Ihara K, Kawate H, Chueh L L, Hayakawa H, Sekiguchi M
Department of Biochemistry, Faculty of Medicine, Kyushu University, Fukuoka, Japan.
Mol Gen Genet. 1994 May 25;243(4):379-89. doi: 10.1007/BF00280468.
O6-Methylguanine-DNA methyltransferase catalyzes transfer of a methyl group from O6-methylguanine and O4-methylthymine of DNA to a cysteine residue of the enzyme protein, thereby repairing the mutagenic and carcinogenic lesions in a single-step reaction. There are highly conserved amino acid sequences around the methyl-accepting cysteine site in eleven molecular species of methyltransferases. To elucidate the significance of the conserved sequence, amino acid substitutions were introduced by site-directed mutagenesis of the cloned DNA for Escherichia coli Ogt methyltransferase, and the activity and stability of mutant forms of the enzyme were examined. When cysteine-139, to which methyl transfer occurs, was replaced by other amino acids, all of the mutants showed the methyltransferase-negative phenotype. Methyltransferase-positive revertants, isolated from one of the negative mutants, had restored codons for cysteine. Thus the cysteine residue is essential for acceptance of the methyl group and is not replaceable by other amino acids. Using this negative and positive selection procedure, the analysis was extended to other residues near the acceptor site. At the histidine-140 and arginine-141 sites, all the positive revertants isolated carried codons for amino acids identical to those of the wild-type protein. At proline-138, five substitutions (serine, glutamine, threonine, histidine, and alanine) exhibited the positive phenotype but levels of methyltransferase activity in extracts of cells harboring these mutant forms were very low. This suggests that the proline residue at this site is important for maintaining the proper conformation of the protein. With valine-142 substitutions there were seven types of positive revertants, among which mutants carrying isoleucine, cysteine, leucine, and alanine showed relatively high levels of methyltransferase activity. These results indicate that the sequence Pro-Cys-His-Arg is a sine qua non for methyltransferase to exert its function.
O6-甲基鸟嘌呤-DNA甲基转移酶催化DNA的O6-甲基鸟嘌呤和O4-甲基胸腺嘧啶上的甲基基团转移至酶蛋白的半胱氨酸残基，从而在一步反应中修复诱变和致癌损伤。在11种甲基转移酶分子物种的甲基接受半胱氨酸位点周围存在高度保守的氨基酸序列。为了阐明保守序列的意义，通过对大肠杆菌Ogt甲基转移酶的克隆DNA进行定点诱变引入氨基酸替换，并检测了该酶突变形式的活性和稳定性。当发生甲基转移的半胱氨酸-139被其他氨基酸取代时，所有突变体均表现出甲基转移酶阴性表型。从其中一个阴性突变体中分离出的甲基转移酶阳性回复突变体具有恢复的半胱氨酸密码子。因此，半胱氨酸残基对于接受甲基基团至关重要，且不能被其他氨基酸替代。使用这种阴性和阳性选择程序，分析扩展至受体位点附近的其他残基。在组氨酸-140和精氨酸-141位点，分离出的所有阳性回复突变体携带的氨基酸密码子与野生型蛋白相同。在脯氨酸-138位点，五个替换（丝氨酸、谷氨酰胺、苏氨酸、组氨酸和丙氨酸）表现出阳性表型，但携带这些突变形式的细胞提取物中的甲基转移酶活性水平非常低。这表明该位点的脯氨酸残基对于维持蛋白质的正确构象很重要。对于缬氨酸-142替换，有七种类型的阳性回复突变体，其中携带异亮氨酸、半胱氨酸、亮氨酸和丙氨酸的突变体表现出相对较高水平的甲基转移酶活性。这些结果表明，序列Pro-Cys-His-Arg是甲基转移酶发挥其功能的必要条件。
