Shaping Substrate Selectivity in a Broad-Spectrum Metallo-β-Lactamase.
Shaping Substrate Selectivity in a Broad-Spectrum Metallo-β-Lactamase.
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塑造广谱金属-β-内酰胺酶的底物选择性。
DOI：
10.1128/aac.02079-17
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发表时间：
2018
影响因子：
4.9
通讯作者：
Vila,AlejandroJ
中科院分区：
文献类型：
--
作者：
González,LisandroJ;Stival,Cintia;Puzzolo,JuanL;Moreno,DiegoM;Vila,AlejandroJ
金属-β-内酰胺酶（MBL）是由细菌病原体产生的主要的碳青霉烯酶。MBL抑制剂的设计受到限制，除其他问题外，关于这些酶如何调节底物识别的知识不完整。虽然大多数MBL是广谱酶，但B2 MBL是唯一的碳青霉烯酶。这种较窄的底物分布归因于B2酶中存在的限制活性位点可及性的序列插入。在这项工作中，我们评估的作用，序列插入自然发生在B2酶Sfh-I和广谱B1酶SPM-1。我们设计了一种嵌合蛋白，其中SPM-1的序列插入被Sfh-I中存在的序列取代。嵌合变体是选择性头孢菌素酶，揭示了MBL的底物谱可以根据蛋白质背景进一步调整。这些结果还表明，MBL的稳定支架允许比在自然界中观察到的更丰富的模块化工程。
Metallo-β-lactamases (MBLs) are the major group of carbapenemases produced by bacterial pathogens. The design of MBL inhibitors has been limited by, among other issues, incomplete knowledge about how these enzymes modulate substrate recognition. While most MBLs are broad-spectrum enzymes, B2 MBLs are exclusive carbapenemases. This narrower substrate profile has been attributed to a sequence insertion present in B2 enzymes that limits accessibility to the active site. In this work, we evaluate the role of sequence insertions naturally occurring in the B2 enzyme Sfh-I and in the broad-spectrum B1 enzyme SPM-1. We engineered a chimeric protein in which the sequence insertion of SPM-1 was replaced by the one present in Sfh-I. The chimeric variant is a selective cephalosporinase, revealing that the substrate profile of MBLs can be further tuned depending on the protein context. These results also show that the stable scaffold of MBLs allows a modular engineering much richer than the one observed in nature.