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 ² Mohan Amaratunga, ‡,§ Kerry Fluhr, ‡,| Joseph T. Jarrett, ‡ Catherine L. Drennan, ‡,| Martha L. Ludwig, ‡,| Rowena G. Matthews,* ,‡,| and Jeffrey D. Scholten ⊥ Biophysics Research DiVision and Department of Biological Chemistry, UniVersity of Michigan, Ann Arbor, Michigan 48109, and Parke-DaVis Research DiVision, Warner-Lambert Co., Ann Arbor, Michigan 48105 ReceiVed October 5, 1995; ReVised Manuscript ReceiVed December 13, 1995 X ABSTRACT: 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 R-helical domain that contacts the upper face of the cobalamin and an R/ (Rossmann) domain that interacts with the lower face. An unexpected conformational change accompanies binding of the methylcobalamin cofactor. The dimeth- ylbenzimidazole 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 contains unique restriction sites at ∼80 base pair intervals and was synthesized by overlap extension of 22 synthetic oligonucleotides ranging in length from 70 to 105 nt and subsequent amplification using two sets of primers. Expression of methionine synthase from a plasmid containing the modified gene was shown to be unaffected by the introduction of the synthetic module. E. coli does not synthesize cobalamin, and overexpression of MetH holoenzyme requires accelerated cobalamin transport. Growth conditions are described that enable the production of holoenzyme rather than apoenzyme. We describe the construction and initial characterization of seven mutants. Four mutations (His759Gly, Asp757Glu, Asp757Asn, and Ser810Ala) alter residues in the hydrogen-bonded network His-Asp-Ser that connects the histidine ligand of the cobalt to solvent. Three mutations (Phe708Ala, Phe714Ala, and Leu715Ala) alter residues in the cap region that covers the upper face of the cobalamin. The His759Gly mutation has profound effects, essentially abolishing steady-state activity, while the Asp757, Ser810, Phe708, and Leu715 mutations lead to decreases in activity. These mutations assess the importance of individual residues in modulating cobalamin reactivity. Cobalamin-dependent methionine synthase from Escheri- chia coli catalyzes the transfer of a methyl group from CH 3 - H 4 folate 1 to homocysteine, generating H 4 folate and methio- nine. This enzyme employs a methylcobalamin cofactor that plays an essential role in the methyl transfer mechanism, being alternately demethylated by homocysteine and re- methylated by CH 3 -H 4 folate. Demethylation of the meth- ylcob(III)alamin cofactor during the catalytic cycle results in the formation of a cob(I)alamin prosthetic group, and this highly reduced form of cobalamin is occasionally oxidized to the inactive cob(II)alamin form of the enzyme. Return of the cob(II)alamin enzyme to the catalytic cycle requires a reductive methylation, in which the electron is supplied by reduced flavodoxin (Fujii & Huennekens, 1974) and the methyl group comes from AdoMet (Mangum & Scrimgeour, 1962). The enzyme must catalyze three different methyl transfer reactions involving different substrates and different oxidation states of the cobalamin prosthetic group [see Scheme 1 in the accompanying paper (Jarrett et al., 1996)]. Recently the X-ray structure of a fragment containing the 27 kDa cobalamin-binding region of E. coli methionine synthase was determined (Drennan et al., 1994a). The ² This research has been supported by NIH Research Grants GM24908 (R.G.M.), and GM16429 (M.L.L.) and by the Parke-Davis Research Division, Warner Lambert Co. (J.D.S.). J.T.J. was supported in part by an NIH postdoctoral fellowship (GM17455). C.L.D. was supported by a Molecular Biophysics Training Grant (GM08570). M.A. was supported by a University of Michigan/Warner Lambert post- doctoral fellowship. * Correspondence should be addressed to this author at: Biophysics Research Division, University of Michigan, 4024 Chemistry, 930 N. University Ave., Ann Arbor, MI 48109-1055. ‡ Biophysics Research Division, University of Michigan. § Present address: Biological Sciences Laboratory, General Electric Co., Schenectady, NY 12301. | Department of Biological Chemistry, University of Michigan. ⊥ Parke-Davis Research Division, Warner-Lambert Co. X Abstract published in AdVance ACS Abstracts, February 1, 1996. 1 Abbreviations: AdoMet, S-adenosyl-L-methionine; bp, base pair(s); EDTA, ethylenediaminetetraacetic acid; Hcy, L-homocysteine; IPTG, isopropyl -D-thiogalactopyranoside; LB, Luria broth; CH + dH4folate, 5,10-methenyltetrahydrofolate; CH3-H4folate, 5-methyltetrahydrofolate; MOPS, 3-(N-morpholino)propanesulfonic acid; nt, nucleotide(s); ORF, open reading frame; PCR, polymerase chain reaction; H4folate, 5,6,7,8- tetrahydrofolate. 2453 Biochemistry 1996, 35, 2453-2463 0006-2960/96/0435-2453$12.00/0 © 1996 American Chemical Society