Shenzhen Key Lab for Food Biological Safety Control, Food Safety and Technology Research Center, Hong Kong PolyU Shen Zhen Research Institute, Shenzhen, People's Republic of China.
State Key Lab of Chirosciences, Department of Applied Biology and Chemical Technology, The Hong Kong Polytechnic University, Hung Hom, Kowloon, Hong Kong.
Antimicrob Agents Chemother. 2017 Nov 22;61(12). doi: 10.1128/AAC.00891-17. Print 2017 Dec.
This work investigated the molecular events driving the evolution of the CTX-M-type β-lactamases by the use of DNA shuffling of fragments of the and genes. Analysis of a total of 51 hybrid enzymes showed that enzymatic activity could be maintained in most cases, yet hybrids that were active possessed fewer amino acid substitutions than those that were inactive, suggesting that point mutations in the constructs rather than reshuffling of the fragments of the two target genes would more likely cause disruption of CTX-M activity. For example, the PL and LP changes in a CTX-M-14 fragment could completely abolish the activity of the enzyme on all antibiotics tested. Structural analysis showed that L was located in the active-site β sheet and might interact with the adjacent hydrophobic residues to stabilize the active-site β sheet and maintain the integrity of the enzyme active site. Likewise, a single amino acid substitution, EK, was found to exhibit a significant suppressive effect on CTX-M-15 activity. Structural analysis showed that E might form a salt bridge with R, disruption of which might affect CTX-M-15 activity. Further analysis of the structure-function relationship of a range of mutant enzymes confirmed that, as can be expected, unstable enzymes lose their activity and avoid selective events. These findings suggest that the distal pockets could also contribute to the activity of the enzymes and may be regarded as alternative targets for inhibitor development.
这项工作通过对 和 基因片段的 DNA 重排来研究推动 CTX-M 型β-内酰胺酶进化的分子事件。对总共 51 种杂交酶的分析表明，在大多数情况下可以保持酶活性，但活性杂交酶的氨基酸取代比非活性杂交酶少，这表明构建体中的点突变而不是两个靶基因片段的重排更可能导致 CTX-M 活性的破坏。例如，CTX-M-14 片段中的 PL 和 LP 变化可以完全使该酶对所有测试的抗生素失去活性。结构分析表明，L 位于活性位点β片层上，可能与相邻的疏水性残基相互作用，稳定活性位点β片层并保持酶活性位点的完整性。同样，发现单个氨基酸取代 EK 对 CTX-M-15 活性具有显著的抑制作用。结构分析表明，E 可能与 R 形成盐桥，其破坏可能影响 CTX-M-15 活性。对一系列突变酶的结构-功能关系的进一步分析证实，可以预期不稳定的酶会失去活性并避免选择事件。这些发现表明，远端口袋也可能对酶的活性有贡献，并且可以被视为抑制剂开发的替代靶标。
