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
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- 作者：Mohan Amaratunga ; Kerry Fluhr ; Joseph T. Jarrett ; Catherine L. Drennan ; Martha L. Ludwig ; Rowena G. Matthews ; and Jeffrey D. Scholten
- 刊名：Biochemistry
- 出版年：1996
- 出版时间：February 20, 1996
- 年：1996
- 卷：35
- 期：7
- 页码：2453 - 2463
- 全文大小：554K
- 年卷期：v.35,no.7(February 20, 1996)
- ISSN：1520-4995
文摘
Cobalamin-dependent methionine synthase fromEscherichia coli is a monomeric 136 kDaprotein composed of multiple functional regions. The X-raystructure of the cobalamin-binding region ofmethionine synthase reveals that the cofactor is sandwiched between an
-helical domain that contactsthe upper face of the cobalamin and an /
(Rossmann) domain thatinteracts with the lower face. Anunexpected conformational change accompanies binding of themethylcobalamin cofactor. The dimethylbenzimidazole ligand to the lower axial position of the cobalt in thefree cofactor is displaced by histidine759 from the Rossmann domain [Drennan, C. L., Huang, S., Drummond, J.T., Matthews, R. G., & Ludwig,M. L. (1994) Science 266, 1669]. In orderto facilitate studies of the roles of amino acid residues inthecobalamin-binding region of methionine synthase, we have constructed asynthetic module correspondingto nucleotides (nt) 1741-2668 in the metH gene andincorporated it into the wild-type metH gene.Thismodule contains unique restriction sites at ~80 base pair intervalsand was synthesized by overlap extensionof 22 synthetic oligonucleotides ranging in length from 70 to 105 ntand subsequent amplification usingtwo sets of primers. Expression of methionine synthase from aplasmid containing the modified genewas shown to be unaffected by the introduction of the synthetic module.E. coli does not synthesizecobalamin, and overexpression of MetH holoenzyme requires acceleratedcobalamin transport. Growthconditions are described that enable the production of holoenzymerather than apoenzyme. We describethe construction and initial characterization of seven mutants.Four mutations (His759Gly, Asp757Glu,Asp757Asn, and Ser810Ala) alter residues in the hydrogen-bonded networkHis-Asp-Ser that connectsthe 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 thecobalamin. The His759Gly mutation hasprofound effects, essentially abolishing steady-state activity, whilethe Asp757, Ser810, Phe708, and Leu715mutations lead to decreases in activity. These mutations assessthe importance of individual residues inmodulating cobalamin reactivity.
-helical domain that contactsthe upper face of the cobalamin and an / (Rossmann) domain thatinteracts with the lower face. Anunexpected conformational change accompanies binding of themethylcobalamin cofactor. The dimethylbenzimidazole ligand to the lower axial position of the cobalt in thefree cofactor is displaced by histidine759 from the Rossmann domain [Drennan, C. L., Huang, S., Drummond, J.T., Matthews, R. G., & Ludwig,M. L. (1994) Science 266, 1669]. In orderto facilitate studies of the roles of amino acid residues inthecobalamin-binding region of methionine synthase, we have constructed asynthetic module correspondingto nucleotides (nt) 1741-2668 in the metH gene andincorporated it into the wild-type metH gene.Thismodule contains unique restriction sites at ~80 base pair intervalsand was synthesized by overlap extensionof 22 synthetic oligonucleotides ranging in length from 70 to 105 ntand subsequent amplification usingtwo sets of primers. Expression of methionine synthase from aplasmid containing the modified genewas shown to be unaffected by the introduction of the synthetic module.E. coli does not synthesizecobalamin, and overexpression of MetH holoenzyme requires acceleratedcobalamin transport. Growthconditions are described that enable the production of holoenzymerather than apoenzyme. We describethe construction and initial characterization of seven mutants.Four mutations (His759Gly, Asp757Glu,Asp757Asn, and Ser810Ala) alter residues in the hydrogen-bonded networkHis-Asp-Ser that connectsthe 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 thecobalamin. The His759Gly mutation hasprofound effects, essentially abolishing steady-state activity, whilethe Asp757, Ser810, Phe708, and Leu715mutations lead to decreases in activity. These mutations assessthe importance of individual residues inmodulating cobalamin reactivity.