Methylmalonyl-CoA decarboxylase (YgfG) for Escherichia coli: A new activity for the crotonase superfamily

Using information obtained from alignments of protein sequences, a gene, ygfG, encoding a new member of the crotonase superfamily was located in the Escherichia coli chromosome. The sequence of the encoded protein, YgfG, possessed the conserved motifs in structurally characterized members of the crotonase superfamily. I assigned methylmalonyl-CoA decarboxylase (MMDC) activity to YgfG.; The gene encoding YgfG is located in an operon that encodes three additional proteins: Sbm, YgfD, and, YgfH. My studies demonstrated that Sbm is a coenzyme B₁₂-dependent methylmalonyl-CoA mutase and YgfH is a propionyl-CoA: succinate CoA transferase. These results provided the metabolic context for the MMDC reaction: Sbm produces the substrate ((R)-methylmalonyl-CoA), and YgfH converts the propionyl CoA product into succinyl-CoA. These three enzymes constitute a metabolic cycle of three reactions in which succinate is converted to propionate and CO₂. The function of YgfD is unknown, although this putative enzyme has an ATP binding site and may be involved in regulation of the cycle.; The crystal structure of MMDC was determined in collaboration with Dr. M. M. Benning and Dr. H. M. Holden at the University of Wisconsin. YgfG has a structure homologous to the other members of the crotonase superfamily but shows differences in the arrangement of the N-terminal α-helices. The crystal structure identified four potential catalytic residues in the active site. Mutational studies suggest that Glu113 is the general acid in the YgfG reaction, Tyr140 is important in binding the substrate.; The E113Q mutant exhibited significantly larger isotope effect for both k_cat and k_cat/K_m than wild-type, suggesting differences in reaction mechanism between wild type and E113Q. Although experimental difficulties encountered did not allow quantitative studies of stereochemistry of the MMDC reaction, the reaction catalyzed by wild-type MMDC proceeds with retention of configuration using (S)-methylmalonyl-CoA as a substrate, and with inversion of configuration using (R)-methylmalonyl-CoA as a substrate. H66F catalyzed the MMDC reaction with at least partial racemization. I postulated that His66 is important in retaining structure of the active site.