Pentapeptide repeat protein QnrB1 requires ATP hydrolysis to rejuvenate poisoned gyrase complexes.

Łukasz Mazurek,Jonathan G Heddle,Iraj Behroz,Zuzanna Pakosz,Dmitry Ghilarov,Karolina Wawro,Wojciech Czyszczoń,Elizabeth Michalczyk,Mikhail Metelev,Svetlana Dubiley,Roderich D Süssmuth

doi:10.1093/nar/gkaa1266

Abstract

DNA gyrase, a type II topoisomerase found predominantly in bacteria, is the target for a variety of ‘poisons’, namely natural product toxins (e.g. albicidin, microcin B17) and clinically important synthetic molecules (e.g. fluoroquinolones). Resistance to both groups can be mediated by pentapeptide repeat proteins (PRPs). Despite long-term studies, the mechanism of action of these protective PRPs is not known. We show that a PRP, QnrB1 provides specific protection against fluoroquinolones, which strictly requires ATP hydrolysis by gyrase. QnrB1 binds to the GyrB protein and stimulates ATPase activity of the isolated N-terminal ATPase domain of GyrB (GyrB43). We probed the QnrB1 binding site using site-specific incorporation of a photoreactive amino acid and mapped the crosslinks to the GyrB43 protein. We propose a model in which QnrB1 binding allosterically promotes dissociation of the fluoroquinolone molecule from the cleavage complex.

Highlights

DNA gyrase is an essential enzyme responsible for the maintenance of the bacterial chromosome in a negatively supercoiled state and for removal of torsion accumulated in front of DNA and RNA polymerases [1,2]
The MICs for microcin B17 (MccB17) were increased 37-fold for the McbG producing strain, whereas AlbG and QnrB1 induction resulted in 6-fold and 3-fold increase of MIC, respectively. These results clearly indicated that all three pentapeptide repeat proteins (PRPs) provide specific protection against their cognate toxins
Apart from supercoiling, DNA gyrase is capable of ATPindependent relaxation of negatively supercoiled DNA, where strand passage is believed to proceed in the reverse direction

Summary

Introduction

DNA gyrase is an essential enzyme responsible for the maintenance of the bacterial chromosome in a negatively supercoiled state and for removal of torsion accumulated in front of DNA and RNA polymerases [1,2]. Gyrase functions as a heterotetramer consisting of two GyrA and two GyrB subunits, with three interfaces (‘gates’) between them (Figure 1A). The supercoiling mechanism of gyrase has been extensively studied and is understood in some detail [1,3,4] (Figure 1A).

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Results

Conclusion