Analysis of metallo-beta-lactamase sequence constraints at high resolution

描述（由申请人提供）：高分辨率金属内酰胺酶序列约束分析-内酰胺类抗生素是最常用的抗菌剂，对这些药物的耐药性日益增加是一个重要的公共卫生问题。细菌耐药最常见的机制是内酰胺酶催化这些药物的水解。锌金属内酰胺酶（MBLs）催化水解包括碳青霉烯类在内的多种内酰胺类抗生素，并已成为耐药的重要来源。含锌酶可进一步细分为B1、B2和B3亚类，其中B1 -内酰胺酶在细菌中分布最广。在B1亚群中，IMP-1、VIM-2和NDM-1酶是与抗生素耐药性最相关的临床来源。为了了解突变如何影响其功能和进化以及设计MBLs抑制剂，有必要了解氨基酸序列如何决定这些酶的结构和功能。这个问题将使用密码子随机化和选择策略，然后进行深度测序。IMP-1、VIM-2、NDM-1 MBLs以及B2亚类CphA酶中的单个密码子将被随机化，以创建包含所有可能取代残基位置的文库。每个位置对酶结构和功能的重要性将通过从每个文库中选择功能克隆来确定，这些克隆基于赋予大肠杆菌对感兴趣的-内酰胺抗生素的抗性的能力。功能突变体的超高通量DNA测序将用于提供每个残基位置的氨基酸取代范围的高分辨率信息，这些信息与用于选择的抗生素耐药性一致，从而确定酶功能的序列要求。实验将对几种内酰胺类抗生素进行，并比较每种药物的序列要求，以确定所有内酰胺类抗生素水解需要野生型氨基酸的残基位置，以及根据用于选择的抗生素表现出改变序列要求的位置，即控制底物特异性的残基。由于对每个活性位点位置进行了这些比较，MBL的催化决定因素和底物特异性的高分辨率图像将出现。这将允许准确估计任何活性位点残基上的任何氨基酸取代对MBL提供的底物特异性和抗生素耐药性谱的影响。深度测序产生的信息将通过对一些特异性改变的酶进行酶动力学和x射线晶体学来扩展，以提供不仅是哪些残基位置控制特异性，而且是如何在分子水平上控制特异性的见解。对活性位点残基位置如何促进¿-内酰胺水解的详细了解将有助于设计与至关重要的MBL残基相互作用的抑制剂。

DESCRIPTION (provided by applicant): Analysis of Metallo-¿-lactamase Sequence Constraints at High Resolution ¿-lactam antibiotics are the most often used antibacterial agents and an increasing incidence of resistance to these drugs is a critical public health concern. The most common mechanism of bacterial resistance is ¿-lactamase-catalyzed hydrolysis of these drugs. The zinc metallo-¿-lactamases (MBLs) catalyze the hydrolysis of a wide range of ¿-lactam antibiotics including carbapenems and have emerged as an important source of resistance. The zinc-containing enzymes can be further subdivided into subclasses B1, B2 and B3 with subclass B1 ¿-lactamases being the most widespread among bacteria. Within subgroup B1, the IMP-1, VIM-2 and NDM-1 enzymes are the most clinically relevant as sources of antibiotic resistance. In order to understand how mutations influence their function and evolution and to design inhibitors of MBLs, it is necessary to understand how the amino acid sequence determines the structure and function of these enzymes. This question will be addressed using a strategy of codon randomization and selection followed by deep sequencing. Individual codons in the IMP-1, VIM-2, NDM-1 MBLs as well as the CphA enzyme from subclass B2 will be randomized to create libraries containing all possible substitutions for the residue position. The importance of each position for enzyme structure and function will be determined by selecting functional clones from each library based on their ability to confer Escherichia coli resistance to a ¿-lactam antibiotic of interest. Ultra-high throughput DNA sequencing of functional mutants will be used to provide high resolution information on the range of amino acid substitutions at each residue position that are consistent with resistance to the antibiotic used for selection and thereby determine the sequence requirements for enzyme function. The experiment will be performed for several ¿-lactam antibiotics and the sequence requirements for each drug will be compared to identify residue positions where the wild type amino acid is required for hydrolysis of all ¿-lactams as well as positions that exhibit altered sequence requirements depending on the antibiotic used for selection, i.e., residues that control substrate specificity. As these comparisons are made for each of the active site positions, a high resolution picture of the determinants of catalysis and substrate specificity for an MBL will emerge. This will allow an accurate estimate of the impact of any amino acid substitution at any active site residue on the substrate specificity and antibiotic resistance profile provided by an MBL. The information generated from deep sequencing will be extended by performing enzyme kinetics and X-ray crystallography for a number of enzymes with altered specificity to provide insights into not only which residue positions control specificity but also how they do so at the molecular level. The detailed knowledge of how active site residue positions contribute to ¿-lactam hydrolysis will facilitate the design of inhibitors that interact with the critically importnt MBL residues.