A synthetic module for the metH gene permits facile mutagenesis of the cobalamin-binding region of Escherichia coli methionine synthase: Initial characterization of seven mutant proteins

Cobalamin-dependent methionine synthase from Escherichia coli is a monomeric 136 kDa protein composed of multiple functional regions. The X-ray structure of the cobalamin-binding region of methionine synthase reveals that the cofactor is sandwiched between an α-helical domain that contacts the upper face of the cobalamin and an α/β (Rossmann) domain that interacts with the lower face. An unexpected conformational change accompanies binding of the methylcobalamin cofactor. The dimethylbenzimidazole ligand to the lower axial position of the cobalt in the free cofactor is displaced by histidine 759 from the Rossmann domain [Drennan, C. L., Huang, S., Drummond, J. T., Matthews, R. G., & Ludwig, M. L. (1994) Science 266, 1669]. In order to facilitate studies of the roles of amino acid residues in the cobalamin-binding region of methionine synthase, we have constructed a synthetic module corresponding to nucleotides (nt) 1741−2668 in the metH gene and incorporated it into the wild-type metH gene. This module co...