Recombinant Human Liver Betaine-homocysteine S-Methyltransferase: Identification of Three Cysteine Residues Critical for Zinc Binding
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- 作者：Andrew P. Breksa ; III and Timothy A. Garrow
- 刊名：Biochemistry
- 出版年：1999
- 出版时间：October 19, 1999
- 年：1999
- 卷：38
- 期：42
- 页码：13991 - 13998
- 全文大小：121K
- 年卷期：v.38,no.42(October 19, 1999)
- ISSN：1520-4995
文摘
Betaine-homocysteine S-methyltransferase (BHMT; EC 2.1.1.5) catalyzes the transfer of anN-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 forfurther physical and mechanistic studies, including characterizing its metal binding properties, we haveoverexpressed the enzyme in E. coli as a fusion construct which facilitated its subsequent purification bya self-cleavable affinity tag system (IMPACT T7). Using this expression and purification system inconjunction 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 reductionin the ability of the protein to bind zinc. Comparing the regions of BHMT amino acid sequence surroundingthese Cys residues with similar amino acid sequences retrievable from protein databases, we have identifiedthe following motif: G[ILV]NCX(20,100)[ALV]X(2)[ILV]GGCCX(3)PX(2)I, which we propose to be asignature 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 B12-dependent methionine synthases, E. coliS-methylmethionine-S-homocysteine methyltransferase, and A. bisulcatus S-methylmethionine-selenocysteine methyltransferase.