The Pentapeptide Repeat Proteins MfpA _Mt and QnrB4 Exhibit Opposite Effects on DNA Gyrase Catalytic Reactions and on the Ternary Gyrase-DNA-Quinolone Complex

Abstract

MfpA Mt and QnrB4 are two newly characterized pentapeptide repeat proteins (PRPs) that interact with DNA gyrase. The mfpA Mt gene is chromosome borne in Mycobacterium tuberculosis, while qnrB4 is plasmid borne in enterobacteria. We expressed and purified the two PRPs and compared their effects on DNA gyrase, taking into account host specificity, i.e., the effect of MfpA Mt on M. tuberculosis gyrase and the effect of QnrB4 on Escherichia coli gyrase. Whereas QnrB4 inhibited E. coli gyrase activity only at concentrations higher than 30 M, MfpA Mt inhibited all catalytic reactions of the M. tuberculosis gyrase described for this enzyme (supercoiling, cleavage, relaxation, and decatenation) with a 50% inhibitory concentration of 2 M. We showed that the D87 residue in GyrA has a major role in the MfpA Mt 6), which results in a right-handed ␤-helical structure (8, 17). The functions of the majority of the members of this large and heterogeneous family remain unknown, but three PRPs, McbG (from Escherichia coli), MfpA Mt (from Mycobacterium tuberculosis), and Qnr (from Klebsiella pneumoniae and other enterobacteria) were reported to interact with DNA gyrase, at least with the E. coli enzyme (17,33,35,44). McbG was shown to protect E. coli DNA gyrase from the toxic action of microcin B17 (33). Qnr and MfpA Mt were involved in resistance to fluoroquinolones, which are synthetic antibacterial agents prescribed worldwide for the treatment of various infectious diseases, including tuberculosis (7).DNA gyrase is an essential ATP-dependent enzyme that transiently cleaves a segment of double-stranded DNA, passes another piece of DNA through the break, and reseals it (12). DNA gyrase is unique in catalyzing the negative supercoiling of DNA in order to facilitate the progression of RNA polymerase. Most eubacteria, such as E. coli, have two type II DNA topoisomerases, i.e., DNA gyrase and topoisomerase IV, but a few, such as M. tuberculosis, harbor only DNA gyrase (11).Quinolones target type II topoisomerases, and their activity is measured by the inhibition of supercoiling by gyrase or decatenation by topoisomerase IV and stabilization of complexes composed of topoisomerase covalently linked to cleaved DNA (16). The DNA gyrase active enzyme is a GyrA 2 GyrB 2 heterotetramer. The quinolone-gyrase interaction site in gyrase is thought to be located at the so-called quinolone resistance-determining regions (QRDR) in the A subunit (amino acids 57 to 196 in GyrA) and the B subunit (amino acids 426 to 466 in GyrB), which contain the majority of mutations conferring quinolone resistance (19). The GyrB QRDR is thought to interact with the GyrA QRDR to form a drug-binding pocket (18). Resistance to quinolones is usually due to chromosomal mutations either in the structural genes encoding type II topoisomerases (QRDR) (19,22) or in regulatory genes producing decreased cell wall permeability or enhancement of efflux pumps (36). The recent emergence of plasmid-borne resistance genes, such as qnr (9,13,31,38,46), aac(6Ј)- 39) and qepA (34...

Plasmid-Mediated Quinolone Resistance: a Multifaceted Threat

“…The effect of purified MfpA Mt and QnrB4 was investigated using various catalytic and noncatalytic type II topoisomerase enzyme assays (125). Histidine-tagged MfpA Mt inhibited the catalytic activity of M. tuberculosis gyrase; IC 50 s for supercoiling, relaxation, and decatenation were 1.75 M, 2 M, and 2 M, respectively, similar to previously reported results (77).…”

“…QnrA-His 6 alone did not itself effect DNA supercoiling (197), nor did it inhibit DNA supercoiling even at a high concentration, unlike MfpA (77). QnrB1-His 6 or QnrB4-His 6 could also protect DNA gyrase in vitro, although at high concentrations, an inhibitory effect was seen (91,125).…”

High prevalence of plasmid-mediated quinolone resistance determinants in Enterobacter cloacae isolated from hospitals of the Qazvin, Alborz, and Tehran provinces, Iran

“…Qnr proteins are members of a pentapeptide repeat protein family, which is capable of protecting DNA gyrase and DNA topoisomerase IV from quinolone compounds (17) . For example, QnrB4 is a characterized pentapeptide repeat protein that interacts with DNA gyrase (18) . Antibiotic treatment against infections caused by qnr-positive isolates is more complicated because of the remarkable ability of these organisms to develop resistance to different antibiotic classes as well as their high potential for transmitting antibiotic resistance between different bacterial species (19) (20) .…”

Structural and Biochemical Analysis of the Pentapeptide Repeat Protein Efs Qnr, a Potent DNA Gyrase Inhibitor

“…No significant inhibition of ATP-independent relaxation was observed at concentrations as high as 100 M. Interestingly, EfsQnr failed to inhibit ATP-dependent relaxation and decatenation catalyzed by E. coli topoisomerase IV. In a recent finding it was reported that MtMfpA inhibited both E. coli and M. tuberculosis gyrases but failed to protect these gyrases from quinolone inhibition, while QnrB4 reversed the quinolone-mediated inhibition of E. coli gyrase but not of M. tuberculosis gyrase, indicating that qnr-mediated inhibition/ protection are species and protein specific (30).…”

“…However, they seem to differ in their abilities to inhibit gyrase. MtMfpA inhibits both supercoiling and relaxation activities of gyrase but does not protect gyrase from quinolone inhibition (12,30). QnrA 1 binds and protects both gyrase and topoisomerase IV from quinolone inhibition but does not inhibit the enzyme activities (45,46).…”