Faculty of Biology - Microbiology, University of Freiburg, 79104, Freiburg, Germany.
Department of Molecular Microbiology and Biotechnology, Institute of Biochemistry, Life Sciences Center, Vilnius University, 10257, Vilnius, Lithuania.
Commun Biol. 2023 Oct 27;6(1):1092. doi: 10.1038/s42003-023-05450-5.
In all domains of life, transfer RNAs (tRNAs) contain post-transcriptionally sulfur-modified nucleosides such as 2- and 4-thiouridine. We have previously reported that a recombinant [4Fe-4S] cluster-containing bacterial desulfidase (TudS) from an uncultured bacterium catalyzes the desulfuration of 2- and 4-thiouracil via a [4Fe-5S] cluster intermediate. However, the in vivo function of TudS enzymes has remained unclear and direct evidence for substrate binding to the [4Fe-4S] cluster during catalysis was lacking. Here, we provide kinetic evidence that 4-thiouridine-5'-monophosphate rather than sulfurated tRNA, thiouracil, thiouridine or 4-thiouridine-5'-triphosphate is the preferred substrate of TudS. The occurrence of sulfur- and substrate-bound catalytic intermediates was uncovered from the observed switch of the S = 3/2 spin state of the catalytic [4Fe-4S] cluster to a S = 1/2 spin state upon substrate addition. We show that a putative gene product from Pseudomonas putida KT2440 acts as a TudS desulfidase in vivo and conclude that TudS-like enzymes are widespread desulfidases involved in recycling and detoxifying tRNA-derived 4-thiouridine monophosphate nucleosides for RNA synthesis.
在所有生命领域中，转移 RNA（tRNA）都含有转录后硫修饰的核苷，如 2-和 4-硫尿嘧啶。我们之前曾报道过，一种来自未培养细菌的含有[4Fe-4S]簇的重组脱硫酶（TudS）通过[4Fe-5S]簇中间体能催化 2-和 4-硫代尿嘧啶的脱硫反应。然而，TudS 酶的体内功能仍不清楚，并且缺乏催化过程中底物与[4Fe-4S]簇结合的直接证据。在这里，我们提供了动力学证据表明 4-硫代尿嘧啶-5'-单磷酸而不是硫代化 tRNA、硫代尿嘧啶、硫代尿苷或 4-硫代尿嘧啶-5'-三磷酸是 TudS 的首选底物。观察到催化[4Fe-4S]簇的 S = 3/2 自旋状态向 S = 1/2 自旋状态的转变，这揭示了硫和底物结合的催化中间产物的存在。我们表明，来自恶臭假单胞菌 KT2440 的假定基因产物在体内充当 TudS 脱硫酶，并得出结论，TudS 样酶是广泛存在的脱硫酶，参与回收和解毒 tRNA 衍生的 4-硫代尿嘧啶单核苷酸用于 RNA 合成。
