The antibiotic microcin B17 is a DNA gyrase poison: characterisation of the mode of inhibition11Edited by J. Karn

Heddle, Jonathan G.; Blance, Stephen J; Zamble, Deborah B.; Hollfelder, Florian; Miller, Deborah A.; Wentzell, Lois M.; Walsh, Christopher T.; Maxwell, Anthony

doi:10.1006/jmbi.2001.4562

Cited by 114 publications

(121 citation statements)

References 45 publications

(32 reference statements)

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“…In comparison, experiments also were performed with ciprofloxacin, which was much more potent than any of the MccB17 analogs, with faster reaction times and a higher amount of cleavage at saturation. This result is consistent with our previously reported experiments (17).…”

Section: Inhibition Of Gyrase By Mccb17supporting

confidence: 94%

“…The treatment of gyrase-DNA reactions with MccB17 followed by SDS and proteinase K previously revealed gyrase-dependent accumulation of linear DNA, attributed to trapping of the cleavable complex by the antibiotic during supercoiling of closed-circular DNA (17). However, under the previous experimental conditions used, the initial rate of formation of this intermediate was too fast to measure with the standard in vitro cleavage assay.…”

Section: Steady-state Parameters For the Formation Of The Cleavable Cmentioning

confidence: 99%

“…The solubility of the analogs provided a constraint on the concentrations that could be used in the reactions and prohibited the determination of rate constants for the least potent compounds B-GCG and B-GGG. Furthermore, this assay is limited by the minimum amount of (17). One possible explanation for this result is that MccB17 acts inefficiently and a number of enzyme turnovers occur before the antibiotic stabilizes the cleaved complex, allowing the DNA to become supercoiled.…”

Section: Inhibition Of Gyrase By Mccb17mentioning

confidence: 99%

“…As an alternative method of slowing down the gyrase reaction (Fig. 5B), the reactions were performed at 37°C but the amount of enzyme used was decreased and the amount of DNA was increased from the standard reaction conditions previously reported (17). Under these conditions, MccB17 causes premature cessation of the enzyme activity, producing a difference in the endpoint levels of supercoiled DNA.…”

Section: Inhibition Of Gyrase By Mccb17mentioning

confidence: 99%

“…The ternary complex blocks passage of the replication fork (14,15), which may initiate the bactericidal activity of the quinolones, although the exact irreversible lethal event has not been firmly established (16). Recent in vitro analysis of the interaction between gyrase and MccB17 revealed that this antibiotic also causes accumulation of the cleavable complex in a manner similar to that of the quinolone drugs (17).…”

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In vitro characterization of DNA gyrase inhibition by microcin B17 analogs with altered bisheterocyclic sites

Zamble

Miller

Heddle

et al. 2001

Proc. Natl. Acad. Sci. U.S.A.

