Analysis of the Functional Contributions of Asn233 in Metallo-β-Lactamase IMP-1
Analysis of the Functional Contributions of Asn233 in Metallo-β-Lactamase IMP-1
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DOI：
10.1128/aac.00340-11
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发表时间：
2011-12-01
影响因子：
4.9
通讯作者：
Palzkill, Timothy
中科院分区：
文献类型：
--
作者：
Brown, Nicholas G.;Horton, Lori B.;Palzkill, Timothy
金属-β-内酰胺酶，例如 IMP-1，是全球主要的健康威胁，因为它们使细菌对多种 β-内酰胺抗生素（包括碳青霉烯类）产生耐药性。了解酶促过程的分子细节和功能的序列要求对于克服 β-内酰胺酶介导的耐药性至关重要。在大约 67% 的所有金属-β-内酰胺酶中，天冬酰胺残基在第 233 位是保守的。尽管 Asn233 具有保守性，但人们对 Asn233 功能的分子基础知之甚少，并且仍然存在争议。先前已表明，该位点的突变表现出上下文依赖性序列要求，因为给定氨基酸的重要性取决于所测试的抗生素。为了对该位置的功能和序列要求进行更彻底的检查，生成了编码 19 个可能的氨基酸取代中每一个的 IMP-1 突变体的集合。测量了含有每种突变体的大肠杆菌对四种 β-内酰胺抗生素的耐药水平。对两种头孢菌素的野生型水平的抗性的233位的序列要求是最宽松的，而对氨苄西林或亚胺培南的抗性则有更严格的序列要求。酶动力学分析和稳态蛋白质水平的测定表明，取代对抗性的影响是由于酶动力学参数的变化造成的。总而言之，结果表明 233 位的取代显着改变了酶的动力学参数，但大多数取代的酶能够对多种 β-内酰胺提供高水平的抗性。
Metallo-beta-lactamases, such as IMP-1, are a major global health threat, as they provide for bacterial resistance to a wide range of beta-lactam antibiotics, including carbapenems. Understanding the molecular details of the enzymatic process and the sequence requirements for function are essential aids in overcoming beta-lactamase-mediated resistance. An asparagine residue is conserved at position 233 in approximately 67% of all metallo-beta-lactamases. Despite its conservation, the molecular basis of Asn233 function is poorly understood and remains controversial. It has previously been shown that mutations at this site exhibit context-dependent sequence requirements in that the importance of a given amino acid depends on the antibiotic being tested. To provide a more thorough examination as to the function and sequence requirements at this position, a collection of IMP-1 mutants encoding each of the 19 possible amino acid substitutions was generated. The resistance levels toward four beta-lactam antibiotics were measured for Escherichia coli containing each of these mutants. The sequence requirements at position 233 for wild-type levels of resistance toward two cephalosporins were the most relaxed, while there were more stringent sequence requirements for resistance to ampicillin or imipenem. Enzyme kinetic analysis and determinations of steady-state protein levels indicated that the effects of the substitutions on resistance are due to changes in the kinetic parameters of the enzyme. Taken together, the results indicate that substitutions at position 233 significantly alter the kinetic parameters of the enzyme, but most substituted enzymes are able to provide for a high level of resistance to a broad range of beta-lactams.