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

描述（由申请方提供）：高分辨率下金属-<$-内酰胺酶序列限制的分析<$-内酰胺抗生素是最常用的抗菌药物，对这些药物的耐药性发生率不断增加是一个严重的公共卫生问题。细菌耐药的最常见机制是这些药物的内酰胺酶催化水解。锌金属β-内酰胺酶（MBL）催化包括碳青霉烯类在内的多种β-内酰胺类抗生素的水解，并已成为耐药性的重要来源。含锌酶可进一步细分为B1、B2和B3亚类，其中B1 <$-内酰胺酶亚类在细菌中分布最广。在亚组B1中，IMP-1、Vim-2和NDM-1酶作为抗生素耐药性的来源最具临床相关性。为了了解突变如何影响它们的功能和进化，并设计MBL的抑制剂，有必要了解氨基酸序列如何决定这些酶的结构和功能。这个问题将使用密码子随机化和选择，然后进行深度测序的策略来解决。IMP-1、Vim-2、NDM-1 MBL以及B2亚类的CphA酶中的单个密码子将被随机化，以创建包含残基位置的所有可能取代的文库。每个位置对于酶结构和功能的重要性将通过基于其赋予大肠杆菌对感兴趣的内酰胺抗生素的抗性的能力从每个文库中选择功能性克隆来确定。功能突变体的超高通量DNA测序将用于提供关于每个残基位置处的氨基酸取代范围的高分辨率信息，所述氨基酸取代范围与用于选择的抗生素的抗性一致，从而确定酶功能的序列要求。将对几种<$-内酰胺抗生素进行实验，并将比较每种药物的序列要求，以鉴定所有<$-内酰胺水解所需的野生型氨基酸的残基位置，以及根据用于选择的抗生素显示改变的序列要求的位置，即，控制底物特异性的残基。由于这些比较是为每个活性位点的位置，一个高分辨率的图片的催化和底物特异性的MBL的决定因素将出现。这将允许准确估计任何活性位点残基处的任何氨基酸取代对MBL提供的底物特异性和抗生素抗性谱的影响。深度测序产生的信息将通过对许多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，而且它们如何在分子水平上这样做的见解。详细了解活性位点残基的位置如何促进β-内酰胺水解，将有助于设计与极其重要的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.