Biophysical/Bioinorganic Group and ‡Synthetic Organic Group, Department of Chemistry and Biochemistry, College of Science, The University of Texas at Arlington , Arlington, Texas 76019, United States.
Biochemistry. 2013 Sep 10;52(36):6182-96. doi: 10.1021/bi4000832. Epub 2013 Aug 27.
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 O2-dependent hydroxylation of a hypermodified-tRNA nucleoside at position 37 (2-methylthio-N(6)-isopentenyl-adenosine(37)-tRNA) [designated ms(2)i(6)A37]. 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(6)A, Cl(2)i(6)A, and ms(2)i(6)A) and their corresponding hydroxylated products (io(6)A, Cl(2)io(6)A, and ms(2)io(6)A) 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(6)A, Cl(2)i(6)A, and ms(2)i(6)A) 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 (v0/[E] for ms(2)i(6)A > i(6)A > Cl(2)i(6)A). Indeed, the >3-fold rate enhancement exhibited by MiaE for the hydroxylation of the free ms(2)i(6)A nucleoside relative to i(6)A is consistent with previous whole cell assays reporting the ms(2)io(6)A and io(6)A product distribution within native tRNA-substrates. This observation suggests that the nucleoside C2-substituent is a key point of interaction regulating MiaE substrate specificity.
tRNA 的转录后修饰可使 tRNA 结构多样化。这些修饰改变核糖体机制中的非共价相互作用，导致与细胞代谢、生长和毒力相关的表型变化。MiaE 是一种羧酸桥接的非血红素二铁单加氧酶，可催化位置 37（2-甲基硫代-N(6)-异戊烯基-腺苷(37)-tRNA）的超修饰 tRNA 核苷的 O2 依赖性羟化（指定为 ms(2)i(6)A37）。在这项工作中，从鼠伤寒沙门氏菌中克隆了重组 MiaE，将其纯化至均相，并通过紫外可见和双模 X 波段 EPR 光谱进行表征，与其他非血红素二铁酶进行比较。此外，还合成了三种核苷底物类似物（i(6)A、Cl(2)i(6)A 和 ms(2)i(6)A）及其相应的羟化产物（io(6)A、Cl(2)io(6)A 和 ms(2)io(6)A），以研究该酶的化学和立体特异性。在不存在天然电子传递链的情况下，利用过氧化物分流来监测底物羟化的速率。值得注意的是，无论在过氧化物分流测定中使用何种底物（i(6)A、Cl(2)i(6)A 和 ms(2)i(6)A），都观察到末端异戊烯基-C4-位置的羟化，E-立体选择性>97%。在酶促测定中未观察到其他非特异性羟化产物。稳态动力学实验还表明，MiaE 羟化的初始速率受核苷碱基 C2 位取代基的高度影响（v0/[E]对于 ms(2)i(6)A > i(6)A > Cl(2)i(6)A）。事实上，MiaE 对游离 ms(2)i(6)A 核苷的羟化速率比 i(6)A 提高了>3 倍，这与先前报道的天然 tRNA 底物中 ms(2)io(6)A 和 io(6)A 产物分布的全细胞测定结果一致。这一观察结果表明，核苷 C2 取代基是调节 MiaE 底物特异性的关键相互作用点。
