Vakulenko Sergei, Golemi Dasantila
Departments of Biochemistry and Molecular Biology, Wayne State University, Detroit, Michigan 48202, USA.
Antimicrob Agents Chemother. 2002 Mar;46(3):646-53. doi: 10.1128/AAC.46.3.646-653.2002.
A derivative of the TEM-1 beta-lactamase producing clinically significant levels of resistance to ceftazidime and beta-lactamase inhibitors in the presence of penicillins was generated following five rounds of DNA shuffling and selection. This complex mutant enzyme contained three amino acid substitutions including those of residues 104 and 276 that are known to produce extended-spectrum resistance and, correspondingly, resistance to beta-lactamase inhibitors. Although the Glu104Lys substitution by itself produced low levels of ceftazidime resistance, additional amino acid replacements in the enzyme with the triple mutation resulted in further enhancement of resistance to ceftazidime. Kinetic studies of the purified beta-lactamase enzyme with the triple mutation indicated enhancement of the catalytic efficiency for turnover (kcat/Km) of ceftazidime. The increases in the Ki values of both clavulanic acid and tazobactam for the enzyme with the triple mutation were consistent with the observed bacterial resistance to the reversibility of beta-lactam resistance with these inhibitors.
经过五轮DNA改组和筛选，产生了一种TEM-1β-内酰胺酶的衍生物，在有青霉素存在的情况下，它对头孢他啶和β-内酰胺酶抑制剂产生临床上显著水平的耐药性。这种复杂的突变酶包含三个氨基酸取代，包括已知产生超广谱耐药性以及相应地对β-内酰胺酶抑制剂耐药的104位和276位残基的取代。虽然Glu104Lys取代本身产生的头孢他啶耐药性水平较低，但该酶中具有三重突变的其他氨基酸替换导致对头孢他啶的耐药性进一步增强。对具有三重突变的纯化β-内酰胺酶进行的动力学研究表明，头孢他啶周转的催化效率（kcat/Km）有所提高。克拉维酸和他唑巴坦对具有三重突变的酶的Ki值增加，与观察到的细菌对这些抑制剂逆转β-内酰胺耐药性的耐药性一致。
