STRUCTURAL STUDIES OF BACTERIAL METALLO-B-LACTAMASES

本子项目是利用由NIH/NCRR资助的中心赠款提供的资源的众多研究子项目之一。子项目和研究者（PI）可能已经从另一个NIH来源获得了主要资金，因此可以在其他CRISP条目中表示。列出的机构是中心的，不一定是研究者的机构。细菌对b-内酰胺类抗生素（青霉素类和头孢菌素类）的耐药性自几年前首次在临床上出现以来，已经以惊人的速度发展。药物的反作用是通过胞外酶水解N-C（=O）内酰胺键来完成的。大约20种已知的b-内酰胺酶需要一个或两个等效的锌（II）来维持活性。其中，大多数在两个等效Zn（II）时达到最大活性。然而，有三种金属-b-内酰胺酶被证明与一种Zn（II）结合时完全活跃，而与另一种Zn（II）结合时则被抑制。几种金属-b-内酰胺酶已经从人类病原体中分离出来，迄今为止还没有临床可行的抑制剂。我们的目的是利用XAS来补充磁共振实验，结合快速冷冻淬火技术，表征稳定静息状态和与金属-b-内酰胺酶催化水解相关的催化中间体。短期目标是确定控制金属-b-内酰胺酶活性的结构特征。我们的长期重点是详细了解由第二金属离子决定的控制反应性的因素。

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Bacterial resistance to the b-lactam family of antibiotics (penicillins and cephalosporins) has progressed at a tremendous rate since it first appeared clinically several years ago. Counteraction of the drugs is accomplished by the expression of extracellular enzymes which hydrolyze the N-C(=O) lactam bond. Approximately 20 of the known b-lactamases require one or two equivalents of Zn(II) for activity. Of these, the majority reach maximal activity with two equivalents of Zn(II). However, three metallo-b-lactamases have been shown to be fully active with one Zn(II) bound, and these are inhibited by binding of a second Zn(II). Several metallo-b-lactamases have been isolated from human pathogens, and to date there is no clinically viable inhibitor available. Our purpose is to use XAS to complement magnetic resonance experiments, in combination with rapid-freeze-quench techniques, to characterize stable resting states and catalytic intermediates relevant to metallo-b-lactamase catalyzed hydrolysis. The short-term goal is to determine the structural features that govern metallo-b-lactamase activity. Our long-term focus is a detailed understanding of the factors controlling reactivity that are determined by the second metal ion.