific binding pathways for gaseous substrates or products is debated. The crystal structure of the redox enzyme cholesterol oxidase, determined at sub-angstrom resolution, revealed a hydrophobic tunnel that may serve as a binding pathway for oxygen and hydrogen peroxide. This tunnel is formed by a cascade of conformational rearrangements and connects the active site with the exterior surface of the protein. To elucidate the relationship between this tunnel and gas binding and release, three mutant enzymes were constructed to block the tunnel or its putative gate. Mutation of the proposed gating residue Asn485 to Asp or tunnel residue Phe359 or Gly347 to Trp or Asn reduces the catalytic efficiency of oxidation. The K mO 2 increases from 300 +/- 35 microM for the wild-type enzyme to 617 +/- 15 microM for the F359W mutant. The k cat for the F359W mutant-catalyzed reaction decreases 13-fold relative to that of the wild-type-catalyzed reaction. The N485D and G347N mutants could not be saturated with oxygen. Transfer of hydride from the sterol to the flavin prosthetic group is no longer rate-limiting for these tunnel mutants. The steady-state kinetics of both wild-type and tunnel mutant enzymes are consistent with formation of a ternary complex of steroid and oxygen during catalysis. Furthermore, kinetic cooperativity with respect to molecular oxygen is observed with the tunnel mutants, but not with the wild-type enzyme. A rate-limiting conformational change for binding and release of oxygen and hydrogen peroxide, respectively, is consistent with the cooperative kinetics. In the atomic-resolution structure of F359W, the indole ring of the tryptophan completely fills the tunnel and is observed in only a single conformation. The size of the indole is proposed to limit conformational rearrangement of residue 359 that leads to tunnel opening in the wild-type enzyme. Overall, these results substantiate the functional importance of the tunnel for substrate binding and product release.

Two high-resolution structures of a double mutant of bacterial cholesterol oxidase in the presence or absence of a ligand, glycerol, are presented, showing the trajectory of glycerol as it binds in a Michaelis complex-like position in the active site. A group of three aromatic residues forces the oxidized isoalloxazine moiety to bend along the N5-N10 axis as a response to the binding of glycerol in the active site. Movement of these aromatic residues is only observed in the glycerol-bound structure, indicating that some tuning of the FAD redox potential is caused by the formation of the Michaelis complex during regular catalysis. This structural study suggests a possible mechanism of substrate-assisted flavin activation, improves our understanding of the interplay between the enzyme, its flavin cofactor and its substrate, and is of use to the future design of effective cholesterol oxidase inhibitors.

L-Amino acid oxidase is a dimeric glycosylated flavoenzyme, a major constituent of the venom-from the snake Calloselasma rhodostoma. The enzyme exhibits apoptosis inducing effects as well as antibacterial and anti-HIV activities. The structure of l-amino acid oxidase with its substrate (L-phenylalanine) has been refined to a resolution of 1.8 A. The complex structure reveals the substrate bound to the reduced flavin (FADred). Alternative conformations for the key residues His223 and Arg322 are evident, suggesting a dynamic active site. Furthermore, conformational changes are apparent for the isoalloxazine ring; the three-ring system exhibits more bending around the N5-N10 axis compared to the oxidized flavin. The implications of the observed dynamics on the mechanism of catalysis are discussed. Inspection of buried surfaces in the enzyme reveals a Y-shaped channel system extending from the external surface of the protein to the active site. One portion of this channel may serve as the entry path for O2 during the oxidative half-reaction. The second region, separated from the proposed O2 channel by the N terminus (residues 8-16) of the protein, may play a role in H2O2 release. Interestingly, the latter portion of the channel would direct the H2O2 product to the exterior surface of the protein, near the glycan moiety, thought to anchor the enzyme to the host cell. This channel location may explain the ability of the enzyme to localize H2O2 to the targeted cell and thus induce the apoptotic effect.

Hydrogen atoms are a vital component of enzyme structure and function. In recent years, atomic resolution crystallography (>or=1.2 A) has been successfully used to investigate the role of the hydrogen atom in enzymatic catalysis. Here, atomic resolution crystallography was used to study the effect of pH on cholesterol oxidase from Streptomyces sp., a flavoenzyme oxidoreductase. Crystallographic observations of the anionic oxidized flavin cofactor at basic pH are consistent with the UV-visible absorption profile of the enzyme and readily explain the reversible pH-dependent loss of oxidation activity. Furthermore, a hydrogen atom, positioned at an unusually short distance from the main chain carbonyl oxygen of Met122 at high pH, was observed, suggesting a previously unknown mechanism of cofactor stabilization. This study shows how a redox active site responds to changes in the enzyme's environment and how these changes are able to influence the mechanism of enzymatic catalysis.

