Impact of remote mutations on metallo-β-lactamase substrate specificity:: Implications for the evolution of antibiotic resistance
Impact of remote mutations on metallo-β-lactamase substrate specificity:: Implications for the evolution of antibiotic resistance
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DOI：
10.1110/ps.041093405
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发表时间：
2005-03-01
期刊：
影响因子：
8
通讯作者：
Pleiss, J
中科院分区：
文献类型：
--
作者：
Oelschlaeger, P;Mayo, SL;Pleiss, J
金属-β-内酰胺酶由于其水解广谱β-内酰胺抗生素的能力而引起关注。区分金属β-内酰胺酶IMP-1和IMP-6的G262 S点突变对头孢噻吩和头孢噻肟的水解没有影响，但显著提高了对头孢噻啶、头孢他啶、苄青霉素、氨苄青霉素和亚胺培南的催化效率。即使残基262远离活性位点，特异性也会发生这种变化。我们研究了其他五个点突变体的底物特异性，这些突变体是由残基262附近的位置处的单核苷酸取代引起的：G262 A、G262 V、S121 G、F218 Y和F218 I。结果表明两种类型的底物：I型（头孢硝肟，头孢噻吩和头孢噻肟），这是同样好地转换IMP-6，IMP-1，和G262 A，但更有效地由其他突变体，和II型（头孢他啶，苄青霉素，氨苄青霉素和亚胺培南），这是水解效率低得多的所有突变体。G262 V、S121 G、F218 Y和F218 I改善I型底物的转化，而G262 A和IMP-1改善II型底物的转化，表明IMP-6的两种不同的进化适应。衬底结构可以解释所观察到的催化效率。I型底物具有R-2电子供体，其可以稳定结合口袋中的底物中间体。相反，与II型底物的这些稳定相互作用的缺乏可能导致差的转化。这一发现可能有助于未来的药物设计。由于G262 A和F218 Y突变体赋予对大肠杆菌BL 21（DE 3）细胞的有效抗性（高最小抑制浓度）。它们很可能会自然进化。
Metallo-beta-lactamases have raised concerns due to their ability to hydrolyze a broad spectrum of P-lactam antibiotics. The G262S point mutation distinguishing the metallo-beta-lactamase IMP-1 from IMP-6 has no effect on the hydrolysis of the drugs cephalothin and cefotaxime, but significantly improves catalytic efficiency toward cephaloridine, ceftazidime, benzylpenicillin, ampicillin, and imipenem. This change in specificity occurs even though residue 262 is remote from the active site. We investigated the substrate specificities of five other point mutants resulting from single-nucleotide substitutions at positions near residue 262: G262A, G262V, S121G, F218Y, and F218I. The results suggest two types of substrates: type I (nitrocefin, cephalothin, and cefotaxime), which are converted equally well by IMP-6, IMP-1, and G262A, but even more efficiently by the other mutants, and type II (ceftazidime, benzylpenicillin, ampicillin, and imipenem), which are hydrolyzed much less efficiently by all the mutants. G262V, S121G, F218Y, and F218I improve conversion of type I substrates, whereas G262A and IMP-1 improve conversion of type II substrates, indicating two distinct evolutionary adaptations from IMP-6. Substrate structure may explain the catalytic efficiencies observed. Type I substrates have R-2 electron donors, which may stabilize the substrate intermediate in the binding pocket. In contrast, the absence of these stabilizing interactions with type II substrates may result in poor conversion. This observation may assist future drug design. As the G262A and F218Y mutants confer effective resistance to Escherichia coli BL21(DE3) cells (high minimal inhibitory concentrations). they are likely to evolve naturally.