Substitution of Arg230 and Arg233 in Escherichia coli elongation factor Tu strongly enhances its pulvomycin resistance.
Pulvomycin is a strong inhibitor of protein synthesis, known to prevent the binding of aminoacyl-tRNA to elongation factor Tu.GTP (EF-Tu.GTP). Recently, three pulvomycin-resistant mutant strains have been isolated by targeted mutagenesis of the tufA gene resulting in EF-Tu substitutions at positions 230, 333 or 334. In order to analyze the functions of arginine residues located in domain II, with respect to pulvomycin resistance and the interaction with aminoacyl-tRNA, we have investigated the effect of the substitutions of the highly conserved residues Arg230 and Arg233 by site-directed mutagenesis. We have purified two mutants species, [R233S]EF-TuHis and [R230V, R233F]EF-TuHis, both with a C-terminal histidine extension to enable purification by Ni2+ affinity chromatography. In this study, we describe the in vitro characterization of these mutant proteins. The results show that the concomitant substitution of residues at positions 230 and 233, dramatically increases the pulvomycin resistance. Preliminary evidence is presented that protein synthesis is inhibited by an EF-Tu.GDP.pulvomycin complex rather than by EF-Tu.GTP.pulvomycin. Moreover, the mutant [R230V, R233F]EF-TuHis shows a stronger protection of the ester bond of aminoacyl-tRNA than wild-type EF-Tu.
Boon
K
K
Leiden Institute of Chemistry, Gorleaus Laboratories, Department of Biochemistry, The Netherlands.
Krab
I
I
Parmeggiani
A
A
Bosch
L
L
Kraal
B
B
D000617:Aminoglycosides; D000900:Anti-Bacterial Agents / Q000378:metabolism / Q000494:pharmacology*; D001483:Base Sequence; D017931:DNA Primers / Q000235:genetics; D004269:DNA, Bacterial / Q000235:genetics; D004352:Drug Resistance, Microbial / Q000235:genetics; D004926:Escherichia coli / Q000187:drug effects* / Q000235:genetics* / Q000378:metabolism; D005798:Genes, Bacterial; D006153:Guanosine Diphosphate / Q000378:metabolism; D006160:Guanosine Triphosphate / Q000378:metabolism; D007700:Kinetics; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010452:Peptide Biosynthesis; D010444:Peptide Elongation Factor Tu / Q000235:genetics* / Q000378:metabolism; D010455:Peptides; D011728:Pyridones / Q000378:metabolism / Q000494:pharmacology
D000617:Aminoglycosides; D000900:Anti-Bacterial Agents; D017931:DNA Primers; D004269:DNA, Bacterial; D010455:Peptides; D011728:Pyridones; C012521:pulvomycin; D006153:Guanosine Diphosphate; C012001:polyphenylalanine; D006160:Guanosine Triphosphate; D010444:Peptide Elongation Factor Tu; C008536:mocimycin