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Organophosphonic acids are unique as natural products in terms of stability and mimicry. The C-P bond that defines these compounds resists hydrolytic cleavage, while the phosphonyl group is a versatile mimic of transition-states, intermediates, and primary metabolites. This versatility may explain why a variety of organisms have extensively explored the use organophosphonic acids as bioactive secondary metabolites. Several of these compounds, such as fosfomycin and bialaphos, figure prominently in human health and agriculture. The enzyme reactions that create these molecules are an interesting mix of chemistry that has been adopted from primary metabolism as well as those with no chemical precedent. Additionally, the phosphonate moiety represents a source of inorganic phosphate to microorganisms that live in environments that lack this nutrient; thus, unusual enzyme reactions have also evolved to cleave the C-P bond. This review is a comprehensive summary of the occurrence and function of organophosphonic acids natural products along with the mechanisms of the enzymes that synthesize and catabolize these molecules. [ABSTRACT FROM AUTHOR]

meta-Cleavage product (MCP) hydrolases catalyze C-C bond fission in the aerobic catabolism of aromatic compounds by bacteria. These enzymes utilize a Ser-His-Asp triad to catalyze hydrolysis via an acyl-enzyme intermediate. BphD, which catalyzes the hydrolysis of 2-hydroxy-6-oxo-6-phenylhexa-2,4-dienoic acid (HOPDA) in biphenyl degradation: catalyzed the hydrolysis of an ester analogue, p-nitrophenyl benzoate (pNPB), with a kcat, value (6.3 ± 0.5 s-1) similar to that of HOPDA (6.5 ± 0.5 s-1). Consistent with the breakdown of a shared intermediate, product analyses revealed that BphD catalyzed the methanolysis of both HOPDA and pNPB, partitioning the products to benzoic acid and methyl benzoate in similar ratios. Turnover of HOPDA was accelerated up to 4-fold in the presence of short, primary alcohols (methanol > ethanol > n-propanol), suggesting that deacylation is rate-limiting during catalysis. In the steady-state hydrolysis of HOPDA, kcat,/Km, values were independent of methanol concentration, while both kcat and Km values increased with methanol concentration. This result was consistent with a simple model of nucleophilic catalysis. Although the enzyme could not be saturated with pNPB at methanol concentrations of >250 mM, kobs, values from the steady.state turnover of pNPB at low methanol concentrations were also consistent with a nucleophilic mechanism of catalysis. Finally, transient-state kinetic analysis of pNPB hydrolysis by BphD variants established that substitution of the catalytic His reduced the rate of acylation by more than 3 orders of magnitude. This suggests that for pNPB hydrolysis, the serine nudeophile is activated by the His-Asp dyad. In contrast, rapid acylation of the H265Q variant during C-C bond deavage suggests that the serinate forms via a substrate-assisted mechanism. Overall, the data indicate that ester hydrolysis proceeds via the same acyl-enzyme intermediate as that of the physiological substrate but that the scrinc nucleophile is activated via a different mechanism. [ABSTRACT FROM AUTHOR]

Enediynes are potent antitumor antibiotics that are classified as 9- or 10-membered according to the size of the enediyne core structure. However, almost nothing is known about enediyne core biosynthesis, and the determinants of 9- versus 10-membered enediyne core biosynthetic divergence remain elusive. Previous work identified enediyne-specific polyketide synthases (PKSEs) that can be phylogenetically distinguished as being involved in 9- versus 10-membered enediyne biosynthesis, suggesting that biosynthetic divergence might originate from differing PKSE chemistries. Recent in vitro studies have identified several compounds produced by the PKSE and associated thioesterase (TE), but condition-dependent product profiles make it difficult to ascertain a true catalytic difference between 9- and 10-membered PKSE-TE systems. Here we report that PKSE chemistry does not direct 9- versus 10-membered enediyne core biosynthetic divergence as revealed by comparing the products from three 9-membered and two 10-membered PKSE-TE systems under identical conditions using robust in vivo assays. Three independent experiments support a common catalytic function for 9- and 10-membered PKSEs by the production of a heptaene metabolite from: (i) all five cognate PKSE-TE pairs in Escherichia coli; (ii) the C-1027 and calicheamicin cognate PKSE-TEs in Streptomyces lividans K4-114; and (iii) selected native producers of both 9- and 10-membered enediynes. Furthermore, PKSEs and TEs from different 9- and 10-membered enediyne biosynthetic machineries are freely interchangeable, revealing that 9- versus 10-membered enediyne core biosynthetic divergence occurs beyond the PKSE-TE level. These findings establish a starting point for determining the origins of this biosynthetic divergence. [ABSTRACT FROM AUTHOR]

