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
2017
LeVieux, Jake A. | Baas, Bert-Jan | Kaoud, Tamer S. | Davidson, Rebecca | Babbitt, Patricia C. | Zhang, Yan Jessie | Whitman, Christian P.
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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