Cholesterol oxidase is a monomeric flavoenzyme that catalyses the oxidation of cholesterol to cholest-5-en-3-one followed by isomerization to cholest-4-en-3-one. The enzyme from Brevibacterium sterolicum contains the FAD cofactor covalently bound to His121. It was previously demonstrated that the H121A substitution results in a approximately 100 mV decrease in the midpoint redox potential and a approximately 40-fold decrease in turnover number compared to wild-type enzyme [Motteran, Pilone, Molla, Ghisla and Pollegioni (2001) Journal of Biological Chemistry 276, 18024-18030]. A detailed kinetic analysis of the H121A mutant enzyme shows that the decrease in turnover number is largely due to a corresponding decrease in the rate constant of flavin reduction, whilst the re-oxidation reaction is only marginally altered and the isomerization reaction is not affected by the substitution and precedes product dissociation. The X-ray structure of the mutant protein, determined to 1.7 A resolution (1 A identical with 0.1 nm), reveals only minor changes in the overall fold of the protein, namely: two loops have slight movements and a tryptophan residue changes conformation by a rotation of 180 degrees about chi1 compared to the native enzyme. Comparison of the isoalloxazine ring moiety of the FAD cofactor between the structures of the native and mutant proteins shows a change from a non-planar to a planar geometry (resulting in a more tetrahedral-like geometry for N5). This change is proposed to be a major factor contributing to the observed alteration in redox potential. Since a similar distortion of the flavin has not been observed in other covalent flavoproteins, it is proposed to represent a specific mode to facilitate flavin reduction in covalent cholesterol oxidase.

Single crystals of cholesterol oxidase from Brevibacterium sterolicum containing a covalently bound form of the FAD cofactor have been obtained. The crystals are grown by vapor diffusion using the hanging drop technique from 12% polyethylene glycol, Mr 8000, and 75 mM MnSO4 as the precipitant at pH 5.2. In order to obtain large diffraction quality crystals, nucleation must occur at 22 degrees C with subsequent growth at 17 degrees C. The crystals belong to the monoclinic space group P21 with cell dimensions a = 78. 5 A, b = 126.7 A, c = 82.4 A and beta = 108.9 degrees with two protein molecules per asymmetric unit. Diffraction of these crystals has been observed to at least 2.2 A resolution and they are suitable for an X-ray structure analysis.

The secretory protein SecB found in Escherichia coli is a molecular chaperone that binds to precursor forms of a number of proteins targeted for export to the periplasmic space. SecB maintains these proteins in a translocation-competent conformation facilitating the translocation process. The material has been cloned and expressed in E. coli. Crystals have been grown from polyethylene glycol 8000 by vapor diffusion using the hanging drop technique. These crystals are monoclinic, belonging to space group C2 with unit cell dimensions a = 56.0 A, b = 111.1 A, c = 134.7 A, and beta = 104 degrees. The crystals diffract to 8 A resolution on a Rigaku imaging plate detector. Dynamic light scattering experiments suggest that SecB exhibits aggregation behavior with a number of different precipitating agents. These results may explain resistance of SecB to forming ordered crystals.

