Structure, function, and inhibition along the reaction coordinate of CTX-M β-lactamases
Structure, function, and inhibition along the reaction coordinate of CTX-M β-lactamases
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DOI：
10.1021/ja042850a
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发表时间：
2005-04-20
影响因子：
15
通讯作者：
Bonnet, R
中科院分区：
文献类型：
--
作者：
Chen, Y;Shoichet, B;Bonnet, R
CTX-M酶是一组新兴的超广谱β-内酰胺酶（ESBLs），其不仅水解青霉素，而且水解第一、第二和第三代头孢菌素。虽然它们已成为某些领域最常见的ESBLs，但有效的抑制剂很少，对其详细机制的了解相对较少。在这里，我们描述的X-射线晶体结构的CTX-M酶在复杂的不同的过渡态类似物和β-内酰胺抑制剂，代表酶，因为它的进展，从它的酰化过渡态，其酰基酶复合物的脱酰过渡态。当酶沿着这个反应坐标移动时，两个关键的催化残基Lys 73和Glu 166改变构象，跟踪反应的状态。出乎意料的是，酰基酶与β-内酰胺抑制剂头孢西丁的复合物仍然具有催化水结合;这种水已经被预测为被抑制剂的不寻常的7 α-甲氧基取代。相反，7 α-基团似乎通过空间位阻阻止脱酰过渡态的形成来抑制。从抑制剂设计的角度来看，我们注意到最好的可逆抑制剂，头孢他啶样硼酸化合物，以4 nM的Ki值与CTX-M-16结合。当在细胞培养中一起使用时，这种抑制剂逆转了产CTX-M细菌中的头孢噻肟耐药性。其与CTX-M酶的复合物的结构和本文描述的反应坐标的结构视图为抑制剂设计和干预提供了模板，以对抗该抗生素耐药性酶家族。
CTX-M enzymes are an emerging group of extended spectrum beta-lactamases (ESBLs) that hydrolyze not only the penicillins but also the first-, second-, and third-generation cephalosporins. Although they have become the most frequently observed ESBLs in certain areas, there are few effective inhibitors and relatively little is known about their detailed mechanism. Here we describe the X-ray crystal structures of CTX-M enzymes in complex with different transition-state analogues and beta-lactam inhibitors, representing the enzyme as it progresses from its acylation transition state to its acyl enzyme complex to the deacylation transition state. As the enzyme moves along this reaction coordinate, two key catalytic residues, Lys73 and Glu166, change conformations, tracking the state of the reaction. Unexpectedly, the acyl enzyme complex with the beta-lactam inhibitor cefoxitin still has the catalytic water bound; this water had been predicted to be displaced by the unusual 7 alpha-methoxy of the inhibitor. Instead, the 7 alpha-group appears to inhibit by preventing the formation of the deacylation transition state through steric hindrance. From an inhibitor design standpoint, we note that the best of the reversible inhibitors, a ceftazidime-like boronic acid compound, binds to CTX-M-16 with a K-i value of 4 nM. When used together in cell culture, this inhibitor reversed cefotaxime resistance in CTX-M-producing bacteria. The structure of its complex with CTX-M enzyme and the structural view of the reaction coordinate described here provide templates for inhibitor design and intervention to combat this family of antibiotic resistance enzymes.