Role of a solvent-exposed tryptophan in the recognition and binding of antibiotic substrates for a metallo-β-lactamase
Role of a solvent-exposed tryptophan in the recognition and binding of antibiotic substrates for a metallo-β-lactamase
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DOI：
10.1110/ps.0305303
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发表时间：
2003-07-01
期刊：
影响因子：
8
通讯作者：
Dyson, HJ
中科院分区：
文献类型：
--
作者：
Huntley, JJA;Fast, W;Dyson, HJ
来自脆弱拟杆菌和相关生物体的金属β-内酰胺酶的许多X射线晶体结构显示出紧邻活性位点锌原子的β-发夹环。晶体学和NMR信息都表明，该β-发夹环的末端含有溶剂暴露的色氨酸残基Trp49，在不存在底物或其他配体的情况下具有高度柔性，从而在一些X射线结构中导致该区域缺乏可观察到的电子密度。我们报告的调查的作用，这移动的，溶剂暴露色氨酸使用定点诱变，稳态动力学测量和表征核磁共振。Trp49似乎在底物结合和促进催化中都有作用。用许多不同的侧链取代该残基表明，结合相互作用取决于吲哚环的庞大疏水性和芳香性，这可以提供与各种抗生素底物的相对非特异性相互作用。以这种方式，在该位置的色氨酸为金属-β-内酰胺酶提供了很大程度的底物特异性宽度。先前的研究确定，抗生素结合位点具有足够的可塑性，现有抗生素的衍生化不太可能导致成功治疗包含该耐药元件的细菌感染。相反，更有效的方法可能是设计针对这种溶剂暴露的色氨酸残基的治疗剂。
Numerous X-ray crystal structures of the metallo-beta-lactamase from Bacteroides fragilis and related organisms show a beta-hairpin loop immediately adjacent to the active-site zinc atom(s). Both crystallographic and NMR information show that the end of this beta-hairpin loop, which contains a solvent exposed tryptophan residue, Trp49, is highly flexible in the absence of substrates or other ligands, giving rise in some of the X-ray structures to a lack of observable electron density in this region. We report an investigation of the role of this mobile, solvent-exposed tryptophan using site-directed mutagenesis, steady state kinetics measurements and characterization by NMR. Trp49 appears to have a role both in substrate binding and in promotion of catalysis. Substitution of this residue with a number of different side chains indicates that the binding interaction depends on the bulky hydrophobic and aromatic nature of the indole ring, which can provide relatively non-specific interactions with a variety of antibiotic substrates. In this way, the tryptophan at this position provides a large degree of the breadth of substrate specificity for the metallo-beta-lactamase. Previous studies established that the antibiotic binding site was sufficiently plastic that the derivatization of existing antibiotics is unlikely to result in the successful treatment of bacterial infections incorporating this resistance element. Rather, a more productive approach may be to design therapeutics directed towards this solvent-exposed tryptophan residue.