The microbial degradation of polychlorinated biphenyls (PCBs) by the biphenyl catabolic (Bph) pathway is limited in part by the pathway's fourth enzyme, BphD. BphD catalyzes an unusual carbon-carbon bond hydrolysis of 2-hydroxy-6-oxo-6-phenylhexa-2,4-dienoic acid (HOPDA), in which the substrate is subject to histidine-mediated enol-keto tautomerization prior to hydrolysis. Chlorinated HOPDAs such as 3-C1 HOPDA inhibit BphD. Here we report that BphD preferentially hydrolyzed a series of 3-substituted HOPDAs in the order H > F > Cl > Me, suggesting that catalysis is affected by steric, not electronic, determinants. Transient state kinetic studies performed using wild-type BphD and the hydrolysis-defective S112A variant indicated that large 3-substituents inhibited His-265-catalyzed tautomerization by 5 orders of magnitude. Structural analyses of S112A.3-Cl HOPDA and S112A·3,10-diF HOPDA complexes revealed a non-productive binding mode in which the plane defined by the carbon atoms of the dienoate moiety of HOPDA is nearly orthogonal to that of the proposed keto tautomer observed in the S112A·HOPDA complex. Moreover, in the 3-Cl HOPDA complex, the 2-hydroxo group is moved by 3.6 A from its position near the catalytic His-265 to hydrogen bond with Arg-190 and access of His-265 is blocked by the 3-Cl substituent. Nonproductive binding may be stabilized by interactions involving the 3-substituent with non-polar side chains. Solvent molecules have poor access to C6 in the S112A·3-Cl HOPDA structure, more consistent with hydrolysis occurring via an acyl-enzyme than a gem-diol intermediate. These results provide insight into engineering BphD for PCB degradation. [ABSTRACT FROM AUTHOR]

BphD of Burkholderia xenovorans LB400 catalyzes an unusual C-C bond hydrolysis of 2-hydroxy-6-oxo-6-phenyl-hexa-2,4-dienoic acid (HOPDA) to afford benzoic acid and 2-hydroxy-2,4-pentadienoic acid (HPD). An enol-keto tautomerization has been proposed to precede hydrolysis via a gemdiol intermediate. The role of the canonical catalytic triad (Ser- 112, His-265, Asp-237) in mediating these two half-reactions remains unclear. We previously reported that the BphD-catalyzed hydrolysis of HOPDA (λmax is 434 nm for the free enolate) proceeds via an unidentified intermediate with a red-shifted absorption spectrum (λmax is 492 nm) (Horsman, G. P., Ke, J., Dai, S., Seah, S. Y. K., Bolin, J. 1., and Eltis, L. D. (2006) Biochemistry 45, 11071-11086). Here we demonstrate that the S112A variant generates and traps a similar intermediate (λmax is 506 nm) with a similar rate, 1/τ ~ 500 s-1. The crystal structure of the S112A;HOPDA complex at 1.8-Å resolution identified this intermediate as the keto tautomer, (E)-2,6-dioxo-6-phenyl-hex-3-enoate. This keto tautomer did not accumulate in either the H265A or the S112A/H265A double variants, indicating that His-265 catalyzes tautomerization. Consistent with this role, the wild type and S112A enzymes catalyzed tautomerization of the product HPD, whereas H265A variants did not. This study thus identifies a keto intermediate, and demonstrates that the catalytic triad histidine catalyzes the tautomerization half-reaction, expanding the role of this residue from its purely hydrolytic function in other serine hydrolases. Finally, the S112A:HOPDA crystal structure is more consistent with hydrolysis occurring via an acyl-enzyme intermediate than a gem-diol intermediate as solvent molecules have poor access to C6, and the closest ordered water is 7 Å away. [ABSTRACT FROM AUTHOR]

