10 research outputs found
Kinetic characterization of GES-22 beta-lactamase harboring the M169L clinical mutation
The class A p-lactamase GES-22 has been identified in Acinetobacter baumannii isolates in Turkey, and subsequently shown to differ from GES-11 by a single substitution (M169L). Because M169 is part of the omega loop, a structure that is known to have major effects on substrate selectivity in class A beta-lactamases, we expressed, purified and kinetically characterized this novel variant. Our results show that compared with GES-11(6xHis), GES-22(6xHis) displays more efficient hydrolysis of penicillins, and aztreonam, but a loss of efficiency against ceftazidime. In addition, the M169L substitution confers on GES-22 more efficient hydrolysis of the mechanistic inhibitors clavulanic acid and sulbactam. These effects are highly similar to other mutations at the homologous position in other class A beta-lactamases, suggesting that this methionine has a key structural role in aligning active site residues and in substrate selectivity across the class.Recep Tayyip Erdogan University:BAP-2013.102.03.12
Turkiye Bilimsel ve Teknolojik Arastirma Kurumu (TUBITAK): TUBITAK-113Z054
United States Department of Health & Human Services
National Institutes of Health (NIH) - USA
1R15AI082416
Turkiye Bilimsel ve Teknolojik Arastirma Kurumu (TUBITAK)
2214-
Antimicrobial resistance (AMR) nanomachines: mechanisms for fluoroquinolone and glycopeptide recognition, efflux and/or deactivation
In this review, we discuss mechanisms of resistance identified in bacterial agents Staphylococcus aureus and the enterococci towards two priority classes of antibiotics—the fluoroquinolones and the glycopeptides. Members of both classes interact with a number of components in the cells of these bacteria, so the cellular targets are also considered. Fluoroquinolone resistance mechanisms include efflux pumps (MepA, NorA, NorB, NorC, MdeA, LmrS or SdrM in S. aureus and EfmA or EfrAB in the enterococci) for removal of fluoroquinolone from the intracellular environment of bacterial cells and/or protection of the gyrase and topoisomerase IV target sites in Enterococcus faecalis by Qnr-like proteins. Expression of efflux systems is regulated by GntR-like (S. aureus NorG), MarR-like (MgrA, MepR) regulators or a two-component signal transduction system (TCS) (S. aureus ArlSR). Resistance to the glycopeptide antibiotic teicoplanin occurs via efflux regulated by the TcaR regulator in S. aureus. Resistance to vancomycin occurs through modification of the D-Ala-D-Ala target in the cell wall peptidoglycan and removal of high affinity precursors, or by target protection via cell wall thickening. Of the six Van resistance types (VanA-E, VanG), the VanA resistance type is considered in this review, including its regulation by the VanSR TCS. We describe the recent application of biophysical approaches such as the hydrodynamic technique of analytical ultracentrifugation and circular dichroism spectroscopy to identify the possible molecular effector of the VanS receptor that activates expression of the Van resistance genes; both approaches demonstrated that vancomycin interacts with VanS, suggesting that vancomycin itself (or vancomycin with an accessory factor) may be an effector of vancomycin resistance. With 16 and 19 proteins or protein complexes involved in fluoroquinolone and glycopeptide resistances, respectively, and the complexities of bacterial sensing mechanisms that trigger and regulate a wide variety of possible resistance mechanisms, we propose that these antimicrobial resistance mechanisms might be considered complex ‘nanomachines’ that drive survival of bacterial cells in antibiotic environments
Genome-wide association mapping of partial resistance to Phytophthora sojae in soybean plant introductions from the Republic of Korea
Acyl-intermediate Structures of an Extended Spectrum Clinically-Derived Class D β-lactamase Variant, OXA-160, in Complex with Cefotaxime, Ceftazidime, and Aztreonam
OXA-24 is a carbapenem-hydrolyzing class D β-lactamase (CHDL) that poses a serious medical threat by destroying carbapenem class antibiotics. OXA-160 is a clinically-derived OXA-24 variant with a Pro→Ser substitution. Previously, it was shown that OXA-160 has higher catalytic activity against third-generation cephalosporins compared to OXA-24 and is able to maintain normal activity against penicillins and carbapenems. To slow deacylation, we introduced a second mutation (Val130Asp) to allow us to capture a drug-complex structure. We examined the OXA-160/Val130Asp variant in complex with the substrates cefotaxime, ceftazidime, and aztreonam using X-ray crystallography. Our analysis shows that all three of these bulky antibiotics require β5-β6 and/or omega loop deviations, and we propose that these conformational changes are made possible by replacing the restricted proline with the more flexible serine. These crystallographic structures reveal that a Pro227Ser mutation enlarges the active site, better accommodating advanced cephalosporin drugs
Structural Basis of Activity against Aztreonam and Extended Spectrum Cephalosporins for Two Carbapenem-Hydrolyzing Class D β‑Lactamases from <i>Acinetobacter baumannii</i>
The carbapenem-hydrolyzing class
D β-lactamases OXA-23 and
OXA-24/40 have emerged worldwide as causative agents for β-lactam
antibiotic resistance in <i>Acinetobacter</i> species. Many
variants of these enzymes have appeared clinically, including OXA-160
and OXA-225, which both contain a P → S substitution at homologous
positions in the OXA-24/40 and OXA-23 backgrounds, respectively. We
purified OXA-160 and OXA-225 and used steady-state kinetic analysis
to compare the substrate profiles of these variants to their parental
enzymes, OXA-24/40 and OXA-23. OXA-160 and OXA-225 possess greatly
enhanced hydrolytic activities against aztreonam, ceftazidime, cefotaxime,
and ceftriaxone when compared to OXA-24/40 and OXA-23. These enhanced
activities are the result of much lower <i>K</i><sub>m</sub> values, suggesting that the P → S substitution enhances the
binding affinity of these drugs. We have determined the structures
of the acylated forms of OXA-160 (with ceftazidime and aztreonam)
and OXA-225 (ceftazidime). These structures show that the R1 oxyimino
side-chain of these drugs occupies a space near the β5-β6
loop and the omega loop of the enzymes. The P → S substitution
found in OXA-160 and OXA-225 results in a deviation of the β5-β6
loop, relieving the steric clash with the R1 side-chain carboxypropyl
group of aztreonam and ceftazidime. These results reveal worrying
trends in the enhancement of substrate spectrum of class D β-lactamases
but may also provide a map for β-lactam improvement