title: Active site analysis of sortase A from Staphylococcus simulans indicates function in cleavage of putative cell wall proteins.
abstract: Sortase mediated transpeptidation reactions play a significant role in covalent attachment of surface proteins to the cell wall of Gram-positive bacteria. Earlier studies have shown that sortase A (StrA) is required for the virulence of Staphylococci. The human pathogen Staphylococcus simulans CJ16 carries a putative sortase A (SsiStrA) encoding gene, but neither transpeptidation activity nor biochemical characteristics of SsiStrA have been investigated. Here, we identified and characterized StrA from coagulase-negative Staphylococci. SsiStrA was cloned and overexpressed in Escherichia coli BL21 in a soluble form. Size-exclusion chromatography, cross-linking and dynamic light scattering demonstrated that SsiStrA existed as monomer-dimer equilibrium in vitro. We further demonstrated that SsiStrA has sortase activity, and it recognized and cleaved the sorting motif LXPTG. H117, C180 and R193 residues were critical for enzyme activity, and calcium ions enhanced activity.
authors: Intensive Care Unit, The First Affiliated Hospital, College of Medicine, Zhejiang University, Hangzhou, China.
authors: Jian
authors: J
authors: Chen
authors: State Key Laboratory for Diagnosis and Treatment of Infectious Disease, Collaborative Innovation Center for Diagnosis and Treatment of Infectious Diseases, The First Affiliated Hospital, College of Medicine, Zhejiang University, Hangzhou, China.
authors: Huihui
authors: H
authors: Dong
authors: Section of Infectious Diseases, Department of Internal Medicine, Yale University School of Medicine, New Haven, USA.
authors: Kristen E
authors: KE
authors: Murfin
authors: Institute of Animal Quarantine, Chinese Academy of Inspection and Quarantine, Beijing, China.
authors: Chunyan
authors: C
authors: Feng
authors: Institute of Animal Quarantine, Chinese Academy of Inspection and Quarantine, Beijing, China. Electronic address: sqwu@sina.com.
authors: Shaoqiang
authors: S
authors: Wu
authors: State Key Laboratory for Diagnosis and Treatment of Infectious Disease, Collaborative Innovation Center for Diagnosis and Treatment of Infectious Diseases, The First Affiliated Hospital, College of Medicine, Zhejiang University, Hangzhou, China. Electronic address: zhengbw@zju.edu.cn.
authors: Beiwen
authors: B
authors: Zheng
mesh_terms: D020816:Amino Acid Motifs / Q000235:genetics; D000595:Amino Acid Sequence; D019881:Aminoacyltransferases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D001665:Binding Sites / Q000235:genetics; D002118:Calcium / Q000378:metabolism; D020134:Catalytic Domain; D002473:Cell Wall / Q000235:genetics / Q000378:metabolism*; D002850:Chromatography, Gel; D002942:Circular Dichroism; D003546:Cysteine Endopeptidases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D004926:Escherichia coli / Q000235:genetics; D015151:Immunoblotting; D007700:Kinetics; D008958:Models, Molecular; D000072417:Protein Domains; D055503:Protein Multimerization; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D013210:Staphylococcus / Q000201:enzymology* / Q000235:genetics; D013379:Substrate Specificity
chemical_list: D001426:Bacterial Proteins; D011994:Recombinant Proteins; D019881:Aminoacyltransferases; C120542:sortase A; D003546:Cysteine Endopeptidases; D002118:Calcium
keywords: Active site; Calcium ions; LPXTG-Motif; Sortase A; Staphylococcus simulans