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

高分辨率金属-β-内酰胺酶序列限制分析

• 批准号: 8660631
• 负责人: Timothy Palzkill
• 金额: $ 38万
• 依托单位: BAYLOR COLLEGE OF MEDICINE
• 依托单位国家: 美国
• 财政年份: 2013
• 资助国家: 美国
• 起止时间: 2013-05-15 至 2018-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8660631
• 关键词: Active Sites; Address; Amino Acid Sequence; Amino Acid Substitution; Amino Acids; Ampicillin; Anti-Bacterial Agents; Antibiotic Resistance; Antibiotic Therapy; Antibiotics; Bacteria; Bacterial Drug Resistance; Biochemical; Carbapenems; Catalysis; Cefotaxime; Characteristics; Codon Nucleotides; Coupled; DNA Sequence; Data; Drug resistance; Enzyme Kinetics; Enzymes; Escherichia coli; Evolution; Exhibits; Family; High-Throughput Nucleotide Sequencing; Hydrolysis; Incidence; Individual; Kinetics; Knowledge; Lactamase; Lactams; Libraries; Methods; Molecular; Monobactams; Mutation; Pharmaceutical Preparations; Positioning Attribute; Process; Property; Public Health; Randomized; Reagent; Research; Resistance; Resistance profile; Resolution; Sampling; Site-Directed Mutagenesis; Source; Specificity; Structure; Subgroup; Substrate Specificity; Technology; Testing; Thermodynamics; X-Ray Crystallography; Zinc; bacterial resistance; base; beta-Lactamase; clinically relevant; combinatorial; deep sequencing; design; enzyme structure; inhibitor/antagonist; insight; interest; member; mutant; public health relevance; pyrosequencing; research study

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.

描述（由申请人提供）：Metallo-¿ 的分析-高分辨率内酰胺酶序列约束 ¿ -内酰胺抗生素是最常用的抗菌剂，对这些药物的耐药性发生率不断增加是一个重要的公共卫生问题，细菌耐药性的最常见机制是 ¿ -内酰胺酶催化这些药物的水解。 -内酰胺酶 (MBL) 催化多种 ¿ -内酰胺类抗生素，包括碳青霉烯类，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 亚类 ¿ -内酰胺酶是细菌中最常见的酶，在 B1 亚组中，IMP-1、VIM-2 和 NDM-1 酶作为抗生素耐药性的来源在临床上最相关。设计 MBL 抑制剂时，有必要了解氨基酸序列如何决定这些酶的结构和功能。这个问题将通过密码子随机化和选择策略以及随后的单个密码子的深度测序来解决。 IMP-1、VIM-2、NDM-1 MBL 以及来自 B2 亚类的 CphA 酶将被随机化，以创建包含所有可能的残基位置取代的文库。每个位置对于酶结构和功能的重要性将由下式确定。根据赋予大肠杆菌抗性的能力从每个文库中选择功能克隆-功能性突变体的超高通量DNA测序将用于提供与用于选择的抗生素抗性一致的每个残基位置的氨基酸取代范围的高分辨率信息，从而确定序列。实验将进行几个 ¿ -将比较内酰胺抗生素和每种药物的序列要求，以确定所有水解所需野生型氨基酸的残基位置 ¿ -内酰胺以及根据用于选择的抗生素而表现出改变的序列要求的位置，即控制底物特异性的残基当对每个活性位点位置进行这些比较时，可以得到催化决定因素的高分辨率图像和MBL 的底物特异性将出现，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的影响。深度测序生成的信息将得到扩展。执行酶对许多具有改变的特异性的酶进行动力学和 X 射线晶体学分析，不仅可以深入了解哪些残基位置控制特异性，还可以了解它们如何在分子水平上控制特异性。 -内酰胺水解将有助于设计与至关重要的 MBL 残基相互作用的抑制剂。