Inagaki Nobuya, Iguchi Akinori, Yokoyama Takahiro, Yokoi Ken-ji, Ono Yasushi, Yamakawa Ayanori, Taketo Akira, Kodaira Ken-Ichi
Molecular Biology Group, Graduate School of Science and Engineering, University of Toyama, Gofuku, Toyama, Japan.
Gene. 2009 Nov 15;447(2):61-71. doi: 10.1016/j.gene.2009.08.004. Epub 2009 Aug 15.
The major autolysin AcmA of Lactococcus lactis ssp. cremoris MG1363 is a modular protein consisting of an N-terminal signal sequence, a central enzymatic region (glu(acma) as a glucosaminidase), and a C-terminal cell-recognition domain (LysM123). glu(acma) (about 160 amino acids) belongs to the glycoside hydrolase (GH) 73 family, and the two acidic residues E128 and D153 have been thought to be catalytically important. In this study, amino-acid substitution analysis of AcmA was first carried out in the Escherichia coli system. Point mutations E94A, E94Q, E128A, D153A, and Y191A markedly reduced cell-lytic activity (3.8%, 1.1%, 4.2%, 4.8%, and 2.4%, respectively), whereas E128Q and D153N retained significant residual activities (32.1% and 44.0%, respectively). On the other hand, Y191F and Y191W mutations retained high activities (66.2% and 46.0%, respectively). These results showed that E94 (rather than E128 and D153) and the aromatic residue Y191 probably play important roles in catalysis of AcmA. Together with mutational analysis of another GH73 glucoaminidase Glu(atlwm) from the Staphylococcus warneri M autolysin Atl(WM), these results suggested that the GH73 members cleave a glycosidic bond via a substrate-assisted mechanism, as postulated in the GH20 members. AcmA and Glu(atlwm) were purified from E. coli recombinant cells, and their enzymatic properties were studied.
乳酸乳球菌乳脂亚种MG1363的主要自溶素AcmA是一种模块化蛋白质，由一个N端信号序列、一个中央酶区域（作为氨基葡萄糖苷酶的glu(acma)）和一个C端细胞识别结构域（LysM123）组成。glu(acma)（约160个氨基酸）属于糖苷水解酶（GH）73家族，两个酸性残基E128和D153被认为具有重要催化作用。在本研究中，首先在大肠杆菌系统中对AcmA进行了氨基酸取代分析。点突变E94A、E94Q、E128A、D153A和Y191A显著降低了细胞裂解活性（分别为3.8%、1.1%、4.2%、4.8%和2.4%），而E128Q和D153N保留了显著的残余活性（分别为32.1%和44.0%）。另一方面，Y191F和Y191W突变保留了高活性（分别为66.2%和46.0%）。这些结果表明，E94（而非E128和D153）和芳香族残基Y191可能在AcmA的催化中起重要作用。与来自沃氏葡萄球菌M自溶素Atl(WM)的另一种GH73氨基葡萄糖苷酶Glu(atlwm)的突变分析一起，这些结果表明，GH73成员通过底物辅助机制切割糖苷键，如GH20成员中所假设的那样。从大肠杆菌重组细胞中纯化了AcmA和Glu(atlwm)，并研究了它们的酶学性质。
