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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 ([k.sub.cat]/[K.sub.m] = 1.2 x [10.sup.7] [M.sup.-1] [s.sup.-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-CI HOPDA ([k.sub.cat]/[K.sub.m]= 1.7 x 106 [M.sup.-1] [s.sup.-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.sup.-]6 phenyl of HOPDA, in contrast to the bulky hydrophobic residues (Phel06, Phe135, Trpl50, and Phe197) found in the class II enzymes that prefer substrates possessing a [C.sup.-]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.

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 ([k.sub.cat]/[K.sub.m], ~[10.sup.4] [M.sup.-1] [s.sup.-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) ([K.sub.m] 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.

A study based is used to demonstrate that in catechol 1,2-dioxygenase (C12O), an intradiol enzyme, and the catechol binds to the Fe(III) as a dianion. It can be concluded that the basis of the respective regiospecificities of intradiol and extradiol dioxydenase linked to the protonation state of the bidentate-bound catechol in the enzyme/substrate complex.

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]

The catalytic mechanism of [(BphD).sub.LB400] was investigated using steady-state and transient kinetic analyses together with structural analyses of the wild-type enzyme and a complex prepared by incubation of the latter with 2-hydroxy-6-oxo-6-phenylhexa-2,4-dienoic acid (HOPDA). The study provides the first full spectra of catalytic intermediates for a meta-cleavage product (MCP) hydrolase and reveals key enzyme-substrate interactions, which allow to predict the catalytic roles of active site residues.

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]

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]

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]

Phosphonates are produced across all domains of life and used widely in medicine and agriculture. Biosynthesis almost universally originates from the enzyme phosphoenolpyruvate mutase (Ppm), EC 5.4.2.9, which catalyzes O-P bond cleavage in phosphoenolpyruvate (PEP) and forms a high energy C-P bond in phosphonopyruvate (PnPy). Mechanistic scrutiny of this unusual intramolecular O-to-C phosphoryl transfer began with the discovery of Ppm in 1988 and concluded in 2008 with computational evidence supporting a concerted phosphoryl transfer via a dissociative metaphosphate-like transition state. This mechanism deviates from the standard 'in-line attack' paradigm for enzymatic phosphoryl transfer that typically involves a phosphoryl-enzyme intermediate, but definitive evidence is sparse. Here we review the experimental evidence leading to our current mechanistic understanding and highlight the roles of previously underappreciated conserved active site residues. We then identify remaining opportunities to evaluate overlooked residues and unexamined substrates/inhibitors., (© 2022 Wiley-VCH GmbH.)