Molecular properties of the glucosaminidase AcmA from Lactococcus lactis MG1363: Mutational and biochemical analyses |
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Authors: | Nobuya Inagaki Akinori Iguchi Takahiro Yokoyama Ken-ji Yokoi Yasushi Ono Ayanori Yamakawa Akira Taketo Ken-Ichi Kodaira |
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Institution: | 1. Molecular Biology Group, Graduate School of Science and Engineering, University of Toyama, 3190 Gofuku, Toyama 930-8555, Japan;2. Toyama Prefectural Food Research Institute, 360 Yoshioka, Toyama 939-8153, Japan;3. Biochemistry Division, Department of Materials Science, Wakayama National College of Technology, 77 Nada, Gobo, Wakayama 644-0023, Japan;4. Department of Applied Physics and Chemistry, Fukui University of Technology, Gakuen, Fukui 910-8505, Japan |
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Abstract: | 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 (gluacma as a glucosaminidase), and a C-terminal cell-recognition domain (LysM123). gluacma (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 Gluatlwm from the Staphylococcus warneri M autolysin AtlWM, these results suggested that the GH73 members cleave a glycosidic bond via a substrate-assisted mechanism, as postulated in the GH20 members. AcmA and Gluatlwm were purified from E. coli recombinant cells, and their enzymatic properties were studied. |
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Keywords: | Amp ampicillin AtlWM autolysin of Staphylococcus warneri M FlgJ peptidoglycan-hydrolyzing protein of Salmonera typhimurium GEWL goose egg-white lysozyme GH20 glycoside hydrolase 20 family gluacma glucosaminidase domain of AcmA gluatlwm glucosaminidase domain of Gluatlwm h hour(s) Hgluacma His-tagged gluacma Hgluatlwm His-tagged gluatlwm IPTG isopropyl-β-d-thiogalacto-pyranoside PAGE polyacrylamide gel electrophoresis PCR polymerase chain reaction Plac promoter of lacZ&prime gene PT7 promoter of T7 r resistant s sensitive wt wild type |
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