title: Catalytic properties of mutant 23 S ribosomes resistant to oxazolidinones.
abstract: Kinetic analysis of ribosomal peptidyltransferase activity in a methanolic puromycin reaction with wild type and drug-resistant 23 S RNA mutants was used to probe the structural basis of catalysis and mechanism of resistance to antibiotics. 23 S RNA mutants G2032A and G2447A are resistant to oxazolidinones both in vitro and in vivo with the latter displaying a 5-fold increase in the value of Km for initiator tRNA and a 100-fold decrease in Vmax in puromycin reaction. Comparison of the Ki values for oxazolidinones, chloramphenicol, and sparsomycin revealed partial cross-resistance between oxazolidinones and chloramphenicol; no cross-resistance was observed with sparsomycin, a known inhibitor of the peptidyltransferase A-site. Inhibition of the mutants using a truncated CCA-Phe-X-Biotin fragment as a P-site substrate is similar to that observed with the intact initiator tRNA, indicating that the inhibition is substrate-independent and that the peptidyltransferase itself is the oxazolidinone target. Mapping of all known mutations that confer resistance to these drugs onto the spatial structure of the 50 S ribosomal subunit allows for docking of an oxazolidinone into a proposed binding pocket. The model suggests that oxazolidinones bind between the P- and A-loops, partially overlapping with the peptidyltransferase P-site. Thus, kinetic, mutagenesis, and structural data suggest that oxazolidinones interfere with initiator fMet-tRNA binding to the P-site of the ribosomal peptidyltransferase center.
authors.affiliation: Bristol-Myers Squibb Pharma Company, Wilmington, Delaware 19880, USA. ebobkova@prdus.jnj.com
authors.forename: Ekaterina V
authors.initials: EV
authors.lastname: Bobkova
authors.forename: Yong Ping
authors.initials: YP
authors.lastname: Yan
authors.forename: Douglas B
authors.initials: DB
authors.lastname: Jordan
authors.forename: Michael G
authors.initials: MG
authors.lastname: Kurilla
authors.forename: David L
authors.initials: DL
authors.lastname: Pompliano
mesh_terms: D000903:Antibiotics, Antineoplastic / Q000494:pharmacology; D001665:Binding Sites; D002384:Catalysis; D020134:Catalytic Domain; D002701:Chloramphenicol / Q000494:pharmacology; D004305:Dose-Response Relationship, Drug; D004351:Drug Resistance; D004926:Escherichia coli / Q000378:metabolism; D020128:Inhibitory Concentration 50; D007700:Kinetics; D008956:Models, Chemical; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D023303:Oxazolidinones / Q000378:metabolism*; D010458:Peptidyl Transferases / Q000378:metabolism; D011485:Protein Binding; D011500:Protein Synthesis Inhibitors / Q000494:pharmacology; D011691:Puromycin / Q000494:pharmacology; D012338:RNA, Ribosomal, 23S / Q000235:genetics*; D012358:RNA, Transfer, Met / Q000378:metabolism; D013033:Sparsomycin / Q000494:pharmacology
chemical_list: D000903:Antibiotics, Antineoplastic; D023303:Oxazolidinones; D011500:Protein Synthesis Inhibitors; D012338:RNA, Ribosomal, 23S; D012358:RNA, Transfer, Met; C089238:fMet-tRNA(fMet); D011691:Puromycin; D002701:Chloramphenicol; D013033:Sparsomycin; D010458:Peptidyl Transferases