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

抗生素耐药细菌病原体的出现和传播已成为一个令人担忧的问题。最近报道的IMP-1金属β-内酰胺酶对几乎所有的β-内酰胺类抗生素都具有耐药性。这项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的催化和底物专一性的决定因素。该系统的开发将利用以下特定目标：1)随机确定IMP-1的关键残基，以测试它们在碳青霉酶底物结合和水解中的作用；2)鉴定IMP-1底物专一性的决定因素；3)确定IMP-1活性是否需要这两个锌结合位点。构建随机定点突变文库，然后进行功能克隆的选择和测序，将评估靶残基在这些目的中的作用。功能是由酶对β-内酰胺类抗生素的水解力来定义的。突变策略将通过识别影响不同底物和锌(II)结合的取代基来帮助确定结合不同底物和锌(II)所需的氨基酸残基。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和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.