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Cg10062 is a cis-3-chloroacrylic acid dehalogenase (cis-CaaD) homologue from Corynebacterium glutamicum with an unknown function and an uninformative genomic context. It shares 53% pairwise sequence similarity with cis-CaaD including the six active site amino acids (Pro-1, His-28, Arg-70, Arg-73, Tyr-103, and Glu-114) that are critical for cis-CaaD activity. However, Cg10062 is a poor cis-CaaD: it lacks catalytic efficiency and isomer specificity. Two acetylene compounds (propiolate and 2-butynoate) and an allene compound, 2,3-butadienoate, were investigated as potential substrates. Cg10062 functions as a hydratase/decarboxylase using propiolate as well as the cis-3-chloro- and 3-bromoacrylates, generating mixtures of malonate semialdehyde and acetaldehyde. The two activities occur sequentially at the active site using the initial substrate. With 2,3-butadienoate and 2-butynoate, Cg10062 functions as a hydratase and converts both to acetoacetate. Mutations of the proposed water-activating residues (E114Q, E114D, and Y103F) have a range of consequences from a reduction in wild type activity to a switch of activities (i.e., hydratase into a hydratase/decarboxylase or vice versa). The intermediates for the hydration and decarboxylation products can be trapped as covalent adducts to Pro-1 when NaCNBH3 is incubated with the E114D mutant and 2,3-butadienoate or 2-butynoate, and the Y103F mutant and 2-butynoate. Three mechanisms are presented to explain these findings. One mechanism involves the direct attack of water on the substrate, whereas the other two mechanisms use covalent catalysis in which a covalent bond forms between Pro-1 and the hydration product or the substrate. The strengths and weaknesses of the mechanisms and the implications for Cg10062 function are discussed. [ABSTRACT FROM AUTHOR]

cis-3-Chloroacrylic acid dehalogenase (cis-CaaD) catalyzes the hydrolytic dehalogenation of cis-3-haloacrylates to yield malonate semialdehyde. The enzyme processes other substrates including an allene (2,3-butadienoate) to produce acetoacetate. In the course of a stereochemical analysis of the cis-CaaD-catalyzed reaction using this allene, the enzyme was unexpectedly inactivated in the presence of NaBH4 by the reduction of a covalent enzyme-substrate bond. Covalent modification was surprising because the accumulated evidence for cis-CaaD dehalogenation favored a mechanism involving direct substrate hydration mediated by Pro-1. However, the results of subsequent mechanistic, pre-steady state and full progress kinetic experiments are consistent with a mechanism in which an enamine forms between Pro-1 and the allene. Hydrolysis of the enamine or an imine tautomer produces acetoacetate. Reduction of the imine species is likely responsible for the observed enzyme inactivation. This is the first reported observation of a tautomerase superfamily member functioning by covalent catalysis. The results may suggest that some fraction of the cis-CaaD-catalyzed dehalogenation of cis-3-haloacrylates also proceeds by covalent catalysis. [ABSTRACT FROM AUTHOR]

The flavoenzyme proline dehydrogenase catalyzes the first step of proline catabolism, the oxidation of proline to pyrroline-5-carboxylate. Here we report the first crystal structure of an irreversibly inactivated proline dehydrogenase. The 1.9 Å resolution structure of Thermus therinophilus proline dehydrogenase inactivated by the mechanism-based inhibitor N-propargylglycine shows that N5 of the flavin cofactor is covalently connected to the &3x20AC;-amino group of Lys99 via a three-carbon linkage, consistent with the mass spectral analysis of the inactivated enzyme. The isoalloxazine ring has a butterfly angle of 25°, which suggests that the flavin cofactor is reduced. Two mechanisms can account for these observations. In both, N-propargylglycine is oxidized to N-propargyliminoglycine. In one mechanism, this α,β-unsaturated iminium compound is attacked by the N5 atom of the now reduced flavin to produce a I ,4-addition product. Schiff base formation between Lys99 and the imine of the 1,4-addition product releases glycine and links the enzyme to the modified flavin. In the second mechanism, hydrolysis of N-propargyliminoglycine yields propynal and glycine. A 1,4-addition reaction with propynal coupled with Schiff base formation between Lys99 and the carbonyl group tethers the enzyme to the flavin via a three-carbon chain. The presumed nonenzymatic hydrolysis of N-propargyliminoglycine and the subsequent rebinding of propynal to the enzyme make the latter mechanism less likely. [ABSTRACT FROM AUTHOR]

