Phenylpyruvate tautomerase activity of trans-3-chloroacrylic acid dehalogenase: evidence for an enol intermediate in the dehalogenase reaction?

Abstract

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.

Figure 1

Partial ¹H NMR spectra (500 MHz, CD₃OD) of (2R)-[3-²H]malate isolated in the stereochemical analysis of the (A) CaaD-catalyzed conversion of 9 to [3-²H]10 in ²H₂O and (B) MIF-catalyzed conversion of 9 to [3-²H]10 in ²H₂O. Both reactions were carried out at pH 9.0. The broadened doublet centered at 2.61 ppm corresponds to (2R,3S)-[3-²H]malate (13) and the broadened doublet centered at 2.77 ppm corresponds to the (2R,3R)-[3-²H]malate (19). The latter broadened doublet appears more like a broadened singlet due to the small coupling constant, which does not permit adequate resolution. For CaaD, the (2R,3R)-[3-²H]isomer predominates by a ratio of ~1.8 to 1, as indicated by integration of the signals. For MIF, the (2R,3S)-[3-²H]isomer predominates by a ratio of ~6.6 to 1. The less prominent doublets centered at 2.64, 2.77, and 2.80 ppm indicate the presence of fully protio malate.

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