Phenylpyruvate Tautomerase Activity of <i>trans</i>-3-Chloroacrylic Acid Dehalogenase:  Evidence for an Enol Intermediate in the Dehalogenase Reaction?

Poelarends, Gerrit J.; Johnson, William H.; Serrano, Héctor; Whitman, Christian P.

doi:10.1021/bi7007189

Cited by 17 publications

(33 citation statements)

References 49 publications

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“…The robust activity argues for the presence of the enol intermediate 14 in the dehalogenase reaction (Scheme 10), and is consistent with the α,β-elimination reaction mechanism shown in route B [55]. A mechanism involving the collapse of the halohydrin 13 (route A) cannot be ruled out because the PPT activity could be a reflection of the ketonization of 12 , an enol, which is common to both mechanisms.…”

Section: Evolution Of Trans- and Cis-3-chloroacrylic Acid Dehalogenassupporting

confidence: 67%

“…Although CaaD does not catalyze the 4-OTreaction (i.e., the rate of production of 2-oxo-3-hexenedioate ( 3 , Scheme 1) cannot be distinguished from the non-enzymatic rate), it exhibits a robust phenylpyruvate tautomerase (PPT) activity, catalyzing the conversion of 4 to 5 (Scheme 2) with a k cat / K m value that is near the one measured for CaaD-catalyzed dehalogenation of 8 (Scheme 4) [55]. CaaD has a k cat / K m value of ~7.1 × 10 4 M −1 s −1 using 8 and a k cat / K m value of ~2.3 × 10 4 M −1 s −1 using 4 [55]. Moreover, the reaction is stereoselective in D 2 O, resulting in the formation of the 3 S -isomer of [3– 2 H]phenylpyruvate in a 1.8:1 ratio.…”

Section: Evolution Of Trans- and Cis-3-chloroacrylic Acid Dehalogenasmentioning

confidence: 99%

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The chemical versatility of the β–α–β fold: Catalytic promiscuity and divergent evolution in the tautomerase superfamily

Poelarends

Veetil

Whitman

2008

Cell. Mol. Life Sci.

163

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Tautomerase superfamily members have an amino-terminal proline and a β–α–β fold, and include 4-oxalocrotonate tautomerase (4-OT), 5-(carboxymethyl)-2-hydroxymuconate isomerase (CHMI), trans- and cis-3-chloroacrylic acid dehalogenase (CaaD and cis-CaaD, respectively), malonate semialdehyde decarboxylase (MSAD), and macrophage migration inhibitory factor (MIF), which exhibits a phenylpyruvate tautomerase (PPT) activity. Pro-1 is a base (4-OT, CHMI, the PPT activity of MIF) or an acid (CaaD, cis-CaaD, MSAD). Components of the catalytic machinery have been identified and mechanistic hypotheses formulated. Characterization of new homologues shows that these mechanisms are incomplete. 4-OT, CaaD, cis-CaaD, and MSAD also have promiscuous activities with a hydratase activity in CaaD, cis-CaaD, and MSAD, PPT activity in CaaD and cis-CaaD, and CaaD and cis-CaaD activities in 4-OT. The shared promiscuous activities provide evidence for divergent evolution from a common ancestor, give hints about mechanistic relationships, and implicate catalytic promiscuity in the emergence of new enzymes.

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Section: Evolution Of Trans- and Cis-3-chloroacrylic Acid Dehalogenassupporting

confidence: 67%

Section: Evolution Of Trans- and Cis-3-chloroacrylic Acid Dehalogenasmentioning

confidence: 99%

The chemical versatility of the β–α–β fold: Catalytic promiscuity and divergent evolution in the tautomerase superfamily

Poelarends

Veetil

Whitman

2008

Cell. Mol. Life Sci.

163

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“…Tautomerization of 8 produces 4 . Experiments showing that the ketonization of phenylenolpyruvate to phenylpyruvate is kinetically competent in the overall CaaD reaction are consistent with route B, but not conclusive (12). …”

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confidence: 99%

Pre-Steady-State Kinetic Analysis of cis-3-Chloroacrylic Acid Dehalogenase: Analysis and Implications

