Shared Catalysis in Virus Entry and Bacterial Cell Wall Depolymerization

JOURNAL OF MOLECULAR BIOLOGY

卷:387

Article

Cohen, Daniel N.^2,3  Sham, Yuk Y.⁴  Haugstad, Greg D.⁵  Xiang, Ye⁶  Rossmann, Michael G.⁶  Anderson, Dwight L.^2,3,7  Popham, David L.¹ 

[1] Virginia Tech, Dept Biol Sci, Blacksburg, VA 24061 USA

[2] Univ Minnesota, Sch Med, Dept Microbiol, Minneapolis, MN 55455 USA

[3] Univ Minnesota, Inst Mol Virol, Minneapolis, MN 55455 USA

[4] Linivers Minnesota, Ctr Drug Design, Minneapolis, MN 55455 USA

[5] Univ Minnesota, Inst Technol, Dept Phys, Characterizat Facil, Minneapolis, MN 55455 USA

[6] Purdue Univ, Dept Biol Sci, W Lafayette, IN 47907 USA

[7] Univ Minnesota, Sch Dent, Dept Diagnost & Biol Sci, Minneapolis, MN USA

关键词: bacteriophage;    metallopeptidase;    autolysin;    phi 29;    gp13;   

DOI  :  10.1016/j.jmb.2009.02.001

来源: Elsevier

【 摘 要 】

Bacterial virus entry and cell wall depolymerization require the breakdown of peptidoglycan (PG), the pep tide-cross-linked polysaccharide matrix that surrounds bacterial cells. Structural studies of lysostaphin, a PG lytic enzyme (autolysin), have suggested that residues in the active site facilitate hydrolysis, but a clear mechanism for this reaction has remained unsolved. The active-site residues and-a structural pattern of beta-sheets are conserved among lysostaphin homologs (such as LytM of Staphylococcus aureus) and the C-terminal domain of gene product 13 (gp13), a protein at the tail tip of the Bacillus subtilis bacteriophage phi 29. gp13 activity on PG and muropeptides was assayed using high-performance liquid chromatography, and gp13 was found to be a D,D-endopeptidase that cleaved the peptide cross-link. Computational modeling of the B. subtilis cross-linked peptide into the gp13 active site suggested that Asp195 may facilitate scissile-bond activation and that His247 is oriented to mediate nucleophile generation. To our knowledge, this is the first model of a Zn2+ metallopeptidase and its substrate. Residue Asp195 of gp13 was found to be critical for Zn2+ binding and catalysis by substitution mutagenesis with Ala or Cys. Circular dichroism and particle-induced X-ray emission spectroscopy showed that the general protein folding and Zn2+ binding were maintained in the Cys mutant but reduced in the Ala mutant. These findings together support a model in which the Asp195 and His247 in gp13 and homologous residues in the LytM and lysostaphin active sites facilitate hydrolysis of the peptide substrate that cross-links PG. Thus, these autolysins and phage-entry enzymes have a shared chemical mechanism of action. (c) 2009 Elsevier Ltd. All rights reserved.

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