YwhB, a 4-oxalocrotonate tautomerase (4-OT) homologue in Bacillus subtilis, has no known biological role, and the gene has no apparent genomic context. The kinetic and stereochemical properties of YwhB have been examined using available enol and dienol compounds. The kinetic analysis shows that YwhB has a relatively nonspecific 1,3- and 1,5-keto-enol tautomerase activity, with the former activity prevailing. Replacement of Pro-1 or Arg-11 with an alanine significantly reduces or abolishes these activities, implicating both residues as critical ones for the activities. In D2O, ketonization of two monoacid substrates (2-hydroxy-2,4-pentadienoate and phenylenolpyruvate) produces a mixture of stereoisomers {2-keto-3-[2H]-4-pentenoate and 3-[2H]-phenylpyruvate}, where the (3R)-isomers predomi- nate. Ketonization of 2-hydroxy-2,4-hexadienedioate, a diacid, in D2O affords mostly the opposite enantiomer, (3S)-2-oxo-[3-2H]-4-hexenedioate. The mono- and diacids apparently bind in different orientations in the active site of YwhB, but the highly stereoselective nature of the YwhB reaction using a diacid suggests that the biological substrate for YwhB may be a diacid. Moreover, of the three dienols examined, 1,3- and 1 ,5-keto-enol tautomerization reactions are only observed for 2-hydroxy-2,4- hexadienedioate, indicating that the C-3 and C-5 positions are accessible for protonation in this compound. Incubation of 4-OT with 2-hydroxy-2,4-hexadienedioate in D2O results in a racemic mixture of 2-oxo- [3-2H]-4-hexenedioate, suggesting that 4-OT may not catalyze a 1,3-keto-enol tautomerization reaction using this dienol. It has previously been shown that 4-OT catalyzes the near stereospecific conversion of 2-hydroxy-2,4-hexadienedioate to (5S)-[5-2H]-2-oxo-3-hexenedioate in D2O. Taken together, these observations suggest that 4-UT might function as a 1,5-keto-enol tautomerase using 2-hydroxy-2,4- hexadienedioate. [ABSTRACT FROM AUTHOR]

The enzymatic conversion of cis- or trans-3-chloroacrylic acid to malonate semialdehyde is a key step in the bacterial degradation of the nematocide 1,3-dichioropropene. Two mechanisms have been proposed for the isomer-specific hydrolytic dehalogenases, cis- and trans-3-chloroacrylic acid dehalogenase (cis-CaaD and CaaD, respectively), responsible for this step. In one mechanism, the enol isomer of malonate semialdehyde is produced by the α,β-elimination of HCI from an initial halohydrin species. Phenylenolpyruvate has now been found to be a substrate for CaaD with a kcat/Km value that approaches the one determined for the CaaD reaction using trans-3-chloroacrylate. Moreover, the reaction is stereoselective, generating the 3S isomer of [3-²H]phenylpyruvate in a 1.8:1 ratio in ²H2O. These two observations and a kinetic analysis of active site mutants of CaaD suggest that the active site of CaaD is responsible for the phenylpyruvate tautomerase (PPT) activity. The activity is a striking example of catalytic promiscuity and could reflect the presence of an enol intermediate in CaaD-mediated dehalogenation of trans-3- chloroacrylate. CaaD and cis-CaaD represent different families in the tautomerase superfamily, a group of structurally homologous proteins characterized by a core β-α-β building block and a catalytic Pro-1. The eukaryotic immunoregulatory protein known as macrophage migration inhibitory factor (MIF), also a tautomerase superfamily member, exhibits a PPT activity, but the biological relevance is unknown. In addition to the mechanistic implications, these results establish a functional link between CaaD and the superfamily tautomerases, highlight the catalytic and binding promiscuity of the β-α-β scaffold, and suggest that the PPT activity of MIF could reflect a partial reaction in an unknown MW-catalyzed reaction. [ABSTRACT FROM AUTHOR]

