Evolution of function in the crotonase superfamily: (3S)-methylglutaconyl-CoA hydratase from Pseudomonas putida.

Members of the enoyl-CoA hydratase (crotonase) superfamily catalyze different overall reactions that utilize a common catalytic strategy delivered by a shared structural scaffold; the substrates are usually acyl esters of coenzyme A, and the intermediates are usually thioester enolate anions stabilized by a conserved oxyanion hole. In many bacterial genomes, orthologous members that contain homologues of acid/base catalyst Glu164 but not of Glu144 in rat mitochondrial crotonase are encoded by operons of which the functions have not been assigned. Focusing on the orthologues from Pseudomonas aeruginosa and P. putida, we have determined that these operons encode enzymes in leucine catabolism with the unknown enzyme assigned as (3S)-methylglutaconyl-CoA hydratase (MGCH), which catalyzes the syn-hydration of (E)-3-methylglutaconyl-CoA to (3S)-hydroxymethylglutaryl-CoA. The discovery that bacterial MGCHs catalyze hydration of enoyl-CoAs utilizing a single active-site residue contrasts with the paradigm crotonases as well as with the recently identified mammalian MGCHs that use homologues of both Glu144 and Glu164 in crotonase. Substrate analogues lacking a gamma-carboxylate have been shown to be competitive inhibitors of the enzyme, and installation of a glutamate for the "missing" homologue of Glu144 fails to introduce hydratase activity with the substrate analogues. Thus, bacterial MGCHs may provide an example of opportunistic evolution in which a carboxylate group of the substrate functionally replaces one of the active site glutamate residues in the reactions catalyzed by crotonases and the eukaryotic MGCHs.