Department of Chemistry and Nano Science, Ewha Womans University, Seoul 03760, Republic of Korea.
Acta Crystallogr D Struct Biol. 2016 Aug;72(Pt 8):976-85. doi: 10.1107/S2059798316011311. Epub 2016 Jul 28.
AmpC BER is an extended substrate spectrum class C β-lactamase with a two-amino-acid insertion in the R2 loop compared with AmpC EC2. The crystal structures of AmpC BER (S64A mutant) and AmpC EC2 were determined. Structural comparison of the two proteins revealed that the insertion increases the conformational flexibility of the R2 loop. Two citrate molecules originating from the crystallization solution were observed in the active site of the S64A mutant. One citrate molecule makes extensive interactions with active-site residues that are highly conserved among class C β-lactamases, whereas the other one is weakly bound. Based on this structural observation, it is demonstrated that citrate, a primary metabolite that is widely used as a food additive, is a competitive inhibitor of two class C β-lactamases (AmpC BER and CMY-10). Consequently, the data indicate enhancement of the flexibility of the R2 loop as an operative strategy for molecular evolution of extended-spectrum class C β-lactamases, and also suggest that the citrate scaffold is recognized by the active sites of class C β-lactamases.
AmpC BER 是一种扩展的底物谱类 Cβ-内酰胺酶，与 AmpC EC2 相比，其 R2 环中有两个氨基酸插入。测定了 AmpC BER（S64A 突变体）和 AmpC EC2 的晶体结构。对这两种蛋白质的结构比较表明，插入增加了 R2 环的构象灵活性。在 S64A 突变体的活性部位观察到两个来自结晶溶液的柠檬酸分子。一个柠檬酸分子与活性位点残基广泛相互作用，这些残基在类 Cβ-内酰胺酶中高度保守，而另一个柠檬酸分子则与活性位点残基弱结合。基于这一结构观察，证明柠檬酸作为食品添加剂广泛使用的主要代谢物是两种类 Cβ-内酰胺酶（AmpC BER 和 CMY-10）的竞争性抑制剂。因此，数据表明 R2 环的灵活性增强是扩展谱类 Cβ-内酰胺酶分子进化的有效策略，并且还表明柠檬酸支架被类 Cβ-内酰胺酶的活性位点识别。