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M icrocins are a class of small antibiotics (Ͻ10 kDa) produced and excreted by some strains of Enterobacteriaceae at the stationary phase of growth (1). Microcin B17 (MccB17) is a ribosomally synthesized compound that is produced in Escherichia coli harboring the plasmid-borne mcb operon, which contains the genes necessary for antibiotic synthesis, dedicated export, and immunity (2). The production of mature MccB17 ( Fig. 1) involves posttranslational modification of a 69-residue polypeptide-precursor to generate two thiazoles, two oxazoles, and two 4,2-fused bisheterocycles (3, 4), followed by cleavage of the 26-residue leader sequence by an unidentified protease (5, 6). Exposure of sensitive bacteria to MccB17 results in inhibition of DNA synthesis, induction of the SOS response in cells with active replication, mutagenic effects, and DNA degradation (7). These observations suggest that MccB17 is either a DNAdamaging agent or targets a key DNA-processing pathway such as replication.Genetic experiments designed to identify the target of MccB17 resulted in the isolation and characterization of a mutation in the B subunit of DNA gyrase (8). Strains carrying this mutation, a Trp-Arg substitution at position 751, exhibited increased resistance to MccB17 and a decrease in MccB17-induced DNA cleavage. Gyrase is a prokaryotic type II topoisomerase that introduces negative supercoils into DNA (9, 10). During the reaction, the enzyme passes through a transient intermediate in which two tyrosine hydroxyl groups form phosphodiester bonds with the exposed 5Ј phosphates of doublestrand cleaved DNA. This intermediate allows the enzyme to pull the ends of the DNA break apart to form a gate through which another DNA segment is passed. The quinolone family of drugs traps this covalent intermediate, known as the cleavable complex, and the accumulation of this complex is monitored in vitro by treatment with SDS and proteinase K, which releases linear DNA (11-13). The ternary complex blocks passage of the replication fork (14, 15), which may initiate the bactericidal activity of the quinolones, although the exact irreversible lethal event has not been firmly established (16). Recent in vitro analysis of the interaction between gyrase and MccB17 revealed that this antibiotic also causes accumulation of the cleavable complex in a manner similar to that of the quinolone drugs (17).Gyrase is the target of several classes of clinically successful drugs, including the quinolones and the coumarins (11, 13); however, the emergence of resistant strains is an incentive to elucidate the mechanism of action of existing drugs and develop new candidates. The identification of gyrase as a target of MccB17 provides an opportunity to analyze the structurefunction relationship of this unusual antibiotic. The experiments described here investigate the effects of modifying MccB17 at the sites of the tandem heterocycles, labeled A and B (Fig. 1). The interaction of MccB17 analogs with DNA gyrase was evaluated by measuring the rate of formation of the ...

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Section: Inhibition Of Gyrase By Mccb17supporting

confidence: 94%

Section: Steady-state Parameters For the Formation Of The Cleavable Cmentioning

confidence: 99%

Section: Inhibition Of Gyrase By Mccb17mentioning

confidence: 99%

Section: Inhibition Of Gyrase By Mccb17mentioning

confidence: 99%

mentioning

confidence: 99%

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In vitro characterization of DNA gyrase inhibition by microcin B17 analogs with altered bisheterocyclic sites

Zamble

Miller

Heddle

et al. 2001

Proc. Natl. Acad. Sci. U.S.A.

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Self‐immunity to antibacterial peptides by ABC transporters

2020

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Bacteria produce under certain stress conditions bacteriocins and microcins that display antibacterial activity against closely related species for survival. Bacteriocins and microcins exert their antibacterial activity by either disrupting the membrane or inhibiting essential intracellular processes of the bacterial target. To this end, they can lyse bacterial membranes and cause subsequent loss of their integrity or nutrients, or hijack membrane receptors for internalisation. Both bacteriocins and microcins are ribosomally synthesised and several are posttranslationally modified, whereas others are not. Such peptides are also toxic to the producer bacteria, which utilise immunity proteins or/and dedicated ATP-binding cassette (ABC) transporters to achieve self-immunity and peptide export. In this review, we discuss the structure and mechanism of self-protection that is conferred by these ABC transporters.

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How Nature Morphs Peptide Scaffolds into Antibiotics

Nolan

Walsh

2008

ChemBioChem

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The conventional notion that peptides are poor candidates for orally available drugs because of protease-sensitive peptide bonds, intrinsic hydrophilicity, and ionic charges contrasts with the diversity of antibiotic natural products with peptide-based frameworks that are synthesized and utilized by Nature. Several of these antibiotics, including penicillin and vancomycin, are employed to treat bacterial infections in humans and have been best-selling therapeutics for decades. Others might provide new platforms for the design of novel therapeutics to combat emerging antibioticresistant bacterial pathogens.

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The antibiotic microcin B17 is a DNA gyrase poison: characterisation of the mode of inhibition11Edited by J. Karn

Cited by 114 publications

References 45 publications

In vitro characterization of DNA gyrase inhibition by microcin B17 analogs with altered bisheterocyclic sites

In vitro characterization of DNA gyrase inhibition by microcin B17 analogs with altered bisheterocyclic sites

Self‐immunity to antibacterial peptides by ABC transporters

How Nature Morphs Peptide Scaffolds into Antibiotics

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