Rider Lance W, Ottosen Mette B, Gattis Samuel G, Palfey Bruce A
Department of Biological Chemistry, The University of Michigan Medical School, Ann Arbor, Michigan 48109-5606, USA.
J Biol Chem. 2009 Apr 17;284(16):10324-33. doi: 10.1074/jbc.M806137200. Epub 2009 Jan 12.
Dihydrouridine synthases (DUSs) are flavin-dependent enzymes that catalyze site-specific reduction of uracils in tRNAs. The mechanism of DUS 2 from Saccharomyces cerevisiae was studied. Previously published turnover rates for this DUS were very low. Our studies show that the catalytic cycle consists of reductive and oxidative half-reactions. The enzyme is reduced by NADPH rapidly but has a very slow oxidative half-reaction using in vitro transcribed tRNA substrates. Using tRNA(Leu) purified from a DUS 2 knockout strain of yeast we obtained reaction rate enhancements of 600-fold over in vitro transcribed substrates, indicating that other RNA modifications are required for rapid uracil reduction. This demonstrates a previously unknown ordering of modifications and indicates that dihydrouridine formation is a later step in tRNA maturation. We also show that an active site cysteine is important for catalysis, likely in the protonation of uracil during tRNA reduction. Dihydrouridine of modified tRNA from Escherichia coli was also oxidized to uridine showing the reaction to be reversible.
二氢尿苷合酶（DUSs）是黄素依赖性酶，可催化tRNA中尿嘧啶的位点特异性还原。对酿酒酵母的DUS 2的机制进行了研究。此前公布的该DUS的周转速率非常低。我们的研究表明，催化循环由还原和氧化半反应组成。该酶可被NADPH快速还原，但使用体外转录的tRNA底物时氧化半反应非常缓慢。使用从酵母DUS 2基因敲除菌株中纯化的tRNA（Leu），我们获得的反应速率比体外转录底物提高了600倍，这表明快速尿嘧啶还原需要其他RNA修饰。这证明了一种以前未知的修饰顺序，并表明二氢尿苷的形成是tRNA成熟过程中的后期步骤。我们还表明，活性位点的半胱氨酸对催化很重要，可能在tRNA还原过程中尿嘧啶的质子化中起作用。来自大肠杆菌的修饰tRNA的二氢尿苷也被氧化为尿苷，表明该反应是可逆的。
