Structural biology of Queuosine biosynthesis

Queuosine(Q)是一种超修饰的RNA碱基，它在5‘-GUN-3’tRNA分子的摆动位置上取代鸟嘌呤。Q完全由细菌制成，相应的队列碱基是真核物种抢救出来的微量营养素。Q生物合成的最后一步是环氧化物前体环氧奎宁的还原，得到Q环戊烯环。负责的环氧奎宁还原酶QueG与钴胺依赖的还原脱卤酶(RdhA)有很远的同源性，然而钴胺在QueG催化中所起的作用仍然不清楚。我们最近报道了嗜热链球菌QueG的溶液和结构特征，揭示了该酶含有一个氧化还原链，该链由两个[4Fe-4S]簇和一个碱基关闭形式的CoB(II)丙胺组成，类似于RDHA。与共享的氧化还原链结构相反，QueG活性中心与RdhA几乎没有同源性，除了一个保守的Tyr，它被认为在还原脱卤化过程中作为质子供体发挥作用。这表明，与RdHA催化的不寻常的碳-卤键化学相反，QueG是通过Co-C键的形成而起作用的。我们的研究建立了III类钴胺依赖酶的共同特征，并揭示了这些酶催化的还原化学中意想不到的多样性。我们现在希望从结构/功能的角度研究更广泛的QueG/RdhA家族，并探索这些酶是否可以用作新的还原酶催化剂，这将具有与更传统的基于黄素/NAD(P)H的体系不同的性质。我们筛选不同的同源物进行表达和结晶，并通过定点突变探索现有的QueG/RdhA系统，旨在揭示其机制。后者将用于介绍生物技术的应用，目的是在体外和最终在体内催化所需的减少(如减少酒精)。后者的一个关键部分将是对假定的电子转移伙伴的研究。

Queuosine (Q) is a hypermodified RNA base that replaces guanine in the wobble positions of 5'-GUN-3' tRNA molecules. Q is exclusively made by bacteria, and the corresponding queuine base is a micronutrient salvaged by eukaryotic species. The final step in Q biosynthesis is the reduction of the epoxide precursor, epoxyqueuosine, to yield the Q cyclopentene ring. The epoxyqueuosine reductase responsible, QueG, shares distant homology with the cobalamin-dependent reductive dehalogenase (RdhA), however the role played by cobalamin in QueG catalysis has remained elusive. We have recently reported the solution and structural characterization of Streptococcus thermophilus QueG, revealing the enzyme harbours a redox chain consisting of two [4Fe-4S] clusters and a cob(II)alamin in the base-off form, similar to RdhAs. In contrast to the shared redox chain architecture, the QueG active site shares little homology with RdhA, with the notable exception of a conserved Tyr that is proposed to function as a proton donor during reductive dehalogenation. This suggests that, in contrast to the unusual carbon-halogen bond chemistry catalyzed by RdhAs, QueG acts via Co-C bond formation. Our study establishes the common features of Class III cobalamin-dependent enzymes, and reveal an unexpected diversity in the reductive chemistry catalyzed by these enzymes. We now wish to study the wider QueG/RdhA family from a structure/function perspective, and probe whether these enzymes can be used as new reductase catalysts, that will have distinct properties from the more traditionally used flavin/NAD(P)H based systems. We screen distinct homologues for expression and crystallization, as well as probe the existing QueG/RdhA systems by site directed mutagenesis aimed at unraveling the mechanism. The latter will be used to inform on biotechnological application with the goal to catalyse desirable reductions (such as alcohol reduction) in vitro and ultimately in vivo. A key part of the latter will be the study of the putative electron transfer partners.