Active site labeling of the gentamicin resistance enzyme AAQ6′)-APH(2′′) by the lipid kinase inhibitor wortmannin
Active site labeling of the gentamicin resistance enzyme AAQ6′)-APH(2′′) by the lipid kinase inhibitor wortmannin
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DOI：
10.1016/s1074-5521(01)00051-5
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发表时间：
2001-08-01
影响因子：
--
通讯作者：
Wright, GD
中科院分区：
文献类型：
--
作者：
Boehr, DD;Lane, WS;Wright, GD
背景：氨基糖苷类抗生素耐药主要是产生共价修饰药物的酶的结果，包括结构和功能与蛋白质和脂质激酶相似的激酶（APHs）。最重要的氨基糖苷抗性酶之一是AAC（6’）-APH（2’），它是一种具有氨基糖苷乙酰转移酶和激酶活性的双功能酶。了解酶活性位点的结构对于破解抗生素耐药的分子机制非常重要，我们探索了活性位点标记技术来研究AAC（6’）-APH（2’）的结构和功能。结果：AAC（6’）-APH（2’）被wortmannin不可逆地失活，wortmannin是一种有效的磷脂酰肌醇3-激酶抑制剂，通过ATP结合口袋中保守赖氨酸的共价修饰。5 '-[p-（氟磺酰基）苯甲酰]腺苷是一种亲电ATP类似物，也是已知的APH(3 ')- iiia等其他APH酶的失活剂，它不能使AAC(6 ')-APH（2 ‘）失活，同样，wortmannin也不能使APH（3 ’）- iiia失活。结论：这些不同的活性位点标记敏感性表明氨基糖苷激酶活性位点结构存在重要差异，并表明设计广泛的、ATP结合位点导向的针对APHs的抑制剂将是困难的。尽管如此，考虑到APH酶对蛋白激酶和脂质激酶抑制剂的敏感性，这种重要抗性酶的有效先导抑制剂很可能在针对其他药理学上重要激酶的化合物文库中被发现。2001爱思唯尔科学有限公司版权所有。
Background: Aminoglycoside antibiotic resistance is largely the result of the production of enzymes that covalently modify the drugs including kinases (APHs) with structural and functional similarity to protein and lipid kinases. One of the most important aminoglycoside resistance enzymes is AAC(6 ')-APH(2 "), a bifunctional enzyme with both aminoglycoside acetyltransferase and kinase activities. Knowledge of enzyme active site structure is important in deciphering the molecular mechanism of antibiotic resistance and here we explored active site labeling techniques to study AAC(6 ')-APH(2 ") structure and function.Results: AAC(6 ')-APH(2 ") was irreversibly inactivated by wortmannin, a potent phosphatidylinositol 3-kinase inhibitor, through the covalent modification of a conserved lysine in the ATP binding pocket. 5 '-[p-(Fluorosulfonyl)benzoyl] adeno sine, an electrophilic ATP analogue and known inactivator of other APH enzymes such as APH(3 ')-IIIa, did not inactivate AAC(6 ')-APH(2 "), and reciprocally, wortmannin did not inactivate APH(3 ')-IIIa.Conclusions: These distinct active site label sensitivities point to important differences in aminoglycoside kinase active site structures and suggest that design of broad range, ATP binding site-directed inhibitors against APHs will be difficult. Nonetheless, given the sensitivity of APH enzymes to both protein and lipid kinase inhibitors, potent lead inhibitors of this important resistance enzyme are likely to be found among the libraries of compounds directed against other pharmacologically important kinases. (C) 2001 Elsevier Science Ltd. All rights reserved.