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

摘要

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-S-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 HC1 from an initial halohydrin species.Phenyle-nolpyruvate has now been found to be a substrate for CaaD with a k_(cat)/K_m 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.