Marcoccia Francesca, Leiros Hanna-Kirsti S, Aschi Massimiliano, Amicosante Gianfranco, Perilli Mariagrazia
Dipartimento di Scienze Cliniche Applicate e Biotecnologiche, Università degli Studi dell'Aquila, L'Aquila, Italy.
The Norwegian Structural Biology Centre (NorStruct), Department of Chemistry, Faculty of Science and Technology, UiT The Arctic University of Norway, Tromsø, Norway.
PLoS One. 2018 Jan 2;13(1):e0189686. doi: 10.1371/journal.pone.0189686. eCollection 2018.
New Delhi Metallo-β-Lactamase (NDM-1) is one of the most recent additions to the β-lactamases family. Since its discovery in 2009, NDM-1 producing Enterobacteriaceae have disseminated globally. With few effective antibiotics against NDM-1 producers, there is an urgent need to design new drug inhibitors through the help of structural and mechanistic information available from mutagenic studies.
RESULTS/CONCLUSIONS: In our study we focus the attention on the non-catalytic residue Leucine 209 by changing it into a Phenylalanine. The L209F laboratory variant of NDM-1 displays a drastic reduction of catalytic efficiency (due to low kcat values) towards penicillins, cephalosporins and carbapenems. Thermofluor-based assay demonstrated that NDM-1 and L209F are stable to the temperature and the zinc content is the same in both enzymes as demonstrated by experiments with PAR in the presence of GdnHCL. Molecular Dynamics (MDs) simulations, carried out on NDM-1 and L209F both complexed and uncomplexed with Benzylpenicillin indicate that the point mutation produces a significant mechanical destabilization of the enzyme and also an increase of water content. These observations clearly show that the single mutation induces drastic changes in the enzyme properties which can be related to the observed different catalytic behavior.
新德里金属β-内酰胺酶（NDM-1）是β-内酰胺酶家族中最新发现的成员之一。自2009年被发现以来，产NDM-1的肠杆菌科细菌已在全球传播。由于针对产NDM-1细菌的有效抗生素很少，迫切需要借助诱变研究获得的结构和机制信息来设计新的药物抑制剂。
结果/结论：在我们的研究中，我们将注意力集中在非催化残基亮氨酸209上，将其替换为苯丙氨酸。NDM-1的L209F实验室变体对青霉素、头孢菌素和碳青霉烯类药物的催化效率大幅降低（由于低kcat值）。基于热荧光的分析表明，NDM-1和L209F对温度稳定，并且在存在盐酸胍的情况下用PAR进行的实验表明，两种酶中的锌含量相同。对与苄青霉素复合和未复合的NDM-1和L209F进行的分子动力学（MDs）模拟表明，点突变导致酶的显著机械不稳定以及含水量增加。这些观察结果清楚地表明，单一突变会引起酶性质的剧烈变化，这可能与观察到的不同催化行为有关。
