The evolution of enzymatic activity in the crotonase superfamily: The mechanism of the reaction catalyzed by 2-ketocyclohexanecarboxyl-CoA hydrolase.

摘要

Members of the mechanistically diverse enoyl-CoA hydratase (crotonase) superfamily catalyze reactions that involve stabilization of an enolate anion derived from an acyl thioester of coenzyme A. 2-Ketocyclohexanecarboxyl-CoA hydrolase (BadI), found in the pathway for anaerobic degradation of benzoate by Rhodopseudomonas palustris, is a member of the crotonase superfamily that catalyzes a reverse Dieckmann reaction in which the substrate is hydrolyzed to pimelyl-CoA. The substrate is the configurationally labile S-2-ketocyclohexanecarboxyl-CoA, and in 2H2O solvent hydrogen is incorporated into the pro-2S position of the pimelyl-CoA product. Therefore, the stereochemical course of the BadI-catalyzed reaction is inversion.;The X-ray crystal structure of BadI revealed that its alpha,beta-fold is superimposable with the seven reported structures of other enzymes of the crotonase superfamily. The structure also facilitated the identification of residues in the active site of BadI: Ser 138, Asp 140, and Tyr 235. Site-specific mutants were generated and assayed. The mutants of Asp 140 (D140A, D140N) and Tyr 235 (Y235F) retained significant catalytic activity, with rates that were only 30-, 170-, and 100-fold reduced, respectively, compared to the rate of reaction catalyzed by wild-type BadI. The mutant of Ser 138 (S138A) was most impaired, with a 2700-fold reduction in rate.;The stereochemical progress of the reaction catalyzed by BadI is important for understanding the catalytic roles of residues in the active site. It was concluded that, unlike other stereochemically characterized crotonase homologs, this reaction must proceed via a Z-enolate. Based on the information derived from stereochemical studies and site-specific mutagenesis, a mechanism of the reaction catalyzed by BadI was proposed, in which Ser 138 plays a central catalytic role. The new data is also related to the homologous 1,4-dihydroxy-2-naphthoyl-CoA synthase (MenB), which has the same active-site configuration as that of BadI but catalyzes a forward Dieckmann reaction in microbial menaquinone biosynthesis.