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首页> 外文期刊>Chemistry & biology >Mutational analysis of active-site residues of the enterococcal D-Ala-D-Ala dipeptidase VanX and comparison with Escherichia coli D-Ala-D-Ala ligase and D-Ala-D-Ala carboxypeptidase VanY
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Mutational analysis of active-site residues of the enterococcal D-Ala-D-Ala dipeptidase VanX and comparison with Escherichia coli D-Ala-D-Ala ligase and D-Ala-D-Ala carboxypeptidase VanY

机译：肠球菌D-Ala-D-Ala二肽酶VanX活性位点残基的突变分析并与大肠杆菌D-Ala-D-Ala连接酶和D-Ala-D-Ala羧肽酶VanY进行比较

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摘要

Background: Vancomycin-resistant enterococci are pathogenic bacteria that attenuate antibiotic sensitivity by producing peptidoglycan precursors that terminate in D-Ala-D-lactate rather than D-Ala-D-Ala. A key enzyme in effecting antibiotic resistance is the metallodipeptidase VanX, which reduces the cellular pool of the D-Ala-D-Ala dipeptide. Results: We constructed eleven mutants, using the recently determined VanX structure as a basis, to investigate residue function. Mutating Asp142 or Ser114 showed a large effect principally on K-M, consistent with roles in recognition of the D-Ala-D-Ala termini. The drastic reduction or absence of activity in the Arg71 mutants correlates with a role in the stabilization of an anionic tetrahedral transition state. Three residues of the Escherichia coli D-Ala-D-Ala ligase (Ddl), Glu15, Ser281 and Arg255, are similarly conserved and have equivalent functions with respect to VanX, consistent with a convergent evolution of active sites to bind D-Ala-D-Ala and tower energy barriers for formation of the tetrahedral intermediate and transition states. In the N-acyl-D-Ala-D-Ala carboxypeptidase VanY, all active-site residues are conserved (except for the two responsible for recognition of the dipeptide amino terminus). Conclusions: The mutagenesis results support structure-based functional predictions and explain why the VanX dipeptidase and Ddl ligase show narrow specificity for the D,D-dipeptide substrate, The results reveal that VanX and Ddl, two enzymes that use the same substrate but proceed in opposite directions driven by distinct cofactors (zinc versus AIP), evolved similar architectural solutions to substrate recognition and catalysis acceleration. VanY sequence analysis predicts an active site and mechanism of reaction similar to VanX. [References: 44]
机译：背景：耐万古霉素的肠球菌是致病性细菌，可通过产生肽聚糖前体终止D-Ala-D-乳酸而不是D-Ala-D-Ala，从而减弱抗生素敏感性。影响抗生素抗性的关键酶是金属二肽酶VanX，它可以减少D-Ala-D-Ala二肽的细胞池。结果：我们以最近确定的VanX结构为基础，构建了11个突变体，以研究残基功能。突变Asp142或Ser114主要表现出对K-M的巨大影响，这与识别D-Ala-D-Ala末端的作用一致。 Arg71突变体中活性的急剧降低或不存在与阴离子四面体过渡态的稳定化有关。大肠杆菌D-Ala-D-Ala连接酶（Ddl）的三个残基，Glu15，Ser281和Arg255类似地保守，并且具有与VanX相同的功能，这与结合D-Ala- D-Ala和塔的能垒，用于形成四面体的中间态和过渡态。在N-酰基-D-Ala-D-Ala羧肽酶VanY中，所有活性位点残基均被保守（除了两个负责识别二肽氨基末端的残基）。结论：诱变结果支持基于结构的功能预测，并解释了为什么VanX二肽酶和Ddl连接酶对D，D-二肽底物显示狭窄的特异性，结果表明VanX和Ddl是两种酶，它们使用相同的底物但继续进行。由不同的辅因子（锌与AIP）驱动的相反方向，演变出类似的体系结构解决方案以实现底物识别和催化加速。 VanY序列分析可预测类似于VanX的活性位点和反应机理。 [参考：44]

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