Crystal Structure of Bacillus anthracis Transpeptidase Enzyme CapD

摘要

Bacillus anthracis elaborates a poly-γ-d-glutamic acid capsule that protects bacilli from phagocytic killing during infection. The enzyme CapD generates amide bonds with peptidoglycan cross-bridges to anchor capsular material within the cell wall envelope of B. anthracis. The capsular biosynthetic pathway is essential for virulence during anthrax infections and can be targeted for anti-infective inhibition with small molecules. Here, we present the crystal structures of the γ-glutamyltranspeptidase CapD with and without α-l-Glu-l-Glu dipeptide, a non-hydrolyzable analog of poly-γ-d-glutamic acid, in the active site. Purified CapD displays transpeptidation activity in vitro, and its structure reveals an active site broadly accessible for poly-γ-glutamate binding and processing. Using structural and biochemical information, we derive a mechanistic model for CapD catalysis whereby Pro427, Gly428, and Gly429 activate the catalytic residue of the enzyme, Thr352, and stabilize an oxyanion hole via main chain amide hydrogen bonds. Bacillus anthracis elaborates a poly-γ-d-glutamic acid capsule that protects bacilli from phagocytic killing during infection. The enzyme CapD generates amide bonds with peptidoglycan cross-bridges to anchor capsular material within the cell wall envelope of B. anthracis. The capsular biosynthetic pathway is essential for virulence during anthrax infections and can be targeted for anti-infective inhibition with small molecules. Here, we present the crystal structures of the γ-glutamyltranspeptidase CapD with and without α-l-Glu-l-Glu dipeptide, a non-hydrolyzable analog of poly-γ-d-glutamic acid, in the active site. Purified CapD displays transpeptidation activity in vitro, and its structure reveals an active site broadly accessible for poly-γ-glutamate binding and processing. Using structural and biochemical information, we derive a mechanistic model for CapD catalysis whereby Pro427, Gly428, and Gly429 activate the catalytic residue of the enzyme, Thr352, and stabilize an oxyanion hole via main chain amide hydrogen bonds. Spores of Bacillus anthracis are the causative agents of anthrax disease (1.Koch R. Beitr. Biol. Pflanz. 1876; 2: 277-310Google Scholar). Upon entry into their hosts, spores germinate and replicate as vegetative bacilli (1.Koch R. Beitr. Biol. Pflanz. 1876; 2: 277-310Google Scholar). The formation of a thick capsule encasing vegetative forms enables bacilli to escape granulocyte0 and macrophage-mediated phagocytosis, and the pathogen eventually disseminates throughout all tissues of an infected host (2.Preisz H. Z. Immunitaetforsch. 1909; 5: 341-452Google Scholar, 3.Zwartouw H.T. Smith H. Biochem. J. 1956; 63: 437-442Crossref PubMed Scopus (64) Google Scholar). Bacilli secrete lethal and edema toxins, which cause macrophage necrosis and precipitate anthrax death (4.Klimpel K.R. Arora N. Leppla S.H. Mol. Microbiol. 1994; 13: 1093-1100Crossref PubMed Scopus (263) Google Scholar, 5.Duesbery N.S. Webb C.P. Leppla S.H. Gordon V.M. Klimpel K.R. Copeland T.D. Ahn N.G. Oskarsson M.K. Fukasawa K. Cleveland D.W. Vande Woude G.F. Science. 1998; 280: 734-737Crossref PubMed Scopus (886) Google Scholar, 6.Vitale G. Pellizzari R. Recchi C. Napolitani G. Mock M. Montecucco C. Biochem. Biophys. Res. Commun. 1998; 248: 706-711Crossref PubMed Scopus (361) Google Scholar, 7.Park J.M. Greten F.R. Li Z.W. Karin M. Science. 2002; 297: 2048-2051Crossref PubMed Scopus (419) Google Scholar). The genes providing for toxin and capsule formation are carried on two large virulence plasmids, pXO1 and pXO2, respectively (8.Mikesell P. Ivins B.E. Ristroph J.D. Dreier T.M. Infect. Immun. 1983; 39: 371-376Crossref PubMed Google Scholar, 9.Green B.D. Battisti L. Koehler T.M. Thorne C.B. Ivins B.E. Infect. Immun. 1985; 49: 291-297Crossref PubMed Google Scholar). Loss of any one plasmid leads to virulence attenuation, a feature that has been exploited for the generation of vaccine-type strains (10.Pasteur L. C.R. Acad. Sci.