Understanding resistance to β-lactams and β-lactamase inhibitors in the SHV β-lactamase -: Lessons from the mutagenesis of Ser-130
Understanding resistance to β-lactams and β-lactamase inhibitors in the SHV β-lactamase -: Lessons from the mutagenesis of Ser-130
复制标题
DOI：
10.1074/jbc.m306059200
复制
发表时间：
2003-12-26
影响因子：
4.8
通讯作者：
Bonomo, RA
中科院分区：
文献类型：
--
作者：
Helfand, MS;Bethel, CR;Bonomo, RA
通过A类β-内酰胺酶中的单个氨基酸突变对β-内酰胺/β-内酰胺酶抑制剂组合的细菌耐药性威胁着我们最有效的临床抗生素。在TEM-1和SHV-1（常见的A类β-内酰胺酶）中，Ser-130的改变赋予对β-内酰胺酶抑制剂克拉维酸和他唑巴坦灭活的抗性。通过使用位点饱和诱变，我们试图确定SHV-1 β-内酰胺酶中Ser-130处的氨基酸取代导致对这些抑制剂的抗性。抗生素敏感性试验显示，氨苄西林和氨苄西林/克拉维酸耐药性只观察到在大肠杆菌中表达的S130 G β-内酰胺酶。S130 G β-内酰胺酶的动力学分析表明，氨苄青霉素的表观Km显著升高，k（cat）/K-m降低。观察到克拉维酸（SHV-1，0.14 μ M; S130 G，46.5 μ M）和他唑巴坦（SHV-1，0.07 μ M; S130 G，4.2 μ M）的预酰化复合物K-I的解离常数显著增加。相比之下，S130 G和SHV-1的k（inact）s对于克拉维酸仅相差17%，对于他唑巴坦仅相差40%。渐进失活研究表明，抑制SHV-1和S130 G所需的抑制剂与酶的比例相似。我们的观察结果表明，酶的活性被保存，尽管氨基酸取代，显着改变β-内酰胺类和β-内酰胺酶抑制剂的活性位点的表观亲和力。这些结果强调了A类β-内酰胺酶的机制的多功能性，并对新型β-内酰胺酶抑制剂的设计产生影响。
Bacterial resistance to beta-lactam/beta-lactamase inhibitor combinations by single amino acid mutations in class A beta-lactamases threatens our most potent clinical antibiotics. In TEM-1 and SHV-1, the common class A beta-lactamases, alterations at Ser-130 confer resistance to inactivation by the beta-lactamase inhibitors, clavulanic acid, and tazobactam. By using site-saturation mutagenesis, we sought to determine the amino acid substitutions at Ser-130 in SHV-1 beta-lactamase that result in resistance to these inhibitors. Antibiotic susceptibility testing revealed that ampicillin and ampicillin/clavulanic acid resistance was observed only for the S130G beta-lactamase expressed in Escherichia coli. Kinetic analysis of the S130G beta-lactamase demonstrated a significant elevation in apparent Km and a reduction in k(cat)/K-m for ampicillin. Marked increases in the dissociation constant for the preacylation complex, K-I, of clavulanic acid (SHV-1, 0.14 muM; S130G, 46.5 muM) and tazobactam (SHV-1, 0.07 muM; S130G, 4.2 muM) were observed. In contrast, the k(inact)s of S130G and SHV-1 differed by only 17% for clavulanic acid and 40% for tazobactam. Progressive inactivation studies showed that the inhibitor to enzyme ratios required to inactivate SHV-1 and S130G were similar. Our observations demonstrate that enzymatic activity is preserved despite amino acid substitutions that significantly alter the apparent affinity of the active site for beta-lactams and beta-lactamase inhibitors. These results underscore the mechanistic versatility of class A beta-lactamases and have implications for the design of novel beta-lactamase inhibitors.