Kinetic and structural analyses of 2-hydroxy-6-oxo-6-phenylhexa-2,4-dienoic acid (HOPDA) hydrolase from Burkholderia xenovorans LB400 (BphDLB400) provide insight into the catalytic mechanism of this unusual serine hydrolase. Single turnover stopped-flow analysis at 25 °C showed that the enzyme rapidly (1/τ1 ∼ 500 s-1) transforms HOPDA λmax = 434 nm) into a species with electronic absorption maxima at 473 and 492 nm. The absorbance of this enzyme-bound species (E:S) decayed in a biphasic manner (1/τ2 = 54 s-1, 1/τ3 = 6 s-1 ∼kcat) with simultaneous biphasic appearance (48 and 8 s-1) of an absorbance band at 270 nm characteristic of one of the products, 2-hydroxypenta-2,4-dienoic acid (HPD). Increasing solution viscosity with glycerol slowed 1/τ1 and 1/τ2 but affected neither 1/τ3 nor kcat, suggesting that 1/τ2 may reflect diffusive HPD dissociation, and 1/τ3 represents an intramolecular event. Product inhibition studies suggested that the other product, benzoate, is released after HPD. Contrary to studies in a related hydrolase, we found no evidence that ketonized HOPDA is partially released prior to hydrolysis, and, therefore, postulate that the biphasic kinetics reflect one of two mechanisms, pending assignment of E:S (λmax = 492 nm). The crystal structures of the wild type, the S112C variant, and S112C incubated with HOPDA were each determined to 1.6 Å resolution. The latter reveals interactions between conserved active site residues and the dienoate moiety of the substrate. Most notably, the catalytic residue His265 is hydrogen-bonded to the 2-hydroxy/oxo substituent of HOPDA, consistent with a role in catalyzing ketonization. The data are more consistent with an acyl-enzyme mechanism than with the formation of a gem-diol intermediate. [ABSTRACT FROM AUTHOR]

The basis of the respective regiospecificities of intradiol and extradiol dioxygenase is poorly understood and may be linked to the protonation state of the bidentate-bound catechol in the enzyme/substrate complex. Previous ultraviolet resonance Raman (UVRR) and UV-visible (UV-vis) difference spectroscopic studies demonstrated that, in extradiol dioxygenases, the catechol is bound to the Fe(II) as a monoanion. In this study, we use the same approaches to demonstrate that, in catechol 1,2-dioxygenase (C12O), an intradiol enzyme, the catechol binds to the Fe(III) as a dianion. Specifically, features at 290 nm and 1550 cm-1 in the UV-vis and UVRR difference spectra, respectively, are assigned to dianionic catechol based on spectra of the model compound, ferric tris(catecholate). The UVRR spectroscopic band assignments are corroborated by density functional theory (DFT) calculations. In addition, negative features at 240 nm in UV-vis difference spectra and at 1600, 1210, and 1175 cm-1 in UVRR difference spectra match those of a tyrosinate model compound, consistent with protonation of the axial tyrosinate ligand when it is displaced from the ferric ion coordination sphere upon substrate binding. The DFT calculations ascribe the asymmetry of the bound dianionic substrate to the trans donor effect of an equatorially ligated tyrosinate ligand. In addition, the computations suggest that trans donation from the tyrosinate ligand may facilitate charge transfer from the substrate to yield the iron-bound semiquinone transition state, which is capable of reacting with dioxygen. in illustrating the importance of ligand trans effects in a biological system, the current study demonstrates the power of combining difference UVRR and optical spectroscopies to probe metal ligation in solution. [ABSTRACT FROM AUTHOR]

