Emergence of Enterobacteriaceae harboring metallo-β-lactamases (MBL) has raised global threats due to their broad antibiotic resistance profiles and the lack of effective inhibitors against them. We have been studied one of the emerging environmental MBL, the L1 from Stenotrophomonas maltophilia K279a. We determined several crystal structures of L1 complexes with three different classes of β-lactam antibiotics (penicillin G, moxalactam, meropenem, and imipenem), with the inhibitor captopril and different metal ions (Zn+2 , Cd+2 , and Cu+2 ). All hydrolyzed antibiotics and the inhibitor were found binding to two Zn+2 ions mainly through the opened lactam ring and some hydrophobic interactions with the binding pocket atoms. Without a metal ion, the active site is very similarly maintained as that of the native form with two Zn+2 ions, however, the protein does not bind the substrate moxalactam. When two Zn+2 ions were replaced with other metal ions, the same di-metal scaffold was maintained and the added moxalactam was found hydrolyzed in the active site. Differential scanning fluorimetry and isothermal titration calorimetry were used to study thermodynamic properties of L1 MBL compared with New Deli Metallo-β-lactamase-1 (NDM-1). Both enzymes are significantly stabilized by Zn+2 and other divalent metals but showed different dependency. These studies also suggest that moxalactam and its hydrolyzed form may bind and dissociate with different kinetic modes with or without Zn+2 for each of L1 and NDM-1. Our analysis implicates metal ions, in forming a distinct di-metal scaffold, which is central to the enzyme stability, promiscuous substrate binding and versatile catalytic activity. STATEMENT: The L1 metallo-β-lactamase from an environmental multidrug-resistant opportunistic pathogen Stenotrophomonas maltophilia K279a has been studied by determining 3D structures of L1 enzyme in the complexes with several β-lactam antibiotics and different divalent metals and characterizing its biochemical and ligand binding properties. We found that the two-metal center in the active site is critical in the enzymatic process including antibiotics recognition and binding, which explains the enzyme's activity toward diverse antibiotic substrates. This study provides the critical information for understanding the ligand recognition and for advanced drug development.

中文翻译：

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对来自新兴病原体嗜麦芽单胞菌的嗜麦芽嗜单胞菌的金属-β-内酰胺酶L1的结构和生化分析显示，其催化活性微妙而独特。

带有金属-β-内酰胺酶（MBL）的肠杆菌科细菌的出现，由于其广泛的抗生素耐药性和缺乏有效的抑制剂而引起了全球性威胁。我们已经研究了一种新兴的环境MBL，即嗜麦芽窄食单胞菌K279a的L1。我们确定了具有三种不同类型的β-内酰胺类抗生素（青霉素G，莫拉西坦，美洛培南和亚胺培南）的L1配合物的几种晶体结构，其中所述抑制剂具有卡托普利和不同的金属离子（Zn + 2，Cd + 2和Cu + 2） ）。发现所有水解的抗生素和抑制剂主要通过打开的内酰胺环以及与结合口袋原子的一些疏水相互作用与两个Zn + 2离子结合。没有金属离子，其活性位点与具有两个Zn + 2离子的天然形式的活性位点非常相似，但是，该蛋白质不结合底物莫西内酰胺。当将两个Zn + 2离子替换为其他金属离子时，可以保持相同的双金属支架，并发现添加的莫西内酰胺在活性位点水解。与New DeliMetallo-β-lactamase-1（NDM-1）相比，差示扫描荧光法和等温滴定热法研究了L1 MBL的热力学性质。两种酶均被Zn + 2和其他二价金属显着稳定，但显示出不同的依赖性。这些研究还表明，对于L1和NDM-1，Moxalactam及其水解形式可能会以不同的动力学模式结合和解离，无论是否添加Zn + 2。我们的分析牵涉到金属离子形成一个独特的双金属支架，这对酶的稳定性，混杂的底物结合和广泛的催化活性至关重要。陈述：通过测定几种β-内酰胺抗生素和不同二价金属的配合物中L1酶的3D结构并表征其生化和配体结合，研究了来自环境多药耐药性病原体嗜麦芽窄食单胞菌K279a的L1金属β-内酰胺酶特性。我们发现活性位点中的双金属中心在酶过程中至关重要，包括抗生素的识别和结合，这解释了酶对多种抗生素底物的活性。这项研究为理解配体识别和高级药物开发提供了关键信息。通过测定几种β-内酰胺抗生素和不同二价金属的配合物中L1酶的3D结构并表征其生化和配体结合特性，研究了一种来自环境多药耐药性致病性嗜麦芽单胞菌K279a的L1金属-β-内酰胺酶。我们发现活性位点中的双金属中心在酶过程中至关重要，包括抗生素的识别和结合，这解释了酶对多种抗生素底物的活性。这项研究为理解配体识别和高级药物开发提供了关键信息。通过测定几种β-内酰胺抗生素和不同二价金属的配合物中L1酶的3D结构并表征其生化和配体结合特性，研究了来自环境多药耐药性致病性嗜麦芽单胞菌K279a的L1金属-β-内酰胺酶。我们发现活性位点中的双金属中心在酶过程中至关重要，包括抗生素的识别和结合，这解释了酶对多种抗生素底物的活性。这项研究为理解配体识别和高级药物开发提供了关键信息。我们发现活性位点中的双金属中心在酶过程中至关重要，包括抗生素的识别和结合，这解释了酶对多种抗生素底物的活性。这项研究为理解配体识别和高级药物开发提供了关键信息。我们发现活性位点中的双金属中心在酶过程中至关重要，包括抗生素的识别和结合，这解释了酶对多种抗生素底物的活性。这项研究为理解配体识别和高级药物开发提供了关键信息。