Characterization of a Newly Identified Mycobacterial Tautomerase with Promiscuous Dehalogenase and Hydratase Activities Reveals a Functional Link to a Recently Diverged cis-3-Chloroacrylic Acid Dehalogenase

摘要

The enzyme cis-3-chloroacrylic acid dehalogen-nase (cis-CaaD) is found in a bacterial pathway that degrades ansynthetic nematocide, cis-1,3-dichloropropene, introduced innthe 20th century. The previously determined crystal structure ofncis-CaaD and its promiscuous phenylpyruvate tautomerasen(PPT) activity link this dehalogenase to the tautomerase super-nfamily, a group of homologous proteins that are characterized byna catalytic amino-terminal proline and a β-R-β structural fold.nThe low-level PPT activity of cis-CaaD, which may be a vestigenof the function of its progenitor, prompted us to search thendatabases for a homologue of cis-CaaD that was annotated as anputative tautomerase and test both its PPT and cis-CaaD activity. We identified a mycobacterial cis-CaaD homologue (designatednMsCCH2) that shares key sequence and active site features with cis-CaaD. Kinetic and 1nH NMR spectroscopic studies show thatnMsCCH2 functions as an efficient PPT and exhibits low-level promiscuous dehalogenase activity, processing both cis- and trans-3-nchloroacrylic acid. To further probe the active site ofMsCCH2, the enzyme was incubated with 2-oxo-3-pentynoate (2-OP). At pHn8.5, MsCCH2 is inactivated by 2-OP due to the covalent modification of Pro-1, suggesting that Pro-1 functions as a nucleophile atnpH 8.5 and attacks 2-OP in aMichael-type reaction. At pH 6.5, however,MsCCH2 exhibits hydratase activity and converts 2-OP tonacetopyruvate, which implies that Pro-1 is cationic at pH 6.5 and not functioning as a nucleophile. At pH 7.5, the hydratase andninactivation reactions occur simultaneously. From these results, it can be inferred that Pro-1 ofMsCCH2 has a pKa value that lies innbetween that of a typical tautomerase (pKa of Pro-1 ∼ 6) and that of cis-CaaD (pKa of Pro-1 ∼ 9). The shared activities andnstructural features, coupled with the intermediate pKa of Pro-1, suggest that MsCCH2 could be characteristic of an evolutionarynintermediate along the past route for the divergence of cis-CaaD from an unknown superfamily tautomerase. This makesMsCCH2nan ideal candidate for laboratory evolution of its promiscuous dehalogenase activity, which could identify additional featuresnnecessary for a fully active cis-CaaD. Such results will provide insight into pathways that could lead to the rapid divergent evolution ofnan efficient cis-CaaD enzyme