Probing the specificity of the subclass B3FEZ-1 metallo-β-lactamase by site-directed mutagenesis
Probing the specificity of the subclass B3FEZ-1 metallo-β-lactamase by site-directed mutagenesis
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DOI：
10.1074/jbc.m403671200
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发表时间：
2004-08-06
影响因子：
4.8
通讯作者：
Galleni, M
中科院分区：
文献类型：
--
作者：
Mercuri, PS;García-Sáez, I;Galleni, M
戈尔曼氏氟杆菌产生的B3Fez-1β-内酰胺酶是一种含锌(II)的酶，能分解青霉素类、头孢菌素类和碳青霉烯类中的β-内酰胺键。利用动力学、计算模拟和X射线结晶学对FeZ-1进行了广泛的研究。为了探索可能参与底物结合和锌结合的残基，我们制备了5个Fez-1的定点突变体(H121A、Y156A、S221A、N225A和Y228A)，并用金属分析和稳态动力学对其进行了表征。H121a的活性与锌离子浓度有关。H121a单锌形式的活性低于双锌形式，后者的活性与野生型酶相似。TYR(156)对于底物的结合和水解不是必需的。残基Ser(221)和Asn(225)的取代通过选择性地降低对碳青霉烯类抗生素的活性来改变底物分布。Y228A突变株被头孢菌素类药物的水解物所抑制。Cys(200)的侧链与头孢菌素的水解物之间的共价键导致形成不活跃且稳定的络合物。
The subclass B3 FEZ-1 beta-lactamase produced by Fluoribacter ( Legionella) gormanii is a Zn(II)-containing enzyme that hydrolyzes the beta-lactam bond in penicillins, cephalosporins, and carbapenems. FEZ-1 has been extensively studied using kinetic, computational modeling and x-ray crystallography. In an effort to probe residues potentially involved in substrate binding and zinc binding, five site-directed mutants of FEZ-1 (H121A, Y156A, S221A, N225A, and Y228A) were prepared and characterized using metal analyses and steady state kinetics. The activity of H121A is dependent on zinc ion concentration. The H121A monozinc form is less active than the dizinc form, which exhibits an activity similar to that of the wild type enzyme. Tyr(156) is not essential for binding and hydrolysis of the substrate. Substitution of residues Ser(221) and Asn(225) modifies the substrate profile by selectively decreasing the activity against carbapenems. The Y228A mutant is inhibited by the product formed upon hydrolysis of cephalosporins. A covalent bond between the side chain of Cys(200) and the hydrolyzed cephalosporins leads to the formation of an inactive and stable complex.