title: Domain function dissection and catalytic properties of Listeria monocytogenes p60 protein with bacteriolytic activity.
abstract: The major extracellular protein p60 of Listeria monocytogenes (Lm-p60) is an autolysin that can hydrolyze the peptidoglycan of bacterial cell wall and has been shown to be required for L. monocytogenes virulence. The predicted three-dimensional structure of Lm-p60 showed that Lm-p60 could be split into two independent structural domains at the amino acid residue 270. Conserved motif analysis showed that V30, D207, S395, and H444 are the key amino acid residues of the corresponding motifs. However, not only the actual functions of these two domains but also the catalytic properties of Lm-p60 are unclear. We try to express recombinant Lm-p60 and identify the functions of two domains by residue substitution (V30A, D207A, S395A, and H444A) and peptide truncation. The C-terminal domain was identified as catalytic element and N-terminal domain as substrate recognition and binding element. Either N-terminal domain truncation or C-terminal domain truncation presents corresponding biological activity. The catalytic activity of Lm-p60 with a malfunctioned substrate-binding domain was decreased, while the substrate binding was not affected by a mulfunctioned catalytic domain. With turbidimetric method, we determined the optimal conditions for the bacteriolytic activity of Lm-p60 against Micrococcus lysodeikficus. The assay for the effect of Lm-p60 on the bacteriolytic activity of lysozyme revealed that the combined use of Lm-p60 protein with lysozyme showed a strong synergistic effect on the bacteriolytic activity.
authors.affiliation: College of Bioscience and Biotechnology, Yangzhou University; Jiangsu Key Laboratory of Zoonosis; Jiangsu Co-innovation Center for Prevention and Control of Important Animal Infectious Diseases and Zoonoses, Yangzhou, 225009, Jiangsu, People's Republic of China.
authors.forename: Minfeng
authors.initials: M
authors.lastname: Yu
authors.affiliation: College of Bioscience and Biotechnology, Yangzhou University; Jiangsu Key Laboratory of Zoonosis; Jiangsu Co-innovation Center for Prevention and Control of Important Animal Infectious Diseases and Zoonoses, Yangzhou, 225009, Jiangsu, People's Republic of China.
authors.forename: Jinrong
authors.initials: J
authors.lastname: Zuo
authors.affiliation: College of Bioscience and Biotechnology, Yangzhou University; Jiangsu Key Laboratory of Zoonosis; Jiangsu Co-innovation Center for Prevention and Control of Important Animal Infectious Diseases and Zoonoses, Yangzhou, 225009, Jiangsu, People's Republic of China.
authors.forename: Hao
authors.initials: H
authors.lastname: Gu
authors.affiliation: College of Bioscience and Biotechnology, Yangzhou University; Jiangsu Key Laboratory of Zoonosis; Jiangsu Co-innovation Center for Prevention and Control of Important Animal Infectious Diseases and Zoonoses, Yangzhou, 225009, Jiangsu, People's Republic of China. guoml@yzu.edu.cn.
authors.forename: Minliang
authors.initials: M
authors.lastname: Guo
authors.affiliation: College of Bioscience and Biotechnology, Yangzhou University; Jiangsu Key Laboratory of Zoonosis; Jiangsu Co-innovation Center for Prevention and Control of Important Animal Infectious Diseases and Zoonoses, Yangzhou, 225009, Jiangsu, People's Republic of China.
authors.forename: Yuelan
authors.initials: Y
authors.lastname: Yin
mesh_terms: D019943:Amino Acid Substitution; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D001433:Bacteriolysis; D020134:Catalytic Domain; D004252:DNA Mutational Analysis; D006868:Hydrolysis; D008089:Listeria monocytogenes / Q000201:enzymology*; D008837:Micrococcus / Q000187:drug effects; D008958:Models, Molecular; D009238:N-Acetylmuramoyl-L-alanine Amidase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D010457:Peptidoglycan / Q000378:metabolism; D017434:Protein Structure, Tertiary; D017384:Sequence Deletion
chemical_list: C078516:60 kDa protein, Listeria monocytogenes; D001426:Bacterial Proteins; D010457:Peptidoglycan; D009238:N-Acetylmuramoyl-L-alanine Amidase
keywords: Domain function; Listeria monocytogenes p60 protein; LysM domain; NlpC/P60 domain; Peptidoglycan hydrolase