Mycobacterium tuberculosis utilizes a unique heterotetrameric structure for dehydrogenation of the cholesterol side chain.
Mycobacterium tuberculosis utilizes a unique heterotetrameric structure for dehydrogenation of the cholesterol side chain.
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结核分枝杆菌利用独特的杂元结构来脱氢侧链。
DOI：
10.1021/bi4002979
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发表时间：
2013-04-30
期刊：
影响因子：
2.9
通讯作者：
Sampson, Nicole S.
中科院分区：
文献类型：
--
作者：
Thomas, Suzanne T.;Sampson, Nicole S.
复合证据支持结核分枝杆菌（M. tuberculosis）代谢胆固醇在发病中的重要作用。肺结核）戏剧。阐明胆固醇被分解代谢的途径对于理解该途径导致感染的分子机制是必要的。基于在多种放线菌中的早期代谢物鉴定研究，已经提出胆固醇侧链代谢需要一个或多个酰基辅酶A脱氢酶（ACADs）。在M.结核病基因组在这里，我们表征了由Rv 3544 c和Rv 3543 c编码的异聚体ACAD，以前分别命名为fadE 28和fadE 29。我们现在将基因Rv 3544 c和Rv 3543 c称为chsE 1和chsE 2，以确认它们在胆固醇侧链脱氢中的经验证的活性。分析超离心和LC/UV实验证实ChsE 1-ChsE 2形成α2β2异四聚体，这是ACAD的新结构。我们的生物信息学分析和诱变研究表明，异源四聚体ChsE 1-ChsE 2只有两个活性位点。ChsE 2中的E241是催化ChsE 1-ChsE 2脱氢所必需的。稳态动力学分析确定，与六氢茚酮底物相比，该酶对完整类固醇环系统具有特异性，在pH 8.5下，特异性常数（kcat/KM）分别为2.5 × 105 ± 0.5 s-1 M-1和9.8 × 102 ± s-1 M-1。由两个不同的顺反子ACAD基因编码的参与甾醇代谢的独特ACAD四级结构的表征为鉴定M中其他甾醇代谢ACADs开辟了道路。结核病和其他放线菌的生物信息学分析。
Compounding evidence supports the important role in pathogenesis that the metabolism of cholesterol by Mycobacterium tuberculosis (M. tuberculosis) plays. Elucidating the pathway by which cholesterol is catabolized is necessary to understand the molecular mechanism by which this pathway contributes to infection. Based on early metabolite identification studies in multiple actinomycetes, it has been proposed that cholesterol side chain metabolism requires one or more acyl-CoA dehydrogenases (ACADs). There are 35 genes annotated as encoding ACADs in the M. tuberculosis genome. Here we characterize a heteromeric ACAD encoded by Rv3544c and Rv3543c, formerly named fadE28 and fadE29, respectively. We now refer to genes Rv3544c and Rv3543c as chsE1 and chsE2 in recognition of their validated activity in cholesterol side chain dehydrogenation. Analytical ultracentrifugation and LC/UV experiments establish that ChsE1-ChsE2 forms an α2β2 heterotetramer, a new architecture for an ACAD. Our bioinformatic analysis and mutagenesis studies reveal that heterotetrameric ChsE1-ChsE2 has only two active sites. E241 in ChsE2 is required for catalysis of dehydrogenation by ChsE1-ChsE2. Steady state kinetic analysis establishes the enzyme is specific for an intact steroid ring system compared to hexahydroindanone substrates with specificity constants (kcat/KM) of 2.5 × 105 ± 0.5 s-1 M-1 vs 9.8 × 102 ± s-1 M-1 respectively, at pH 8.5. The characterization of a unique ACAD quaternary structure involved in sterol metabolism that is encoded by two distinct cistronic ACAD genes opens the way to identification of additional sterol metabolizing ACADs in M. tuberculosis and other actinomycetes through bioinformatic analysis.
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