A series of 2-fluoro-4-alkene and 2-fluoro-4-alkyne substrate analogues were synthesized and examined as potential inhibitors of three enzymes: 4-oxalocrotonate tautomerase (4-OT) and vinylpyruvate hydratase (VPH) from the catechol meta-fission pathway and a closely related 4-OT homologue found in Bacillus subtilis designated YwhB. All of the compounds were potent competitive inhibitors of 4-OT with the monocarboxylated 2E-fluoro-2,4-pentadienoate and the dicarboxylated 2E-fluoro-2-en-4-ynoate being the most potent. Despite the close mechanistic and structural similarities between 4-OT and YwhB, these compounds were significantly less potent inhibitors of YwhB with IC values ranging from 5- to 633-fold lower than those determined for 4-OT. The study of VPH is complicated by the fact that the enzyme is only active as a complex with the metal-dependent 4-oxalocrotonate decarboxylase (4-OD), the enzyme following 4-OT in the catechol meta-fission pathway. A structure-based sequence analysis identified 4-OT as a member of the fumarylacetoacetate hydrolase (FAH) superfamily and implicated Glu-109 and Glu-111 as potential metal-binding ligands. Changing these residues to a glutamine verified their importance for enzymatic activity and enabled the production of soluble E109Q4-OD/VPH or El 11Q4- ODIVPH complexes, which retained full hydratase activity but had little decarboxylase activity. Subsequent incubation of the E109Q4-OD/VPH complex with the substrate analogues identified the 2E and 2Z isomers of the monocarboxylated 2-fluoropent-2-en-4-ynoate as competitive inhibitors. The combined results set the stage for crystallographic studies of 4-OT, YwhB, and VPH using these inhibitors as ligands. [ABSTRACT FROM AUTHOR]

4-Oxalocrotonate tautomerase (4-OT) and YwhB, a 4-OT homologue found in Bacillus subtilis, exhibit a low level hydratase activity that converts trans-3-haloacrylates to acetaldehyde, presumably through a malonate semialdehyde intermediate. The mechanism for the initial transformation of the 3-haloacrylate to malonate semialdehyde involves Pro-1 as well as an arginine, two residues that play critical roles in the 4-OT-catalyzed isomerization reaction and the YwhB-catalyzed tautomerization reaction. These residues are also critical for the trans-3-chloroacrylic acid dehalogenase (CaaD)-catalyzed conversion of trans-3-haloacrylates to malonate semialdehyde. Recently, 3-bromo- and 3-chloropropiolate, the acetylene analogues of 3-haloacrylates, were characterized as potent irreversible inhibitors of CaaD due to the covalent modification of the catalytic proline. In view of these observations, an investigation of the behavior of 4-OT and YwhB with the 3-halopropiolates was undertaken. The results show that these compounds are potent irreversible inhibitors of 4-OT and YwhB with Pro-1 being the sole site of covalent modification by 3-bromopropiolate. The inactivation process could involve the enzyme-catalyzed addition of water to the 3-halopropiolate yielding an acyl halide, which would inactivate the enzyme or be initiated by the nucleophilic attack of Pro-1 at the C-3 position of the 3-halopropiolate in a Michael type reaction. The presence of the halogen along with Arg-11 could facilitate both reactions with the latter causing the polarization of the α,β-unsaturated acids. The 3-halopropiolates are the first identified inhibitors of YwhB and confirm the importance of Pro-1 in its mechanism. In addition, the results set the stage for the use of these compounds as mechanistic probes of the primary as well as low level activities of 4-OT and YwhB. [ABSTRACT FROM AUTHOR]

Malonate semialdehyde decarboxylase (MSAD) has been identified as the protein encoded by the orf130 gene from Pseudomonas pavonaceae 170 on the basis of the genomic context of the gene as well as its ability to catalyze the decarboxylation of malonate semialdehyde to generate acetaldehyde. The enzyme is found in a degradative pathway for the xenobiotic nematocide trans1,3-dichloropropene. MSAD has no sequence homology to previously characterized decarboxylases, but the presence of a conserved motif (Pro¹-(X)[sub 8]-Gly-Arg[sub 11]-XAsp-X-Gln) in its N-terminal region suggested a relationship to the tautomerase superfamily. Sequence analysis identified Pro¹ and Arg[sup 75] as potential active site residues that might be involved in the MSAD activity. The results of site-directed mutagenesis experiments confirmed the importance of these residues to activity and provided further evidence to implicate MSAD as a new member of the tautomerase superfamily. MSAD is the first identified decarboxylase in the superfamily and is possibly the first characterized member of a new and distinct family within this superfamily. Malonate semialdehyde is analogous to a β-keto acid, and enzymes that catalyze the decarboxylation of these acids generally utilize metal ion catalysis, a Schiff base intermediate, or polarization of the carbonyl group by hydrogen bonding and/or electrostatic interactions. A mechanistic analysis shows that the rate of the reaction is not affected by the presence of a metal ion or EDTA while the incubation of MSAD with the substrate in the presence of sodium cyanoborohydride results in the irreversible inactivation of the enzyme. The site of modification is Pro¹. These observations are consistent with the latter two mechanisms, but do not exclude the first mechanism. Based on the sequence analysis, the outcome of the mutagenesis and mechanistic experiments, and the roles determined for Pro¹ and the conserved arginine in all tautomerase superfamily members characterized thus far, two mechartistic scenarios are proposed for the MSAD-catalyzed reaction in which Pro¹ and Arg[sup 75] play prominent roles. [ABSTRACT FROM AUTHOR]

Evaluates the phenylpyruvate tautomerase activity of the macrophage migration inhibitory factor, an immunoregulatory protein. Role of lysine-32 in facilitating substrate binding; Impact of electrostatic interaction on stereoselectivity of the K32A region; Effect of phenyl bond substitution on phenylpyruvate tautomerase activity.

