Phenylpyruvate tautomerase activity of trans-3-chloroacrylic acid dehalogenase: evidence for an enol intermediate in the dehalogenase reaction?
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-dichloropropene. 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 alpha,beta-elimination of HCl 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 beta-alpha-beta 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 beta-alpha-beta scaffold, and suggest that the PPT activity of MIF could reflect a partial reaction in an unknown MIF-catalyzed reaction.
Department of Pharmaceutical Biology, Groningen Research Institute of Pharmacy, University of Groningen, Antonius Deusinglaan 1, 9713 AV Groningen, The Netherlands.
Gerrit J
GJ
Poelarends
William H
WH
Johnson
Hector
H
Serrano
Christian P
CP
Whitman
D000179:Acrylates / Q000737:chemistry / Q000378:metabolism; D000818:Animals; D001665:Binding Sites; D002384:Catalysis; D006867:Hydrolases / Q000737:chemistry* / Q000235:genetics; D019746:Intramolecular Oxidoreductases / Q000737:chemistry* / Q000235:genetics; D007700:Kinetics; D008263:Macrophage Migration-Inhibitory Factors / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D009154:Mutation; D010667:Phenylpyruvic Acids / Q000737:chemistry; D013237:Stereoisomerism; D013379:Substrate Specificity
D000179:Acrylates; D008263:Macrophage Migration-Inhibitory Factors; D010667:Phenylpyruvic Acids; C071773:3-chloroacrylic acid; D006867:Hydrolases; C077721:trans-3-chloroacrylic acid dehalogenase; D019746:Intramolecular Oxidoreductases; C022346:phenylpyruvate tautomerase; C031606:phenylpyruvic acid