STRUCTURE-FUNCTION STUDY OF METALLO-B-LACTAMASE IMP-1

耐药细菌病原体的出现和传播已经成为一个令人担忧的问题。最近发现的IMP-1金属- β -内酰胺酶对几乎所有β -内酰胺类抗生素都具有耐药性。本提案的重点是使用定点诱变来确定B类金属- β -内酰胺酶IMP-1的催化和底物特异性的决定因素。该系统将根据以下具体目标进行开发：1)随机化IMP-1的假定关键残基，以测试其在碳青霉酰胺酶底物结合和水解中的作用；2)确定IMP-1底物特异性的决定因素，3)确定两个锌结合位点是否都是IMP-1活性所必需的。构建随机位点定向诱变文库，然后选择和测序功能克隆，将评估目标残基在这些目标中的作用。功能是由酶水解-内酰胺类抗生素的能力决定的。诱变策略将通过识别影响这些分子结合的取代，帮助确定哪些氨基酸残基是结合不同底物和锌（II）所必需的。动力学研究将用于表征特异性改变的突变酶，金属结合和生化分析将建立锌含量与IMP-1催化活性之间的相关性。

The emergence and spread of antibiotic resistant bacterial pathogens has become an alarming problem. The recently described IMP-1 metallo-beta-lactamase, confers resistance to virtually all beta-lactam antibiotics. The focus of this proposal is to use site-directed mutagenesis to define the determinants of catalysis and substrate specificity of the class B metallo-beta-lactamase, IMP-1. The system will be developed using the following specific aims: 1) randomization of the putative critical residues of IMP-1 to test their role in carbapenamase substrate binding and hydrolysis; 2) identification of the determinants of IMP-1 substrate specificity and 3) determining if both zinc-binding sites are required for IMP-1 activity. The construction of a randomized site-directed mutagenesis library followed by the selection and sequencing of functional clones will assess the role of the target residues in these aims. Function is defined by the ability of the enzyme to hydrolyze beta-lactam antibiotics. The mutagenesis strategy will help define what amino acid residues are necessary for binding different substrates and zinc (II) by identifying substitutions that affect the binding of these molecules. Kinetic studies will be used to characterize mutant enzymes with altered specificity, and metal- binding and biochemical assays will establish a correlation between zinc content and catalytic activity of IMP-1.