Recombinant human liver betaine-homocysteine S-methyltransferase: identification of three cysteine residues critical for zinc binding.
Betaine-homocysteine S-methyltransferase (BHMT; EC 2.1.1.5) catalyzes the transfer of an N-methyl group from betaine to homocysteine to produce dimethylglycine and methionine, respectively. The enzyme is found in the pathway of choline oxidation and is abundantly expressed in liver and kidney. We have recently shown that human BHMT is a zinc metalloenzyme [Millian, N. S., and Garrow, T. A. (1998) Arch. Biochem. Biophys. 356, 93-98]. To facilitate the rapid purification of human BHMT for further physical and mechanistic studies, including characterizing its metal binding properties, we have overexpressed the enzyme in E. coli as a fusion construct which facilitated its subsequent purification by a self-cleavable affinity tag system (IMPACT T7). Using this expression and purification system in conjunction with site-directed mutagenesis, we have identified Cys217, Cys299, and Cys300 as zinc ligands. Mutating any of these Cys residues to Ala results in the complete loss of activity and a significant reduction in the ability of the protein to bind zinc. Comparing the regions of BHMT amino acid sequence surrounding these Cys residues with similar amino acid sequences retrievable from protein databases, we have identified the following motif: G[ILV]NCX(20,100)[ALV]X(2)[ILV]GGCCX(3)PX(2)I, which we propose to be a signature for a family of zinc-dependent methyltransferases that utilize thiols or selenols as methyl acceptors. Some of the members of this family include the vitamin B(12)-dependent methionine synthases, E. coli S-methylmethionine-S-homocysteine methyltransferase, and A. bisulcatus S-methylmethionine-selenocysteine methyltransferase.
Breksa
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AP
Department of Food Science and Human Nutrition, University of Illinois, Urbana 61801, USA.
Garrow
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TA
D001483:Base Sequence; D050940:Betaine-Homocysteine S-Methyltransferase; D001665:Binding Sites / Q000235:genetics; D002686:Chitin / Q000235:genetics; D003545:Cysteine / Q000235:genetics / Q000378:metabolism*; D027081:DNA Gyrase; D004250:DNA Topoisomerases, Type II / Q000235:genetics; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics; D015964:Gene Expression Regulation, Bacterial; D005822:Genetic Vectors / Q000138:chemical synthesis; D006801:Humans; D008099:Liver / Q000201:enzymology*; D008780:Methyltransferases / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D016415:Sequence Alignment; D012984:Software; D015032:Zinc / Q000378:metabolism*
D011993:Recombinant Fusion Proteins; D002686:Chitin; D008780:Methyltransferases; C494548:BHMT protein, human; D050940:Betaine-Homocysteine S-Methyltransferase; D027081:DNA Gyrase; D004250:DNA Topoisomerases, Type II; D015032:Zinc; D003545:Cysteine