Multiple drug resistance (MDR) in Gram-negative bacteria represents one of the most intractable problems facing modern medicine. Not only is antibiotic resistance incrementally increasing during clinical treatment of infections, but also the evolution and acquisition of new mechanisms of antibiotic resistance lead to the sudden loss of the capacity to treat infections. The most recent superbug, MDR-Neisseria gonorrhoeae, causes the untreatable sexually transmitted infection gonorrhoeae. Chronic gonococcal infections have a high morbidity rate and, due to the explosion in cases worldwide, the community burden is enormous. N. gonorrhoeae colonizes the mucosal surfaces of the human body and has a number of virulence mechanisms that prevent clearance by the human immune system. The most important of these mechanisms is decoration of the lipooligosaccharide lipid A headgroups with phosphoethanolamine (PEA) by the enzyme, lipid A PEA transferase (LptA). Inactivation of the LptA results in the complete loss of PEA groups from lipid A, loss of bacterial colonisation of epithelial cells (Takahashi et al., 2008), increased sensitivity to cationic antimicrobial peptides (Tzeng et al., 2005) and reduced resistance to human complement mediated killing (Lewis et al., 2013). LptA knockouts of N. gonorrhoeae also result in the complete loss of virulence in models of human and mouse infections. Based on these findings we have undertaken a structure-guided approach to develop inhibitors of LptA that will assist in controlling infection and transmission by this important human pathogen. LptA is a membrane protein that interacts with two different lipid substrates. We have determined the crystal structure of the enzyme to 2.75Å resolution. The structure provides insights into the mechanism of substrate binding and catalysis and suggests that significant conformational changes occur through its catalytic cycle.

Cholesterol oxidase catalyzes the oxidation and isomerization of cholesterol to cholest-4-en-3-one. An asparagine residue (Asn485) at the active site is believed to play an important role in catalysis. To test the precise role of Asn485, we mutated it to a leucine and carried out kinetic and crystallographic studies. Steady-state kinetic analysis revealed a 1300-fold decrease in the oxidation k(cat)/K(m) for the mutant enzyme whereas the k(cat)/K(m) for isomerization is only 60-fold slower. The primary kinetic isotope effect in the mutant-catalyzed reaction indicates that 3alpha-H transfer remains the rate-determining step. Measurement of the reduction potentials for the wild-type and N485L enzymes reveals a 76 mV decrease in the reduction potential of the FAD for the mutant enzyme relative to wild type. The crystal structure of the mutant, determined to 1.5 A resolution, reveals a repositioning of the side chain of Met122 near Leu485 to form a hydrophobic pocket. Furthermore, the movement of Met122 facilitates the binding of an additional water molecule, possibly mimicking the position of the equatorial hydroxyl group of the steroid substrate. The wild-type enzyme shows a novel N-H...pi interaction between the side chain of Asn485 and the pyrimidine ring of the cofactor. The loss of this interaction in the N485L mutant destabilizes the reduced flavin and accounts for the decreased reduction potential and rate of oxidation. Thus, the observed structural rearrangement of residues at the active site, as well as the kinetic data and thermodynamic data for the mutant, suggests that Asn485 is important for creating an electrostatic potential around the FAD cofactor enhancing the oxidation reaction.

The human PDE4A catalytic domain (PDE4A 330–723 ) expressed in Sf9 cells was found to be heavily phosphorylated on both serines of the conserved SPS motif by mass spectrometric analysis. The purified protein exists as a tetramer at a concentration ∼1 mg/ml from light scattering measurement and has a K m of 2 μM in hydrolyzing cAMP. In comparison, a partially purified PDE4A 330–723 expressed in Escherichia coli has an apparent K m of 10 μM. The EC 50 values for the Mg 2+ - or Co 2+ -mediated cAMP hydrolysis between the two enzymes differed by less than twofold. In addition, both enzymes exhibit similar sensitivities toward inhibition by a diverse set of inhibitors. Together with the fact that its adjacent peptide was covalently labeled by an electrophilic cAMP analogue, these results support that the SPS motif is not part of but is positioned near the active site. An efficient purification protocol that provides a highly purified PDE4A catalytic domain suitable for crystallization study is described.

Cholesterol oxidase is a monomeric flavoenzyme that catalyzes the oxidation and isomerization of cholesterol to cholest-4-en-3-one. Two forms of the enzyme are known, one containing the cofactor non-covalently bound to the protein and one in which the cofactor is covalently linked to a histidine residue. The x-ray structure of the enzyme from Brevibacterium sterolicumcontaining covalently bound FAD has been determined and refined to 1.7-A resolution. The active site consists of a cavity sealed off from the exterior of the protein. A model for the steroid substrate, cholesterol, can be positioned in the pocket revealing the structural factors that result in different substrate binding affinities between the two known forms of the enzyme. The structure suggests that Glu475, located at the active site cavity, may act as the base for both the oxidation and the isomerization steps of the catalytic reaction. A water-filled channel extending toward the flavin moiety, inside the substrate-binding cavity, may act as the entry point for molecular oxygen for the oxidative half-reaction. An arginine and a glutamate residue at the active site, found in two conformations are proposed to control oxygen access to the cavity from the channel. These concerted side chain movements provide an explanation for the biphasic mode of reaction with dioxygen and the ping-pong kinetic mechanism exhibited by the enzyme.

