Instituto de Biología Molecular y Celular de Rosario (IBR, CONICET-UNR), Ocampo y Esmeralda, Rosario, Argentina.
Department of Biochemistry, Case Western Reserve University School of Medicine, Cleveland, Ohio, USA.
Antimicrob Agents Chemother. 2018 Dec 21;63(1). doi: 10.1128/AAC.01754-18. Print 2019 Jan.
Carbapenems are "last resort" β-lactam antibiotics used to treat serious and life-threatening health care-associated infections caused by multidrug-resistant Gram-negative bacteria. Unfortunately, the worldwide spread of genes coding for carbapenemases among these bacteria is threatening these life-saving drugs. Metallo-β-lactamases (MβLs) are the largest family of carbapenemases. These are Zn(II)-dependent hydrolases that are active against almost all β-lactam antibiotics. Their catalytic mechanism and the features driving substrate specificity have been matter of intense debate. The active sites of MβLs are flanked by two loops, one of which, loop L3, was shown to adopt different conformations upon substrate or inhibitor binding, and thus are expected to play a role in substrate recognition. However, the sequence heterogeneity observed in this loop in different MβLs has limited the generalizations about its role. Here, we report the engineering of different loops within the scaffold of the clinically relevant carbapenemase NDM-1. We found that the loop sequence dictates its conformation in the unbound form of the enzyme, eliciting different degrees of active-site exposure. However, these structural changes have a minor impact on the substrate profile. Instead, we report that the loop conformation determines the protonation rate of key reaction intermediates accumulated during the hydrolysis of different β-lactams in all MβLs. This study demonstrates the existence of a direct link between the conformation of this loop and the mechanistic features of the enzyme, bringing to light an unexplored function of active-site loops on MβLs.
碳青霉烯类抗生素是“最后的手段”β-内酰胺类抗生素，用于治疗由多重耐药革兰氏阴性菌引起的严重和危及生命的医疗保健相关感染。不幸的是，这些细菌中编码碳青霉烯酶的基因在全球范围内传播，正在威胁这些救命药物。金属β-内酰胺酶（MβLs）是碳青霉烯酶中最大的家族。这些酶是 Zn(II)依赖性的水解酶，对几乎所有β-内酰胺类抗生素都有活性。它们的催化机制和决定底物特异性的特征一直是激烈争论的话题。MβLs 的活性位点被两个环包围，其中一个环，即环 L3，在底物或抑制剂结合时被证明会采取不同的构象，因此预计在底物识别中发挥作用。然而，不同 MβLs 中观察到的该环的序列异质性限制了对其作用的概括。在这里，我们报告了在临床相关碳青霉烯酶 NDM-1 的支架内对不同环进行的工程改造。我们发现，环序列决定了酶在未结合形式下的构象，引发不同程度的活性位点暴露。然而，这些结构变化对底物谱的影响较小。相反，我们报告说，环构象决定了在所有 MβLs 中水解不同β-内酰胺时积累的关键反应中间体的质子化速率。这项研究证明了该环的构象与酶的机制特征之间存在直接联系，揭示了活性位点环在 MβLs 上的一个未被探索的功能。
