Dipartimento di Scienze Cliniche Applicate e Biotecnologiche, Università degli Studi dell'Aquila, L'Aquila, Italy.
Laboratoire de Macromolécules Biologiques, Centre d'Ingénierie des Protéines, Université de Liège, Liège, Belgium.
Antimicrob Agents Chemother. 2018 Apr 26;62(5). doi: 10.1128/AAC.01851-17. Print 2018 May.
GES-type β-lactamases are a group of enzymes that have evolved their hydrolytic activity against carbapenems. In this study, the role of residue 174 inside the Ω-loop of GES-1 and GES-5 was investigated. GES-1 and GES-5 mutants, selected by site saturation mutagenesis, were purified and kinetically characterized. In comparison with GES-1 and GES-5 wild-type enzymes, GES-1 and GES-5 mutants exhibited lower and / values for cephalosporins and penicillins. Concerning carbapenems, GES-1 shared higher values but lower values than those calculated for GES-1. The GES-1 and GES-5 mutants showed high hydrolytic efficiency for imipenem, with / values 100- and 660-fold higher, respectively, than those of GES-1. Clavulanic acid and tazobactam are good inhibitors for both GES-1 and GES-5 Molecular dynamic (MD) simulations carried out for GES-1, GES-5, GES-1, and GES-5 complexed with imipenem and meropenem have shown that mutation at position 174 induces a drastic increase of enzyme flexibility, in particular in the Ω-loop. The circular dichroism (CD) spectroscopy spectra of the four enzymes indicate that the P174E substitution in GES-1 and GES-5 does not affect the secondary structural content of the enzymes.
GES 型β-内酰胺酶是一组对碳青霉烯类抗生素具有水解活性的酶。本研究探讨了 GES-1 和 GES-5 中 Ω 环内残基 174 的作用。通过定点饱和突变选择 GES-1 和 GES-5 的突变体，对其进行了纯化和动力学特征分析。与 GES-1 和 GES-5 野生型酶相比，GES-1 和 GES-5 突变体对头孢菌素类和青霉素类的 和 / 值较低。对于碳青霉烯类抗生素，GES-1 的 值较高，但 / 值低于 GES-1 的计算值。GES-1 和 GES-5 突变体对亚胺培南具有较高的水解效率， / 值分别比 GES-1 高 100 倍和 660 倍。克拉维酸和他唑巴坦是 GES-1 和 GES-5 的良好抑制剂。对 GES-1、GES-5、GES-1 和 GES-5 与亚胺培南和美罗培南复合物进行的分子动力学（MD）模拟表明，174 位的突变导致酶的灵活性显著增加，特别是在 Ω 环中。四种酶的圆二色（CD）光谱表明，GES-1 和 GES-5 中的 P174E 取代不影响酶的二级结构含量。
