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首页> 外文期刊>Biochemistry >Peroxide-shunt substrate-specificity for the Salmonella typhimurium O _2-dependent tRNA modifying monooxygenase (MiaE)
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Peroxide-shunt substrate-specificity for the Salmonella typhimurium O _2-dependent tRNA modifying monooxygenase (MiaE)

机译：鼠伤寒沙门氏菌O _2依赖的tRNA修饰单加氧酶（MiaE）的过氧化物分流底物特异性

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摘要

Post-transcriptional modifications of tRNA are made to structurally diversify tRNA. These modifications alter noncovalent interactions within the ribosomal machinery, resulting in phenotypic changes related to cell metabolism, growth, and virulence. MiaE is a carboxylate bridged, nonheme diiron monooxygenase, which catalyzes the O_2-dependent hydroxylation of a hypermodified-tRNA nucleoside at position 37 (2-methylthio-N~6- isopentenyl-adenosine(37)-tRNA) [designated ms~2i~6A _(37)]. In this work, recombinant MiaE was cloned from Salmonella typhimurium, purified to homogeneity, and characterized by UV-visible and dual-mode X-band EPR spectroscopy for comparison to other nonheme diiron enzymes. Additionally, three nucleoside substrate-surrogates (i~6A, Cl~2i~6A, and ms~2i~6A) and their corresponding hydroxylated products (io~6A, Cl~2io ~6A, and ms~2io~6A) were synthesized to investigate the chemo- and stereospecificity of this enzyme. In the absence of the native electron transport chain, the peroxide-shunt was utilized to monitor the rate of substrate hydroxylation. Remarkably, regardless of the substrate (i~6A, Cl~2i~6A, and ms~2i~6A) used in peroxide-shunt assays, hydroxylation of the terminal isopentenyl-C4-position was observed with >97% E-stereoselectivity. No other nonspecific hydroxylation products were observed in enzymatic assays. Steady-state kinetic experiments also demonstrate that the initial rate of MiaE hydroxylation is highly influenced by the substituent at the C2-position of the nucleoside base (v_0/[E] for ms~2i~6A > i ~6A > Cl~2i~6A). Indeed, the >3-fold rate enhancement exhibited by MiaE for the hydroxylation of the free ms ~2i~6A nucleoside relative to i~6A is consistent with previous whole cell assays reporting the ms~2io~6A and io~6A product distribution within native tRNA-substrates. This observation suggests that the nucleoside C2-substituent is a key point of interaction regulating MiaE substrate specificity.
机译：对tRNA进行转录后修饰，以在结构上使tRNA多样化。这些修饰改变了核糖体机制内的非共价相互作用，导致与细胞代谢，生长和毒力有关的表型改变。 MiaE是一种羧酸盐桥联的非血红素二铁单加氧酶，可催化位置37（2-甲硫基-N〜6-异戊烯基-腺苷（37）-tRNA）[被指定为MS〜2i〜 6A _（37）]。在这项工作中，从鼠伤寒沙门氏菌中克隆了重组MiaE，纯化至同质，并通过紫外可见和双模式X波段EPR光谱进行了表征，以便与其他非血红素二铁酶进行比较。此外，合成了三种核苷底物替代物（i〜6A，Cl〜2i〜6A和ms〜2i〜6A）及其相应的羟基化产物（io〜6A，Cl〜2io〜6A和ms〜2io〜6A）。研究这种酶的化学和立体特异性。在没有天然电子传输链的情况下，使用过氧化物分流器监测底物羟基化的速率。显着地，不管在过氧化物分流测定中使用的底物（i〜6A，Cl〜2i〜6A和ms〜2i〜6A），末端异戊烯基-C4-位的羟基化均具有> 97％的E-立体选择性。在酶促测定中未观察到其他非特异性羟基化产物。稳态动力学实验还表明，MiaE羟基化反应的初始速率受核苷碱基C2位置的取代基影响很大（对于ms〜2i〜6A> i〜6A> Cl〜2i〜，v_0 / [E] 6A）。实际上，由MiaE表现出的相对于i〜6A的游离ms〜2i〜6A核苷羟基化的> 3倍速率增强与先前报道的ms〜2io〜6A和io〜6A产物分布的全细胞试验一致天然tRNA底物。该观察结果表明核苷C2取代基是相互作用的关键点，其调节MiaE底物特异性。

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