Nicotinamide adenine dinucleotide (NAD+) is a life essential molecule involved in versatile biological processes. To date, only two de novo biosynthetic routes to NAD+ are described, both of which start from a proteinogenic amino acid and are tightly controlled. Here, a de novo quinolinic acid pathway starting from chorismate, which provides an alternative route (named as the C3N pathway) to NAD+ biosynthesis, is established. Significantly, the C3N pathway yields extremely high cellular concentrations of NAD(H) in E. coli. Its utility in cofactor engineering is demonstrated by introducing the four‐gene C3N module to cell factories to achieve higher production of 2,5‐dimethylpyrazine and develop an efficient C3N‐based whole‐cell bioconversion system for preparing chiral amines. The wide distribution and abundance of chorismate in most kingdoms of life implies a general utility of the C3N pathway for modulating cellular levels of NAD(H) in versatile organisms. [ABSTRACT FROM AUTHOR]

BphK is a glutathione S-transferase of unclear physiological function that occurs in some bacterial biphenyl catabolic (bph) pathways. We demonstrated that BphK of Burkholderia xenovorans strain LB400 catalyzes the dehalogenation of 3-chloro 2-hydroxy-6-oxo-6-phenyl-2,4-dienoates (HOPDAs), compounds that are produced by the cometabolism of polychlorinated biphenyls (PCBs) by the bph pathway and that inhibit the pathway's hydrolase. A one-column protocol was developed to purify heterologously produced BphK. The purified enzyme had the greatest specificity for 3-Cl HOPDA (kcat/Km, ∼ 104 M-1 s-1), which it dechlorinated approximately 3 orders of magnitude more efficiently than 4-chlorobenzoate, a previously proposed substrate of BphK. The enzyme also catalyzed the dechlorination of 5-Cl HOPDA and 3,9,11-triCl HOPDA. By contrast, BphK did not detectably transform HOPDA, 4-Cl HOPDA, or chlorinated 2,3-dihydroxybiphenyls. The BphK-catalyzed dehalogenation proceeded via a ternary-complex mechanism and consumed 2 equivalents of glutathione (GSH) (Km for GSH in the presence of 3-Cl HOPDA, ∼0.1 mM). A reaction mechanism consistent with the enzyme's specificity is proposed. The ability of BphK to dehalogenate inhibitory PCB metabolites supports the hypothesis that this enzyme was recruited to facilitate PCB degradation by the bph pathway. [ABSTRACT FROM AUTHOR]

The C-1027 enediyne antitumor antibiotic from Streptomyces globisporus possesses an ( S)-3-chloro-5-hydroxy-β-tyrosine moiety, the chloro- and hydroxy-substituents of which are installed by a flavin-dependent halogenase SgcC3 and monooxygenase SgcC, respectively. Interestingly, a single flavin reductase, SgcE6, can provide reduced flavin to both enzymes. Bioinformatics analysis reveals that, similar to other flavin reductases involved in natural product biosynthesis, SgcE6 belongs to the HpaC-like subfamily of the Class I flavin reductases. The present study describes the steady-state kinetic characterization of SgcE6 as a strictly NADH- and FAD-specific enzyme. [ABSTRACT FROM AUTHOR]

