Latallo M J, Cortina G A, Faham S, Nakamoto R K, Kasson P M
Department of Molecular Physiology , University of Virginia , Box 800886 , Charlottesville , VA 22908 , USA . Email:
Department of Biomedical Engineering , University of Virginia , USA.
Chem Sci. 2017 Sep 1;8(9):6484-6492. doi: 10.1039/c7sc02676e. Epub 2017 Jul 19.
The CTX-M family of beta lactamases mediate broad-spectrum antibiotic resistance and are present in the majority of drug-resistant Gram-negative bacterial infections worldwide. Allosteric mutations that increase catalytic rates of these drug resistance enzymes have been identified in clinical isolates but are challenging to predict prospectively. We have used molecular dynamics simulations to predict allosteric mutants increasing CTX-M9 drug resistance, experimentally testing top mutants using multiple antibiotics. Purified enzymes show an increase in catalytic rate and efficiency, while mutant crystal structures show no detectable changes from wild-type CTX-M9. We hypothesize that increased drug resistance results from changes in the conformational ensemble of an acyl intermediate in hydrolysis. Machine-learning analyses on the three top mutants identify changes to the binding-pocket conformational ensemble by which these allosteric mutations transmit their effect. These findings show how molecular simulation can predict how allosteric mutations alter active-site conformational equilibria to increase catalytic rates and thus resistance against common clinically used antibiotics.
CTX-M 家族的β-内酰胺酶介导广谱抗生素耐药性，在全球大多数耐药革兰氏阴性菌感染中都有发现。已在临床分离株中鉴定出增加这些耐药酶催化速率的变构突变，但前瞻性预测具有挑战性。我们使用分子动力学模拟来预测增加 CTX-M9 耐药性的变构突变体，并使用多种抗生素对顶级突变体进行实验测试。纯化的酶显示催化速率和效率增加，而突变体晶体结构与野生型 CTX-M9 相比没有可检测到的变化。我们假设耐药性增加是由于水解过程中酰基中间体构象集合的变化所致。对三个顶级突变体的机器学习分析确定了结合口袋构象集合的变化，这些变构突变通过这些变化传递其效应。这些发现表明分子模拟如何能够预测变构突变如何改变活性位点的构象平衡以提高催化速率，从而增强对常见临床使用抗生素的耐药性。
