Petrosino J F, Palzkill T
Department of Microbiology and Immunology, Baylor College of Medicine, Houston, Texas 77030, USA.
J Bacteriol. 1996 Apr;178(7):1821-8. doi: 10.1128/jb.178.7.1821-1828.1996.
Beta-Lactamase is a bacterial protein that provides resistance against beta-lactam antibiotics. TEM-1 beta-lactamase is the most prevalent plasmid-mediated beta-lactamase in gram-negative bacteria. Normally, this enzyme has high levels of hydrolytic activity for penicillins, but mutant beta-lactamases have evolved with activity toward a variety of beta-lactam antibiotics. It has been shown that active site substitutions are responsible for changes in the substrate specificity. Since mutant beta-lactamases pose a serious threat to antimicrobial therapy, the mechanisms by which mutations can alter the substrate specificity of TEM-1 beta-lactamase are of interest. Previously, screens of random libraries encompassing 31 of 55 active site amino acid positions enabled the identification of the residues responsible for maintaining the substrate specificity of TEM-1 beta-lactamase. In addition to substitutions found in clinical isolates, many other specificity-altering mutations were also identified. Interestingly, many nonspecific substitutions in the N-terminal half of the active site omega loop were found to increase ceftazidime hydrolytic activity and decrease ampicillin hydrolytic activity. To complete the active sight study, eight additional random libraries were constructed and screened for specificity-altering mutations. All additional substitutions found to alter the substrate specificity were located in the C-terminal half of the active site loop. These mutants, much like the N-terminal omega loop mutants, appear to be less stable than the wild-type enzyme. Further analysis of a 165-YYG-167 triple mutant, selected for high levels of ceftazidime hydrolytic activity, provides an example of the correlation which exists between enzyme instability and increased ceftazidime hydrolytic activity in the ceftazidime-selected omega loop mutants.
β-内酰胺酶是一种细菌蛋白，可提供对β-内酰胺类抗生素的抗性。TEM-1β-内酰胺酶是革兰氏阴性菌中最常见的质粒介导的β-内酰胺酶。通常，这种酶对青霉素具有高水平的水解活性，但突变型β-内酰胺酶已经进化出对多种β-内酰胺类抗生素的活性。已经表明，活性位点取代是底物特异性变化的原因。由于突变型β-内酰胺酶对抗菌治疗构成严重威胁，因此突变可改变TEM-1β-内酰胺酶底物特异性的机制备受关注。此前，对包含55个活性位点氨基酸位置中的31个的随机文库进行筛选，使得能够鉴定出负责维持TEM-1β-内酰胺酶底物特异性的残基。除了在临床分离株中发现的取代外，还鉴定出许多其他改变特异性的突变。有趣的是，在活性位点ω环N端一半中发现的许多非特异性取代增加了头孢他啶的水解活性并降低了氨苄青霉素的水解活性。为了完成活性位点研究，构建了另外八个随机文库并筛选了改变特异性的突变。所有发现改变底物特异性的额外取代都位于活性位点环的C端一半。这些突变体与N端ω环突变体非常相似，似乎比野生型酶更不稳定。对选择具有高水平头孢他啶水解活性的165-YYG-167三重突变体的进一步分析，提供了头孢他啶选择的ω环突变体中酶不稳定性与头孢他啶水解活性增加之间存在相关性的一个例子。
