Identification of the zinc ligands in cobalamin-independent methionine synthase (MetE) from Escherichia coli

Cobalamin-independent methionine synthase (MetE) from Escherichia coli cata lyzes the transfer of a methyl group from methyltetrahydrofolate to homocys teine to form tetrahydrofolate and methionine. It contains 1 equiv of zinc that is essential for its catalytic activity. Extended X-ray absorption fin e structure analysis of the zinc-binding site has suggested tetrahedral coo rdination with two sulfur (cysteine) and one nitrogen or oxygen ligands pro vided by the enzyme and an exchangeable oxygen or nitrogen ligand that is r eplaced by the homocysteine thiol group in the enzyme-substrate complex [Go nzalez, J. C., Peariso, K., Penner-Hahn, J. E., and Matthews, R. G, (1996) Biochemistry 35, 12228-34]. Sequence alignment of MetE homologues shows tha t His641, Cys643, and Cys726 are the only conserved residues. We report her e the construction, expression, and purification of the His641Gln, Cys643Se r, and Cys726Ser mutants of MetE. Each mutant displays significantly impair ed activity and contains less than 1 equiv of zinc upon purification. Furth ermore, each mutant binds zinc with lower binding affinity (K-a approximate to 10(14) M-1) compared to the wild-type enzyme (K-a > 10(16) M-1). All th e MetE mutants are able to bind homocysteine. X-ray absorption spectroscopy analysis of the zinc-binding sites in the mutants indicates that the four- coordinate zinc site is preserved but that the ligand sets are changed. Our results demonstrate that Cys643 and Cys726 are two of the zinc ligands in MetE from E. coli and suggest that His641 is a third endogenous ligand. The effects of the mutations on the specific activities of the mutant proteins suggest that zinc and homocysteine binding alone are not sufficient for ac tivity; the chemical nature of the ligands is also a determining factor for catalytic activity in agreement with model studies of the alkylation of zi nc-thiolate complexes.