Methionine synthase (MetH) catalyzes the conversion of homocysteine to methionine, using a methyl group that is derived from methyltetrahydrofolate. B12 serves as an intermediate methyl group carrier. Patients with mutations in the MetH gene display severe neurological dysfunction, megaloblastic anemia, homocystinuria, or hypomethioninemia. MetH is one of two B12 dependent enzymes that have been identified in mammals. Since human MetH shows substantial sequence identity to the enzyme from Escherichia coli, studies with the bacterial enzyme may provide valuable insights into the catalytic mechanism of the human enzyme. The crystal structure of the B12 binding domain of the E. coli MetH revealed that in the enzyme bound B12, the dimethylbenzimidazole is replaced by the imidazole of His759. The His759 and the nearby Asp757 and Ser810 form a ligand triad and have been suggested to play a role during catalysis. The goal of this proposal is to quantitate and to dissect the role of the ligand triad. The rate enhancements achieved by binding of the cofactor to the protein will be quantitated. To separate the contribution of the ligand triad to conformational changes from those to the chemistry, separately overexpressed fragments of the wild type enzyme and ligand triad variants will be examined for activity. These studies may help clarify the utility of the ligand triad in MetH and in an emerging class of B12 dependent enzymes that prefer base-off coordination of the cofactor.
| Bandarian, Vahe; Matthews, Rowena G (2004) Measurement of energetics of conformational change in cobalamin-dependent methionine synthase. Methods Enzymol 380:152-69 |
| Bandarian, Vahe; Ludwig, Martha L; Matthews, Rowena G (2003) Factors modulating conformational equilibria in large modular proteins: a case study with cobalamin-dependent methionine synthase. Proc Natl Acad Sci U S A 100:8156-63 |
| Bandarian, Vahe; Pattridge, Katherine A; Lennon, Brett W et al. (2002) Domain alternation switches B(12)-dependent methionine synthase to the activation conformation. Nat Struct Biol 9:53-6 |
| Bandarian, V; Matthews, R G (2001) Quantitation of rate enhancements attained by the binding of cobalamin to methionine synthase. Biochemistry 40:5056-64 |
