TAILIEUCHUNG - Báo cáo khoa học: Mode of action of the microbial metabolite GE23077, a novel potent and selective inhibitor of bacterial RNA polymerase

GE23077, a novel microbial metabolite recently isolated from Actinomadurasp. culture media, is a potent and selective inhibitor of bacterial RNApolymerase (RNAP). It inhibitsGram-positive (Bacillus subtilis) andGram-negative (Escherichia coli)RNAPs with IC50 values (. the concen-tration at which the enzyme activity is inhibited by 50%) in the 10 )8 Mrange, whereas it is not active onE. coliDNA polymerase or on eukaryotic (wheat germ) RNAP II (IC50 values10 )4 Min both cases). | Eur. J. Biochem. 271 3146-3154 2004 FEBS 2004 doi Mode of action of the microbial metabolite GE23077 a novel potent and selective inhibitor of bacterial RNA polymerase Edoardo SarubbiỊ Federica Monti Emiliana Corti Anna Mielef and Enrico Selva Vicuron Pharmaceuticals Gerenzano Varese Italy GE23077 a novel microbial metabolite recently isolated from Actinomadura sp. culture media is a potent and selective inhibitor of bacterial RNA polymerase RNAP . It inhibits Gram-positive Bacillus subtilis and Gram-negative Escherichia coli RNAPs with IC50 values . the concentration at which the enzyme activity is inhibited by 50 in the 10 8 M range whereas it is not active on E. coli DNA polymerase or on eukaryotic wheat germ RNAP II IC50 values 10-4 M in both cases . In spite of its potent activity on purified bacterial RNAPs GE23077 shows a narrow spectrum of antimicrobial activity on Gram-positive and Gram-negative bacteria. To investigate the molecular basis of this behaviour the effects of GE23077 on macromolecular biosynthesis were tested in E. coli cells permeabilized under different conditions. The addition of GE23077 to plasmo-lyzed cells resulted in an immediate and specific inhibition of intracellular RNA biosynthesis in a dose-response manner strongly suggesting that cell penetration is the main obstacle for effective antimicrobial activity of the antibiotic. Biochemical studies were also conducted with purified enzymes to obtain further insights into the mode of action of GE23077. Interestingly the compound displays a behaviour similar to that of rifampicin an antibiotic structurally unrelated to GE23077 both compounds act at the level of transcription initiation but not on the Ơ subunit and not on the formation of the promoter DNA-RNAP complex. Tests on different rifampicin-resistant E. coli RNAPs did not show any cross-resistance between the two compounds indicating distinct binding sites on the target enzyme. In conclusion .

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