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首页> 外文期刊>Biochemistry >A Catalytic Intermediate and Several Flavin Redox States Stabilized by Folate-Dependent tRNA Methyltransferase from Bacillus subtilis
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A Catalytic Intermediate and Several Flavin Redox States Stabilized by Folate-Dependent tRNA Methyltransferase from Bacillus subtilis

机译：枯草芽孢杆菌叶酸依赖性tRNA甲基转移酶稳定的催化中间体和几种黄素氧化还原态。

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The flavoprotein TrmFO catalyzes the C5 methylationnof uridine 54 in the TΨC loop of tRNAs using 5,10-nmethylenetetrahydrofolate (CH2THF) as a methylene donor andnFAD as a reducing agent. Here, we report biochemical andnspectroscopic studies that unravel the remarkable capability ofnBacillus subtilis TrmFO to stabilize, in the presence of oxygen,nseveral flavin-reduced forms, including an FADH• radical, and ancatalytic intermediate endowed with methylating activity. ThenFADH• radical was characterized by high-field electron paramagneticnresonance and electron nuclear double-resonance spectroscopies.nInterestingly, the enzyme exhibited tRNA methylation activity in the absence of both an added carbon donor and an externalnreducing agent, indicating that a reaction intermediate, containing presumably CH2THF and FAD hydroquinone, is present in thenfreshly purified enzyme. Isolation by acid treatment, under anaerobic conditions, of noncovalently bound molecules, followed bynmass spectrometry analysis, confirmed the presence in TrmFO of nonmodified FAD. Addition of formaldehyde to the purifiednenzyme protects the reduced flavins from decay by probably preventing degradation of CH2THF. The absence of air-stable reducednFAD species during anaerobic titration of oxidized TrmFO, performed in the absence or presence of addedCH2THF, argues againstntheir thermodynamic stabilization but rather implicates their kinetic trapping by the enzyme. Altogether, the unexpected isolation ofna stable catalytic intermediate suggests that the flavin-binding pocket of TrmFO is a highly insulated environment, diverting thenreduced FAD present in this intermediate from uncoupled reactions.
机译：黄酮蛋白TrmFO使用5，10-亚甲基四氢叶酸（CH2THF）作为亚甲基供体，使用nFAD作为还原剂，在tRNA的TΨC环中催化尿苷54的C5甲基化。在这里，我们报道了生化和光谱学研究，这些研究揭示了枯草芽孢杆菌TrmFO在氧存在下稳定的少量黄素还原形式（包括FADH•自由基）和具有甲基化活性的催化中间体的出色能力。然后，FADH•自由基的特征在于高场电子顺磁共振和电子核双共振谱。n有趣的是，该酶在既不添加碳供体也不在外部还原剂的情况下表现出tRNA甲基化活性，表明该反应中间体可能含有CH2THF FAD对苯二酚存在于新纯化的酶中。在厌氧条件下，通过酸处理分离非共价键合的分子，然后进行质谱分析，证实了在TrmFO中存在未修饰的FAD。将甲醛添加到纯化的酶中可能通过防止CH2THF降解来保护还原的黄素免于降解。在不存在或存在CH2THF的条件下进行的氧化TrmFO的厌氧滴定过程中，缺乏空气稳定的还原nFAD物质，不利于其热力学稳定性，而是暗示了它们的动力学被酶捕获。总之，稳定的催化中间体的意外分离表明TrmFO的黄素结合口袋是高度绝缘的环境，转移了存在于该中间体中的FAD，使其从未偶联的反应中转移出来。

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  • 来源
    《Biochemistry》 |2011年第23期|p.5208-5219|共12页
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    Djemel Hamdane Vincent Gurineau Sun Un and Batrice Golinelli-Pimpaneau;

  • 作者单位

    †Laboratoire d’Enzymologie et Biochimie Structurales, Centre de Recherche de Gif, CNRS, 91198 Gif-sur-Yvette, France‡Institut de Chimie des Substances Naturelles, Centre de Recherche de Gif, CNRS, 91198 Gif-sur-Yvette, France§Service de Biou0001energu0001etique Biologie Structurale et Mu0001ecanismes, CNRS, Institut de Biologie et Technologies de Saclay,CEA Saclay, 91191 Gif-sur-Yvette, France;

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