The Role of the β5-α11 Loop in the Active-Site Dynamics of Acylated Penicillin-Binding Protein A from Mycobacterium tuberculosis
The Role of the β5-α11 Loop in the Active-Site Dynamics of Acylated Penicillin-Binding Protein A from Mycobacterium tuberculosis
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DOI：
10.1016/j.jmb.2012.02.021
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发表时间：
2012-05-18
影响因子：
5.6
通讯作者：
Davies, Christopher
中科院分区：
文献类型：
--
作者：
Fedarovich, Alena;Nicholas, Robert A.;Davies, Christopher
青霉素结合蛋白A （PBPA）是一种B类青霉素结合蛋白，对结核分枝杆菌的细胞分裂起重要作用。我们已经确定了载子形式的PBPA的第二种晶体结构，并将其与早期的载子酶结构进行了比较。活性位点区域的显著结构差异是显而易见的，包括β -发夹环的排序增加和SxN活性位点基序的移位，使其现在占据了一个似乎具有催化能力的位置。使用两种测定法，其中一种使用色氨酸残基的固有荧光，我们还测量了抗生素亚胺培南、青霉素G和头孢曲松的二级酰化速率常数。其中，亚胺培南具有明显的抗结核活性，其酰化效率最高。对PBPA与相同抗生素配合物的晶体结构进行了测定，发现活性位点附近的β 5- α 11环的构象都存在差异，但这些构象对于每个β -内酰胺以及晶体不对称单元中的两个分子都是不同的。总的来说，这些数据揭示了PBPA的β 5- α 11环是一个灵活的区域，对酰化很重要，并进一步证明载脂蛋白形式的青霉素结合蛋白可以占据不同的构象状态。(C) 2012 Elsevier Ltd.版权所有。
Penicillin-binding protein A (PBPA) is a class B penicillin-binding protein that is important for cell division in Mycobacterium tuberculosis. We have determined a second crystal structure of PBPA in apo form and compared it with an earlier structure of apoenzyme. Significant structural differences in the active site region are apparent, including increased ordering of a beta-hairpin loop and a shift of the SxN active site motif such that it now occupies a position that appears catalytically competent. Using two assays, including one that uses the intrinsic fluorescence of a tryptophan residue, we have also measured the Second-order acylation rate constants for the antibiotics imipenem, penicillin G, and ceftriaxone. Of these, imipenem, which has demonstrable anti-tubercular activity, shows the highest acylation efficiency. Crystal structures of PBPA in complex with the same antibiotics were also determined, and all show conformational differences in the beta 5-alpha 11 loop near the active site, but these differ for each beta-lactam and also for each of the two molecules in the crystallographic asymmetric unit. Overall, these data reveal the beta 5-alpha 11 loop of PBPA as a flexible region that appears important for acylation and provide further evidence that penicillin-binding proteins in apo form can occupy different conformational states. (C) 2012 Elsevier Ltd. All rights reserved.