Biochemical and Spectroscopic Studies of Epoxyqueuosine Reductase: A Novel Iron-Sulfur Cluster- and Cobalamin-Containing Protein Involved in the Biosynthesis of Queuosine
Biochemical and Spectroscopic Studies of Epoxyqueuosine Reductase: A Novel Iron-Sulfur Cluster- and Cobalamin-Containing Protein Involved in the Biosynthesis of Queuosine
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DOI：
10.1021/acs.biochem.5b00335
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发表时间：
2015-08-11
期刊：
影响因子：
2.9
通讯作者：
Bandarian, Vahe
中科院分区：
文献类型：
--
作者：
Miles, Zachary D.;Myers, William K.;Bandarian, Vahe
肌苷是存在于tRNA的摆动位置的高度修饰的核苷，其反密码子（His、Asp、Asn和Tyr）中具有5 '-GUN-3'序列。碱基的7-脱氮嘌呤核心是在原核生物中从鸟苷5 '-三磷酸在一系列8个连续的酶促转化中从头合成的，最后3个发生在tRNA上。环氧肌苷还原酶（QueG）催化途径中的最后一步，其需要环氧肌苷的双电子还原以形成肌苷。生化分析表明，这种酶需要钴胺素和两个[4Fe-4S]簇催化。光谱研究表明，钴胺素似乎以碱基关闭构象结合，其中辅因子的二甲基苯并咪唑部分从钴的配位范围中去除，但没有被咪唑侧链取代，这是许多钴胺素的标志依赖性酶。生物信息学鉴定的残基被证明在调节钴胺素的初级协调领域中具有作用。这些研究首次证明了QueG的辅因子要求。
Queuosine is a hypermodified nucleoside present in the wobble position of tRNAs with a 5'-GUN-3' sequence in their anticodon (His, Asp, Asn, and Tyr). The 7-deazapurine core of the base is synthesized de novo in prokaryotes from guanosine 5'-triphosphate in a series of eight sequential enzymatic transformations, the final three occurring on tRNA. Epoxyqueuosine reductase (QueG) catalyzes the final step in the pathway, which entails the two-electron reduction of epoxyqueuosine to form queuosine. Biochemical analyses reveal that this enzyme requires cobalamin and two [4Fe-4S] clusters for catalysis. Spectroscopic studies show that the cobalamin appears to bind in a base-off conformation, whereby the dimethylbenzimidazole moiety of the cofactor is removed from the coordination sphere of the cobalt but not replaced by an imidazole side chain, which is a hallmark of many cobalamin-dependent enzymes. The bioinformatically identified residues are shown to have a role in modulating the primary coordination sphere of cobalamin. These studies provide the first demonstration of the cofactor requirements for QueG.