The members of the crotonase (enoyl CoA hydratase) superfamily catalyze a wide range of reactions related to stabilization of a thioester enolate intermediate. The five Specific Aims focus on previously unstudied reactions, thereby providing novel insights into the structural bases for mechanistic diversity within the superfamily.
Each Specific Aim i nvolves mechanistic and structural studies: the mechanistic studies will be performed in Dr. Gerlt's laboratory at the University of Illinois (P.I.); the structural studies will be performed in Dr. Hazel Holden's laboratory at the University of Wisconsin (Co-P.I.). 1) 3R-Enoyl CoA hydratase (3R-ECH) and carnitinyl CoA racemase (CaiD) likely catalyze hydration reactions of enoyl CoA substrates to generate 3R-OH acyl CoA products; the crotonase in beta-oxidation catalyzes hydration of enoyl CoA substrates to generate 3S-OH acyl CoA products. We will study both 3R-ECH and CaiD so that we can understand the structural basis for the altered stereochemistry. 2) Methylmalonyl CoA decarboxylase (MMCD; former YgfG) catalyzes the decarboxylation of methylmalonyl. We assigned this function based on enzymological studies of three enzymes encoded by an operon and have determined its high resolution x-ray structure. We will study MMCD so that we can define how the active site promotes decarboxylation. 3) 1,4-Dihydroxynaphthoyl CoA synthase (MenB) and 2-ketocyclohexanecarboxyl CoA hydrolase (BadI) share 47 percent sequence identity and catalyze Dieckman and reverse-Dieckman condensation reactions, respectively. We will study both MenB and BadI so that we can understand the structural bases for these difficult reactions. 4) Cis,cis-Muconyl isomerase (PaaG) catalyzes a reaction in phenylacetate catabolism. We will study PaaG to determine whether cis-trans isomerization involves addition of an active site nucleophile to the substrate. 5) 3-OH Isobutyryl CoA hydrolase (HIBCH) catalyzes hydrolysis of a thioester in valine catabolism. We will study HIBCH so that we can determine whether the reaction proceeds via and enolate anion or an anionic tetrahedral intermediate.
