Mechanism and Diversity of the Erythromycin Esterase Family of Enzymes
Mechanism and Diversity of the Erythromycin Esterase Family of Enzymes
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DOI：
10.1021/bi201790u
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发表时间：
2012-02-28
期刊：
影响因子：
2.9
通讯作者：
Wright, Gerard D.
中科院分区：
文献类型：
--
作者：
Morar, Mariya;Pengelly, Kate;Wright, Gerard D.
大环内酯类抗生素如阿奇霉素和红霉素是现代抗菌化疗的主要药物，与所有抗生素一样，它们容易产生耐药性。大环内酯类耐药的一种机制是通过药物灭活：红霉素酯酶EreA和EreB催化的大环内酯环的酶水解。一个基因组酶学的方法被用来深入了解Ere酶的催化机制和起源。我们的分析表明，红霉素酯酶包括一个单独的组中的水解酶超家族，其中包括同源物上发现的蜡状芽孢杆菌，Bcr 135和Bcr 136，其三维结构的染色体上的未表征的功能已被确定。Bcr 136的生化特性证实，它是一种酯酶，但不能降解大环内酯类。使用稳态动力学，同源性为基础的结构建模，定点诱变，溶剂同位素效应的研究，pH值和抑制剂分析进行了各种组合的EreA，EreB和Bcr 136酶，我们确定了活性位点，并深入了解这个新的酶超家族的一些催化功能。我们排除了丝氨酸/苏氨酸亲核试剂的可能性，并表明一个组氨酸，H46（EreB编号），是必不可少的催化功能。该残基被提议用作活化水分子作为反应亲核试剂的通用碱。此外，我们发现，EreA，EreB和Bcr 136是不同的，只有EreA抑制螯合剂和假设含有非催化金属。这些酯酶的详细表征允许直接比较的电阻决定因素，EreA和EreB，与它们的原型，Bcr 136，并讨论它们的潜在连接。
Macrolide antibiotics such as azithromycin and erythromycin are mainstays of modern antibacterial chemotherapy, and like all antibiotics, they are vulnerable to resistance. One mechanism of macrolide resistance is via drug inactivation: enzymatic hydrolysis of the macrolactone ring catalyzed by erythromycin esterases, EreA and EreB. A genomic enzymology approach was taken to gain insight into the catalytic mechanisms and origins of Ere enzymes. Our analysis reveals that erythromycin esterases comprise a separate group in the hydrolase superfamily, which includes homologues of uncharacterized function found on the chromosome of Bacillus cereus, Bcr135 and Bcr136, whose three-dimensional structures have been determined. Biochemical characterization of Bcr136 confirms that it is an esterase that is, however, unable to inactivate macrolides. Using steady-state kinetics, homology-based structure modeling, site-directed mutagenesis, solvent isotope effect studies, pH, and inhibitor profiling performed in various combinations for EreA, EreB, and Bcr136 enzymes, we identified the active site and gained insight into some catalytic features of this novel enzyme superfamily. We rule out the possibility of a Ser/Thr nucleophile and show that one histidine, H46 (EreB numbering), is essential for catalytic function. This residue is proposed to serve as a general base in activation of a water molecule as the reaction nucleophile. Furthermore, we show that EreA, EreB, and Bcr136 are distinct, with only EreA inhibited by chelating agents and hypothesized to contain a noncatalytic metal. Detailed characterization of these esterases allows for a direct comparison of the resistance determinants, EreA and EreB, with their prototype, Bcr136, and for the discussion of their potential connections.