Summary: Rational design of enzymes with improved properties, such as enantioselectivity, usually focuses mutations within the substrate binding site. On the other hand, directed evolution of enzymes usually targets the entire protein and discovers beneficial mutations far from the substrate binding site. In this paper, we propose an explanation for this discrepancy and show that a combined approach—random mutagenesis within the substrate binding site—is better. To increase the enantioselectivity (E) of a Pseudomonas fluorescens esterase (PFE) toward methyl 3-bromo-2-methylpropionate, we focused mutagenesis into the substrate binding site at Trp28, Val121, Phe198, and Val225. Five of the catalytically active mutants (13%) showed better enantioselectivity than wild-type PFE. The increases in enantioselectivity were higher (up to 5-fold, reaching E = 61) than with mutants identified by random mutagenesis of the entire enzyme. [Copyright &y& Elsevier]

In contrast to the well-characterized second messenger adenosine 3 ′ ,5 ′ -cyclic monophosphate (3 ′ ,5 ′ -cAMP), the biological roles of its isomer 2 ′ ,3 ′ -cAMP remain largely unknown, especially in bacteria. Recent work reported that RNase I-dependent elevation of 2 ′ ,3 ′ -cNMP levels in Escherichia coli correlated with reduced biofilm production, and separate studies demonstrated E. coli ribonuclease activation in response to aminoglycoside antibiotics. Here we report that E. coli produced 2 ′ ,3 ′ -cAMP in response to kanamycin at sub-inhibitory levels. Surprisingly, other aminoglycosides like streptomycin or gentamicin did not generate levels of 2 ′ ,3 ′ -cAMP detectable by 31P NMR. Interestingly, because 2 ′ ,3 ′ -cAMP is also produced in E. coli strains expressing a plasmid-encoded kanamycin resistance gene but not by other ribosome-targeting antibiotics, this kanamycin-specific production may not reflect disrupted protein synthesis. Overall, this finding provides a link between aminoglycoside-induced ribonuclease activity and 2 ′ ,3 ′ -cAMP production in E. coli. • Kanamycin was found to induce production of 2′,3′-cyclic AMP in Escherichia coli. • A stable 2′,3′-cAMP tracking system based on31P NMR was devised. • A new link between aminoglycoside-induced ribonuclease activity and 2′,3′-cAMP production in E. coli was proposed. [ABSTRACT FROM AUTHOR]

Rubrolones are tropolonoid natural products with a unique carbon skeleton. Extensive secondary metabolite analysis of the endophytic Streptomyces sp. KIB-H033 revealed a new class of rubrolone analogue possessing a rare benzoic acid-pyridine inner salt moiety. Precursor feeding with [13C]-acetate revealed a labeling pattern consistent with tropolone moiety construction via type-II PKS chemistry followed by complex oxidative rearrangements. This bacterial biosynthetic route represents a surprising departure from fungal tropolone assembly during stipitatic acid biosynthesis. [ABSTRACT FROM AUTHOR]

In the recently identified cholesterol catabolic pathway of Mycobacterium tuberculosis, 2-hydroxy-6-oxo-6-phenylhexa-2,4-dienoate hydrolase (HsaD) is proposed to catalyze the hydrolysis of a carbon-carbon bond in 4,5-9,10-diseco-3-hydroxy-5,9,17-tri-oxoandrosta-1(10),2-diene-4-oic acid (DSHA), the cholesterol meta-cleavage product (MCP) and has been implicated in the intracellular survival of the pathogen. Herein, purified HsaD demonstrated 4-33 times higher specificity for DSHA (kcat/Km = 3.3 ± 0.3 × 104 m-1 s-1) than for the biphenyl MCP 2-hydroxy-6-oxo-6-phenylhexa-2,4-dienoic acid (HOPDA) and the synthetic analogue 8-(2-chlorophenyl)-2-hydroxy-5-methyl-6-oxoocta-2,4-dienoic acid (HOPODA), respectively. The S114A variant of HsaD, in which the active site serine was substituted with alanine, was catalytically impaired and bound DSHA with a Kd of 51 ± 2 μm. The S114A·DSHA species absorbed maximally at 456 nm, 60 nm red-shifted versus the DSHA enolate. Crystal structures of the variant in complex with HOPDA, HOPODA, or DSHA to 1.8-1.9 Åindicate that this shift is due to the enzyme-induced strain of the enolate. These data indicate that the catalytic serine catalyzes tautomerization. A second role for this residue is suggested by a solvent molecule whose position in all structures is consistent with its activation by the serine for the nucleophilic attack of the substrate. Finally, the a-helical lid covering the active site displayed a ligand-dependent conformational change involving differences in side chain carbon positions of up to 6.7 Å, supporting a two-conformation enzymatic mechanism. Overall, these results provide novel insights into the determinants of specificity in a mycobacterial cholesterol-degrading enzyme as well as into the mechanism of MCP hydrolases. [ABSTRACT FROM AUTHOR]

