Catalytic Properties of Mutant 23 S Ribosomes Resistant to Oxazolidinones
| Autor: | Yong Ping Yan, Ekaterina V. Bobkova, David L. Pompliano, Douglas B. Jordan, Michael G. Kurilla |
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| Rok vydání: | 2003 |
| Předmět: |
Models
Molecular RNA Transfer Met Stereochemistry Mutant Drug Resistance Biology Biochemistry Ribosome Catalysis Inhibitory Concentration 50 chemistry.chemical_compound Catalytic Domain Escherichia coli Molecular Biology Oxazolidinones Protein Synthesis Inhibitors Antibiotics Antineoplastic Binding Sites Dose-Response Relationship Drug Wild type Sparsomycin Cell Biology Ribosomal RNA Peptidyltransferase activity Kinetics RNA Ribosomal 23S Chloramphenicol Models Chemical chemistry Puromycin Mutation Peptidyl Transferases Transfer RNA Mutagenesis Site-Directed Protein Binding |
| Zdroj: | Journal of Biological Chemistry. 278:9802-9807 |
| ISSN: | 0021-9258 |
| DOI: | 10.1074/jbc.m209249200 |
| Popis: | 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 K m for initiator tRNA and a 100-fold decrease in V max in puromycin reaction. Comparison of the K i 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. |
| Databáze: | OpenAIRE |
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