The amino‐acid sequence of the Toho‐1 β‐lactamase contains several conserved residues in the active site, including Ser70, Lys73, Ser130 and Glu166, some of which coordinate a catalytic water molecule. This catalytic water molecule is essential in the acylation and deacylation parts of the reaction mechanism through which Toho‐1 inactivates specific antibiotics and provides resistance to its expressing bacterial strains. To investigate the function of Glu166 in the acylation part of the catalytic mechanism, neutron and X‐ray crystallographic studies were performed on a Glu166Gln mutant. The structure of this class A β‐lactamase mutant provides several insights into its previously reported reduced drug‐binding kinetic rates. A joint refinement of both X‐ray and neutron diffraction data was used to study the effects of the Glu166Gln mutation on the active site of Toho‐1. This structure reveals that while the Glu166Gln mutation has a somewhat limited impact on the positions of the conserved amino acids within the active site, it displaces the catalytic water molecule from the active site. These subtle changes offer a structural explanation for the previously observed decreases in the binding of non‐β‐lactam inhibitors such as the recently developed diazobicyclooctane inhibitor avibactam.

中文翻译：

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使用中子和 X 射线蛋白质晶体学探讨 A 类 β-内酰胺酶中保守残基 Glu166 的作用。


Toho-1 β-内酰胺酶的氨基酸序列在活性位点包含多个保守残基，包括 Ser70、Lys73、Ser130 和 Glu166，其中一些残基与催化水分子配位。这种催化水分子在反应机制的酰化和脱酰化部分中至关重要，Toho-1 通过该机制使特定抗生素失活并为其表达的细菌菌株提供抗性。为了研究 Glu166 在催化机制酰化部分中的功能，对 Glu166Gln 突变体进行了中子和 X 射线晶体学研究。这种 A 类 β-内酰胺酶突变体的结构为其先前报道的药物结合动力学速率降低提供了一些见解。 X 射线和中子衍射数据的联合细化用于研究 Glu166Gln 突变对 Toho-1 活性位点的影响。该结构表明，虽然 Glu166Gln 突变对活性位点内保守氨基酸的位置影响有限，但它从活性位点取代了催化水分子。这些微妙的变化为之前观察到的非β-内酰胺抑制剂（例如最近开发的重氮双环辛烷抑制剂阿维巴坦）的结合减少提供了结构解释。