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

抗生素抗性细菌病原体的出现和传播已成为令人担忧的问题。 最近描述的IMP-1金属-β-内酰胺酶赋予对几乎所有β-内酰胺抗生素的抗性。 该建议的重点是使用定点突变来确定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。将使用以下特定目的开发该系统：1）IMP-1的假定关键残基的随机化，以检测其在碳青霉烯酶底物结合和水解中的作用; 2）IMP-1底物特异性决定因素的鉴定和3）确定IMP-1活性是否需要两个锌结合位点。 构建随机定点突变文库，然后选择功能性克隆并测序，将评估靶残基在这些目标中的作用。 功能由酶水解β-内酰胺抗生素的能力定义。 诱变策略将有助于确定哪些氨基酸残基是必要的结合不同的底物和锌（II），通过确定取代，影响这些分子的结合。 动力学研究将用于表征特异性改变的突变酶，金属结合和生物化学测定将建立IMP-1的锌含量和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.