Robertson¹,

Schroeder

Zhang

et al. 2009

Biochemistry

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Isomer-specific 3-chloroacrylic acid dehalogenases catalyze the hydrolytic dehalogenation of the cis-and trans-isomers of 3-chloroacrylate to yield malonate semialdehyde. These reactions represent key steps in the degradation of the nematocide, 1,3-dichloropropene. The kinetic mechanism of cis-3-chloroacrylic acid dehalogenase (cis-CaaD) has now been examined using stopped-flow and chemical-quench techniques. Stopped-flow analysis of the reaction, following the fluorescence of an active site tryptophan, is consistent with a minimal three-step model involving substrate binding, chemistry, and product release. Chemical quench experiments show burst kinetics, indicating that product release is at least partially rate limiting. Global fitting of all of the kinetic results by simulation is best accommodated by a four-step mechanism. In the final kinetic model, the enzyme binds substrate and isomerizes to an alternate fluorescent form, chemistry occurs, and is followed by the ordered release of two products, with the release of the first product as the rate-limiting step. Bromide ion is a competitive inhibitor of the reaction indicating that it binds to the free enzyme rather than to the enzyme with one product still bound. This observation suggests that malonate semialdehyde is the first product released by the enzyme (rate limiting), followed by halide. A comparison of the unliganded cis-CaaD crystal structure with that of an inactivated cis-CaaD where the prolyl nitrogen of Pro-1 is covalently attached to (R)-2-hydroxypropanoate provides a possible explanation for the isomerization step. The structure of the covalently modified enzyme shows that a 7-residue loop comprised of residues 32-38 is closed down on the active site cavity where the backbone amides of two residues (Phe-37 and Leu-38) interact with the carboxylate group of the adduct. In the unliganded form, the same loop points away from the active site cavity. Similarly, substrate binding may cause this loop to close down on the active site and sequester the reaction from the external environment. † This research was supported by the National Institutes of Health Grant GM-65324 and the Robert A. Welch Foundation (F-1334 and SUPPORTING INFORMATION AVAILABLEHere we describe the conventional analysis of the pre-steady state kinetic data (including kinetic schemes and equations used in the fitting process), outline the defining characteristics of the experimental data, evaluate the conventional analysis results, and show why global fitting to a four-step model was required. This is further supported by analysis of the confidence contours and plots for the reversible inhibition of cis-CaaD by bromide ion. This material is available free of charge via the Internet at http://pubs.acs.org. NIH Public Access Author ManuscriptBiochemistry. Author manuscript; available in PMC 2010 December 15. Published in final edited form as:Biochemistry. The catabolism of 1,3-dichloropropene (1, Scheme 1) in Pseudomonas pavonaceae 170 and coryneform bacterium strain FG41 i...

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“…The kinetic parameters are comparable to those obtained for 4-OT and greater than those obtained for cis -CaaD with a 400-fold greater k cat , 2-fold greater K m , and 194-fold greater k cat / K m . 36,37 …”

Section: Resultsmentioning

confidence: 99%

“… a The kinetic parameters were measured using the assay described in the text in 20 mM Na 2 HPO 4 buffer (pH 9) at 22 °C. b The kinetic parameters are from reference 36. c The kinetic parameters are from reference 37. …”

Section: Figurementioning

confidence: 99%

Kinetic and structural characterization of a cis -3-Chloroacrylic acid dehalogenase homologue in Pseudomonas sp. UW4: A potential step between subgroups in the tautomerase superfamily

LeVieux¹,

Baas

Kaoud

et al. 2017

Archives of Biochemistry and Biophysics

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A Pseudomonas sp. UW4_01740 (designated Ps01740) protein of unknown function was identified as similar to 4-oxalocrotonate tautomerase (4-OT)-like and cis-3-chloroacrylic acid dehalogenase (cis-CaaD)-like subgroups of the tautomerase superfamily (TSF). Ps01740 lacks only Tyr-103 of the amino acids critical for cis-CaaD activity (Pro-1, His-28, Arg-70, Arg-73, Tyr-103, Glu-114). As Ps01740 may represent an important variant of these enzymes, its kinetic and structural properties have been determined. Ps01740 shows tautomerase activity with phenylenolpyruvate, but lacks native 4-OT activity and dehalogenase activity with the isomers of 3-chloroacrylic acid. Ps01740 shows mostly low-level hydratase activity at pH 7.0, converting 2-oxo-3-pentynoate to acetopyruvate, consistent with cis-CaaD-like behavior. At pH 9.0, this compound results primarily in covalent modification of Pro-1, which is consistent with 4-OT-like behavior. These observations could reflect a pKa for Pro-1 that is closer to that of cis-CaaD (~9.2) than to 4-OT (~6.4. A structure of the native enzyme, at 2.6 Å resolution, highlights differences at the active site from those of 4-OT and cis-CaaD that add to our understanding of how contemporary TSF reactions and mechanisms may have diverged from a common 4-OT-like ancestor.

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Phenylpyruvate Tautomerase Activity of trans-3-Chloroacrylic Acid Dehalogenase: Evidence for an Enol Intermediate in the Dehalogenase Reaction?

Cited by 17 publications

References 49 publications

The chemical versatility of the β–α–β fold: Catalytic promiscuity and divergent evolution in the tautomerase superfamily

The chemical versatility of the β–α–β fold: Catalytic promiscuity and divergent evolution in the tautomerase superfamily

Pre-Steady-State Kinetic Analysis of cis-3-Chloroacrylic Acid Dehalogenase: Analysis and Implications

Kinetic and structural characterization of a cis -3-Chloroacrylic acid dehalogenase homologue in Pseudomonas sp. UW4: A potential step between subgroups in the tautomerase superfamily

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