保守的 Asp-Asp 对的突变会损害 CTX-M 类 A 型β-内酰胺酶的结构、功能和抑制作用。
Mutation of the conserved Asp-Asp pair impairs the structure, function, and inhibition of CTX-M Class A β-lactamase.
机构信息
Department of Molecular Medicine, University of South Florida College of Medicine, Tampa, FL, USA.
Department of Pharmaceutical Chemistry, University of California San Francisco, San Francisco, CA, USA.
出版信息
FEBS Lett. 2021 Dec;595(24):2981-2994. doi: 10.1002/1873-3468.14215. Epub 2021 Nov 7.
The Asp233-Asp246 pair is highly conserved in Class A β-lactamases, which hydrolyze β-lactam antibiotics. Here, we characterize its function using CTX-M-14 β-lactamase. The D233N mutant displayed decreased activity that is substrate-dependent, with reductions in k /K ranging from 20% for nitrocefin to 6-fold for cefotaxime. In comparison, the mutation reduced the binding of a known reversible inhibitor by 10-fold. The mutant structures showed movement of the 213-219 loop and the loss of the Thr216-Thr235 hydrogen bond, which was restored by inhibitor binding. Mutagenesis of Thr216 further highlighted its contribution to CTX-M activity. These results demonstrate the importance of the aspartate pair to CTX-M hydrolysis of substrates with bulky side chains, while suggesting increased protein flexibility as a means to evolve drug resistance.
Asp233-Asp246 对在水解β-内酰胺抗生素的 A 类β-内酰胺酶中高度保守。在这里，我们使用 CTX-M-14 β-内酰胺酶来描述其功能。D233N 突变体显示出活性降低，这种活性是底物依赖性的，对于硝噻吩的 k /K 值降低了 20%，对于头孢噻肟则降低了 6 倍。相比之下，该突变降低了已知可逆抑制剂结合的 10 倍。突变体结构显示 213-219 环的运动和 Thr216-Thr235 氢键的丢失，抑制剂结合恢复了氢键。Thr216 的突变进一步突出了它对 CTX-M 对具有大侧链底物水解的贡献。这些结果表明，天冬氨酸对 CTX-M 水解具有大侧链的底物非常重要，同时表明增加蛋白质的灵活性是产生耐药性的一种手段。
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