Streptococcus mutans is a major pathogen causing human dental caries. As a Gram-positive bacterium with a small genome (about 2 Mb) it is considered a poor source of natural products. Due to a recent explosion in genomic data available for S. mutans strains, we were motivated to explore the natural product production potential of this organism. Bioinformatic characterization of 169 publically available genomes of S. mutans from human dental caries revealed a surprisingly rich source of natural product biosynthetic gene clusters. Anti-SMASH analysis identified one nonribosomal peptide synthetase (NRPS) gene cluster, seven polyketide synthase (PKS) gene clusters and 136 hybrid PKS/NRPS gene clusters. In addition, 211 ribosomally synthesized and post-translationally modified peptides (RiPPs) clusters and 615 bacteriocin precursors were identified by a combined analysis using BAGEL and anti-SMASH. S. mutans harbors a rich and diverse natural product genetic capacity, which underscores the importance of probing the human microbiome and revisiting species that have traditionally been overlooked as 'poor' sources of natural products. [ABSTRACT FROM AUTHOR]

Sphingomonas wittichii RW1 degrades chlorinated dibenzofurans and dibenzo-p-dioxins via meta cleavage. We used inverse PCR to amplify dxnB2, a gene encoding one of three meta-cleavage product (MCP) hydrolases identified in the organism that are homologues of BphD involved in biphenyl catabolism. Purified DxnB2 catalyzed the hydrolysis of 8-OH 2-hydroxy-6-oxo-6-phenylhexa-2,4-dienoate (HOPDA) approximately six times faster than for HOPDA at saturating substrate concentrations. Moreover, the specificity of DxnB2 for HOPDA (kcat/Km = 1.2 x 107 M-1 s-1) was about half that of the BphDs of Burkholderia xenovorans LB400 and Rhodococcus globerulus P6, two potent polychlorinated biphenyl (PCB)-degrading strains. Interestingly, DxnB2 transformed 3-Cl and 4-OH HOPDAs, compounds that inhibit the BphDs and limit PCB degradation. DxnB2 had a higher specificity for 9-Cl HOPDA than for HOPDA but a lower specificity for 8-Cl HOPDA (kcat/Km = 1.7 x 106 M-1 s-1), the chlorinated analog of 8-OH HOPDA produced during dibenzofuran catabolism. Phylogenetic analyses based on structure-guided sequence alignment revealed that DxnB2 belongs to a previously unrecognized class of MCP hydrolases, evolutionarily divergent from the BphDs although the physiological substrates of both enzyme types are HOPDAs. However, both classes of enzymes have mainly small hydrophobic residues lining the subsite that binds the C-6 phenyl of HOPDA, in contrast to the bulky hydrophobic residues (Phe106, Phe135, Trp150, and Phe197) found in the class II enzymes that prefer substrates possessing a C-6 alkyl. Thr196 and/or Asn203 appears to be an important determinant of specificity for DxnB2, potentially forming hydrogen bonds with the 8-OH substituent. This study demonstrates that the substrate specificities of evolutionarily divergent hydrolases may be useful for degrading mixtures of pollutants, such as PCBs. [ABSTRACT FROM AUTHOR]