title: Structural Insights into Inhibition of Escherichia coli Penicillin-binding Protein 1B.
abstract: In Escherichia coli, the peptidoglycan cell wall is synthesized by bifunctional penicillin-binding proteins such as PBP1b that have both transpeptidase and transglycosylase activities. The PBP1b transpeptidase domain is a major target of β-lactams, and therefore it is important to attain a detailed understanding of its inhibition. The peptidoglycan glycosyltransferase domain of PBP1b is also considered an excellent antibiotic target yet is not exploited by any clinically approved antibacterials. Herein, we adapt a pyrophosphate sensor assay to monitor PBP1b-catalyzed glycosyltransfer and present an improved crystallographic model for inhibition of the PBP1b glycosyltransferase domain by the potent substrate analog moenomycin. We elucidate the structure of a previously disordered region in the glycosyltransferase active site and discuss its implications with regards to peptidoglycan polymerization. Furthermore, we solve the crystal structures of E. coli PBP1b bound to multiple different β-lactams in the transpeptidase active site and complement these data with gel-based competition assays to provide a detailed structural understanding of its inhibition. Taken together, these biochemical and structural data allow us to propose new insights into inhibition of both enzymatic domains in PBP1b.
authors.affiliation: From the Department of Biochemistry and Molecular Biology and Centre for Blood Research, University of British Columbia, Vancouver, British Columbia, V6T 1Z3, Canada.
authors.forename: Dustin T
authors.initials: DT
authors.lastname: King
authors.affiliation: From the Department of Biochemistry and Molecular Biology and Centre for Blood Research, University of British Columbia, Vancouver, British Columbia, V6T 1Z3, Canada.
authors.forename: Gregory A
authors.initials: GA
authors.lastname: Wasney
authors.affiliation: From the Department of Biochemistry and Molecular Biology and Centre for Blood Research, University of British Columbia, Vancouver, British Columbia, V6T 1Z3, Canada.
authors.forename: Michael
authors.initials: M
authors.lastname: Nosella
authors.affiliation: From the Department of Biochemistry and Molecular Biology and Centre for Blood Research, University of British Columbia, Vancouver, British Columbia, V6T 1Z3, Canada.
authors.forename: Anita
authors.initials: A
authors.lastname: Fong
authors.affiliation: From the Department of Biochemistry and Molecular Biology and Centre for Blood Research, University of British Columbia, Vancouver, British Columbia, V6T 1Z3, Canada ncjs@mail.ubc.ca.
authors.forename: Natalie C J
authors.initials: NC
authors.lastname: Strynadka
mesh_terms: D018360:Crystallography, X-Ray; D048168:Escherichia coli K12 / Q000737:chemistry* / Q000235:genetics; D029968:Escherichia coli Proteins / Q000737:chemistry* / Q000235:genetics; D046915:Penicillin-Binding Proteins / Q000737:chemistry* / Q000235:genetics; D046928:Peptidoglycan Glycosyltransferase / Q000737:chemistry* / Q000235:genetics; D000072417:Protein Domains; D046929:Serine-Type D-Ala-D-Ala Carboxypeptidase / Q000737:chemistry* / Q000235:genetics; D047090:beta-Lactams / Q000737:chemistry*
chemical_list: D029968:Escherichia coli Proteins; D046915:Penicillin-Binding Proteins; D047090:beta-Lactams; D046928:Peptidoglycan Glycosyltransferase; C478681:penicillin-binding protein 1B, E coli; D046929:Serine-Type D-Ala-D-Ala Carboxypeptidase
keywords: antibiotics; bacteria; glycosyltransferase; inhibitor; moenomycin; penicillin-binding protein 1b; peptidoglycan; transglycosylation; transpeptidation; β-lactam