The final two steps of the meta-cleavage pathway for catechol degradation in Pseudomonas sp. strain CF600 involve the conversion of 4-hydroxy-2-ketovalerate to pyruvate and acetyl coenzyme A by the enzymes 4-hydroxy-2-ketovalerate aldolase and NAD(+)-dependent acylating aldehyde dehydrogenase. Biochemical studies indicate that these two enzymes comprise a bifunctional heterodimer (DmpFG, molecular mass 71 kDa) and suggest that the product of the aldolase reaction is transferred to the dehydrogenase active site via a channeling mechanism. Crystals of the DmpFG complex grow in multiple fan-like clusters of thin plates by the hanging-drop method and are improved by streak-seeding. The crystals belong to the orthorhombic space group P2(1)2(1)2(1), with unit-cell parameters a = 102.0, b = 140.7, c = 191.3 A, and diffract to 2.1 A resolution. The asymmetric unit contains four DmpFG heterodimers. Heavy-atom derivative screening identified three isomorphous derivatives.

Background: The bifunctional enzyme formiminotransferase-cyclodeaminase (FTCD) contains two active sites at different positions on the protein structure. The enzyme binds a γ-linked polyglutamylated form of the tetrahydrofolate substrate and channels the product of the transferase reaction from the transferase active site to the cyclodeaminase active site. Structural studies of this bifunctional enzyme and its monofunctional domains will provide insight into the mechanism of substrate channeling and the two catalytic reactions. Results : The crystal structure of the formiminotransferase (FT) domain of FTCD has been determined in the presence of a product analog, folinic acid. The overall structure shows that the FT domain comprises two subdomains that adopt a novel α/β fold. Inspection of the folinic acid binding site reveals an electrostatic tunnel traversing the width of the molecule. The distribution of charged residues in the tunnel provides insight into the possible mode of substrate binding and channeling. The electron density reveals that the non-natural stereoisomer, (6 R )-folinic acid, binds to the protein; this observation suggests a mechanism for product release. In addition, a single molecule of glycerol is bound to the enzyme and indicates a putative binding site for formiminoglutamate. Conclusions : The structure of the FT domain in the presence of folinic acid reveals a possible novel mechanism for substrate channeling. The position of the folinic acid and a bound glycerol molecule near to the sidechain of His82 suggests that this residue may act as the catalytic base required for the formiminotransferase mechanism.

The fatty aldehyde dehydrogenase from the luminescent bacterium, Vibrio harveyi (Vh-ALDH), is unique with respect to its high specificity for NADP(+) over NAD(+). By mutation of a single threonine residue (Thr175) immediately downstream of the beta(B) strand in the Rossmann fold, the nucleotide specificity of Vh-ALDH has been changed from NADP(+) to NAD(+). Replacement of Thr175 by a negatively charged residue (Asp or Glu) resulted in an increase in k(cat)/K(m) for NAD(+) relative to that for NADP(+) of up to 5000-fold due to a decrease for NAD(+) and an increase for NADP(+) in their respective Michaelis constants (K(a)). Differential protection by NAD(+) and NADP(+) against thermal inactivation and comparison of the dissociation constants of NMN, 2'-AMP, 2'5'-ADP, and 5'-AMP for these mutants and the wild-type enzyme clearly support the change in nucleotide specificity. Moreover, replacement of Thr175 with polar residues (N, S, or Q) demonstrated that a more efficient NAD(+)-dependent enzyme T175Q could be created without loss of NADP(+)-dependent activity. Analysis of the three-dimensional structure of Vh-ALDH with bound NADP(+) showed that the hydroxyl group of Thr175 forms a hydrogen bond to the 2'-phosphate of NADP(+). Replacement with glutamic acid or glutamine strengthened interactions with NAD(+) and indicated why threonine would be the preferred polar residue at the nucleotide recognition site in NADP(+)-specific aldehyde dehydrogenases. These results have shown that the size and the structure of the residue at the nucleotide recognition site play the key roles in differentiating between NAD(+) and NADP(+) interactions while the presence of a negative charge is responsible for the decrease in interactions with NADP(+) in Vh-ALDH.