探讨 L10 环在新德里金属β-内酰胺酶（NDM-1）中的作用：动力学和动态研究。
Exploring the Role of L10 Loop in New Delhi Metallo-β-lactamase (NDM-1): Kinetic and Dynamic Studies.
机构信息
Department of Biotechnological and Applied Clinical Sciences, University of L'Aquila, 67100 L'Aquila, Italy.
Department of Experimental Medicine, Tor Vergata University of Rome, Via Montpellier 1, 00121 Rome, Italy.
出版信息
Molecules. 2021 Sep 9;26(18):5489. doi: 10.3390/molecules26185489.
Four NDM-1 mutants (L218T, L221T, L269H and L221T/Y229W) were generated in order to investigate the role of leucines positioned in L10 loop. A detailed kinetic analysis stated that these amino acid substitutions modified the hydrolytic profile of NDM-1 against some β-lactams. Significant reduction of k values of L218T and L221T for carbapenems, cefazolin, cefoxitin and cefepime was observed. The stability of the NDM-1 and its mutants was explored by thermofluor assay in real-time PCR. The determination of TB and TD demonstrated that NDM-1 and L218T were the most stable enzymes. Molecular dynamic studies were performed to justify the differences observed in the kinetic behavior of the mutants. In particular, L218T fluctuated more than NDM-1 in L10, whereas L221T would seem to cause a drift between residues 75 and 125. L221T/Y229W double mutant exhibited a decrease in the flexibility with respect to L221T, explaining enzyme activity improvement towards some β-lactams. Distances between Zn1-Zn2 and Zn1-OH- or Zn2-OH- remained unaffected in all systems analysed. Significant changes were found between Zn1/Zn2 and first sphere coordination residues.
为了研究位于 L10 环中的亮氨酸的作用，生成了 4 种 NDM-1 突变体（L218T、L221T、L269H 和 L221T/Y229W）。详细的动力学分析表明，这些氨基酸取代修饰了 NDM-1 对某些β-内酰胺类抗生素的水解特性。观察到 L218T 和 L221T 对碳青霉烯类、头孢唑林、头孢西丁和头孢吡肟的 k 值显著降低。通过实时 PCR 的热荧光测定法研究了 NDM-1 及其突变体的稳定性。TB 和 TD 的测定表明，NDM-1 和 L218T 是最稳定的酶。进行了分子动力学研究，以解释突变体在动力学行为上观察到的差异。特别是，L218T 在 L10 中的波动比 NDM-1 更多，而 L221T 似乎会导致残基 75 和 125 之间的漂移。与 L221T 相比，L221T/Y229W 双突变体的灵活性降低，这解释了其对某些β-内酰胺类抗生素的酶活性提高。在所有分析的系统中，Zn1-Zn2 和 Zn1-OH- 或 Zn2-OH- 之间的距离保持不变。Zn1/Zn2 和第一配体配位残基之间发现了显著变化。
Zotero 插件