A SYNTHETIC MODULE FOR THE METH GENE PERMITS FACILE MUTAGENESIS OF THE COBALAMIN-BINDING REGION OF ESCHERICHIA-COLI METHIONINE SYNTHASE - INITIAL CHARACTERIZATION OF 7 MUTANT PROTEINS

Cobalamin-dependent methionine synthase from Escherichia coli is a mon omeric 136 kDa protein composed of multiple functional regions. The X- ray structure of the cobalamin-binding region of methionine synthase r eveals that the cofactor is sandwiched between an a-helical domain tha t contacts the upper face of the cobalamin and an alpha/beta (Rossmann ) domain that interacts with the lower face. An unexpected conformatio nal change accompanies binding of the methylcobalamin cofactor. The di methylbenzimidazole ligand to the lower axial position of the cobalt i n the free cofactor is displaced by histidine 759 from the Rossmann do main [Drennan, C. L., Huang, S., Drummond, J. T., Matthews, R. G., & L udwig, M. L. (1994) Science 266, 1669]. In order to facilitate studies of the roles of amino acid residues in the cobalamin-binding region o f methionine synthase, we have constructed a synthetic module correspo nding to nucleotides (nt) 1741-2668 in the metH gene and incorporated it into the wild-type metH gene. This module contains unique restricti on sites at similar to 80 base pair intervals and was synthesized by o verlap extension of 22 synthetic oligonucleotides ranging in length fr om 70 to 105 nt and subsequent amplification using two sets of primers , Expression of methionine synthase from a plasmid containing the modi fied gene was shown to be unaffected by the introduction of the synthe tic module. E. coli does not synthesize cobalamin, and overexpression of MetH holoenzyme requires accelerated cobalamin transport. Growth co nditions are described that enable the production of holoenzyme rather than apoenzyme. We describe the construction and initial characteriza tion of seven mutants, Four mutations (His759Gly, Asp757Gla, Asp757Asn , and Ser810Ala) alter residues in the hydrogen-bonded network His-Asp -Ser that connects the histidine ligand of the cobalt to solvent. Thre e mutations (Phe708Ala, Phe714Ala, and Leu715Ala) alter residues in th e cap region that covers the upper face of the cobalamin, The His759Gl y mutation has profound effects, essentially abolishing steady-state a ctivity, while the Asp757, Ser810, Phe708, and Leu715 mutations lead t o decreases in activity. These mutations assess the importance of indi vidual residues in modulating cobalamin reactivity.