Mutational analysis of catalytic sites of the cell wall lytic N-acetylmuramoyl-L-alanine amidases Cw1C and Cw1V
Mutational analysis of catalytic sites of the cell wall lytic N-acetylmuramoyl-L-alanine amidases Cw1C and Cw1V
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DOI：
10.1074/jbc.m103903200
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发表时间：
2001-07-27
影响因子：
4.8
通讯作者：
Sekiguchi, J
中科院分区：
文献类型：
--
作者：
Shida, T;Hattori, H;Sekiguchi, J
对枯草芽孢杆菌CwlC和多粘芽孢杆菌变种（Bacillus polymyxa var.）Colistinus CwIV是CwlB（LytC）家族中的细胞壁裂解性N-乙酰胞壁酰-L-丙氨酸酰胺酶。CwlC酰胺酶从C末端至残基177的缺失不改变酰胺酶活性。然而，当缺失稍微向N末端延伸时，酰胺酶活性完全丧失。此外，没有前19个氨基酸的N-末端缺失突变体不具有酰胺酶活性。这些结果表明，CwlC酰胺酶的N-末端一半（残基1-176），与截短的CwIV（CwlVt）同源的区域，是一个催化结构域。对CwlC的催化结构域内的20个高度保守的氨基酸残基进行定点诱变。在两个残基（Glu-24和Glu-141）处的单个氨基酸取代完全丧失酰胺酶活性。类似地，截短的CwIV（CwIV 1）的两个谷氨酸残基（E26 Q和E142 Q）的取代（其对应于CwIC的Glu-24和Glu-141）对酰胺酶活性是关键的。EDTA处理的CwIV 1.不具有酰胺酶活性。添加Zn ~（2+）、Mn ~（2+）和Co ~（2+）可使EDTA处理的CwIV ~ 1的酰胺酶活性恢复，但添加Me ~（2+）和Ca ~（2+）则不能恢复。这些结果表明CwlB家族中的酰胺酶是含有两个谷氨酸作为催化残基的锌酰胺酶。
The Bacillus subtilis CwlC and the Bacillus polymyxa var. colistinus CwIV are the cell wall lytic N-acetylmuramoyl-L-alanine amidases in the CwlB (LytC) family. Deletion in the CwlC amidase from the C terminus to residue 177 did not change the amidase activity. However, when the deletion was extended slightly toward the N terminus, the amidase activity was entirely lost. Further, the N-terminal deletion mutant without the first 19 amino acids did not have the amidase activity. These results indicate that the N-terminal half (residues 1-176) of the CwlC amidase, the region homologous to the truncated CwIV (CwlVt), is a catalytic domain. Site-directed mutagenesis was performed on 20 highly conserved amino acid residues within the catalytic domain of CwlC. The amidase activity was lost completely on single amino acid substitutions at two residues (Glu-24 and Glu-141). Similarly, the substitution of the two glutamic acid residues (E26Q and E142Q) of the truncated CwIV (CwIV1), which corresponded to Glu-24 and Glu-141 of CwIC, was critical to the amidase activity. The EDTA-treated CwIV1. did not have amidase activity. The amidase activity of the EDTA-treated CwIV1 was restored by the addition of Zn2+, Mn2+, and Co2+ but not by the addition of Me2+ and Ca2+. These results suggest that the amidases in the CwlB family are zinc amidases containing two glutamic acids as catalytic residues.