Studies the kinetic and structural effects of mutations on P1G and P1A mutants of the catalyst 4-oxalocrotonate tautomerase. Production, expression, and characterization of mutants; Kinetic properties of P1 mutants; Stereochemical analysis of P1G and P1A catalyzed reactions; Structural properties of P1G and P1A mutant enzymes.

Presents a study on the relationship between the catechol and homoprocatechuate meta-fission pathways. Mechanistic and structural studies conducted of all enzymes in the two pathways, including 2-oxo-hept-4-ene-1,7-dioate (OHED) hydratase; Kinetic properties of OHED hydratase; Description of experiments conducted in the study; Results of the study.

Examines the catalyzed 5-(Carboxymethyl)-2-hydroxymuconate isomerase and 5-(Carboxymethyl)-2-oxo-3-hexene-1,6-dioate carboxylase of Escherichia coli. Kinetics of the nonenzymatic ketonization; Stoichiometry of 5-(Carboxymethyl)-2-hydroxymuconate isomerase; Experimental procedures.

Studies the chemical and enzymatic ketonization of the conjugated enol, 5-(carboxymethyl)-2-hydroxymuconate. UV and 1H NMR spectroscopic results; Reduction of isomeric ketones; Rapid interconversion in aqueous phosphate buffer; Relevance of findings to related dienol; More.

Nature may exploit the catalytic promiscuity observed in some enzymes in order to create new enzymes. A catalytically promiscuous enzyme has one or more low-level activities in addition to its primary, physiological activity. Duplication of the gene, followed by a series of mutations to amplify the desired activity, generates a new enzyme now having the progenitor's low-level activity as the primary activity. This article reports the observation of low-level hydration activities in the bacterial isomerase, 4-oxalocrotonate tautomerase (4-OT) from the bacteria Pseudomonas putida and a 4-OT homologue, YwhB, found in Bacillus subtilis.

This article reports that malonate semialdehyde decarboxylase (MSAD) from Pseudomonas pavonaceae 170 catalyzes the metal ion-independent decarboxylation of a catabolite generated by the bacteria. The members of this superfamily are structurally homologous proteins that share a characteristic beta-alpha-beta fold as well as a catalytic amino-terminal proline. Pro-1 is a charged species that polarizes the C-3 carbonyl group of 2 by hydrogen bonding and/or an electrostatic interaction. The results clearly show that MSAD has a hydratase activity, from which it can be inferred that Pro-1 is cationic and not functioning as a nucleophile.

The bacterial degradation pathways for the nematocide 1,3-dichloropropene rely on hydrolytic dehalogenation reactions catalyzed by cis- and trans-3-chloroacrylic acid dehalogenases (cis-CaaD and CaaD, respectively). X-ray crystal structures of native cis-CaaD and cis-CaaD inactivated by (R)-oxirane-2-carboxylate were elucidated. They locate four known catalytic residues (Pro-1, Arg-70, Arg-73, and Glu-114) and two previously unknown, potential catalytic residues (His-28 and Tyr-103′). The Y103F and H28A mutants of these latter two residues displayed reductions in cis-CaaD activity confirming their importance in catalysis. The structure of the inactivated enzyme shows covalent modification of the Pro-1 nitrogen atom by (R)-2-hydroxypropanoate at the C3 position. The interactions in the complex implicate Arg-70 or a water molecule bound to Arg-70 as the proton donor for the epoxide ring-opening reaction and Arg-73 and His-28 as primary binding contacts for the carboxylate group. This proposed binding mode places the (R)-enantiomer, but not the (S)-enantiomer, in position to covalently modify Pro-1. The absence of His-28 (or an equivalent) in CaaD could account for the fact that CaaD is not inactivated by either enantiomer. The cis-CaaD structures support a mechanism in which Glu-114 and Tyr-103′ activate a water molecule for addition to C3 of the substrate and His-28, Arg-70, and Arg-73 interact with the C1 carboxylate group to assist in substrate binding and polarization. Pro-1 provides a proton at C2. The involvement of His-28 and Tyr-103′ distinguishes the cis-CaaD mechanism from the otherwise parallel CaaD mechanism. The two mechanisms probably evolved independently as the result of an early gene duplication of a common ancestor. [ABSTRACT FROM AUTHOR]