Crystal Structure and Catalysis of the Selenoprotein Thioredoxin Reductase 1
Crystal Structure and Catalysis of the Selenoprotein Thioredoxin Reductase 1
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DOI：
10.1074/jbc.m807068200
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发表时间：
2009-02-06
影响因子：
4.8
通讯作者：
Arner, Elias S. J.
中科院分区：
文献类型：
--
作者：
Cheng, Qing;Sandalova, Tatyana;Arner, Elias S. J.
硒蛋白含有高度反应性的第21个氨基酸硒代半胱氨酸（Sec）编码的预定义的UGA密码子的重新编码。由于缺乏硒蛋白的供应，高化学反应性的Sec，和复杂的翻译机制，硒蛋白的晶体结构是很难获得。因此，SEC参与酶催化的结构先决条件知之甚少。在这里，我们提出了催化活性的大鼠硫氧还蛋白还原酶1（TrxR 1）的晶体结构，揭示了惊喜的C-末端SEC的活性位点，鉴于以前的文献。氧化酶呈现反式构型的硒基硫化物基序，Sec-498的硒原子位于Tyr-116的侧链下方，从而远离被提议参与电子传递到含Sec活性位点的氧化还原活性部分。在减少硒硫醇基序，Sec残基移向溶剂暴露，与其推测的作用，在减少TrxR 1底物或作为目标的亲电子试剂抑制酶。Y116 I突变降低了硫氧还蛋白还原的催化效率，但令人惊讶地增加了使用5-羟基-1，4-萘醌（胡桃醌）作为底物的周转率。同样的突变也降低了对顺铂抑制的敏感性。结果表明，Tyr-116通过与氧化的TrxR 1的硒基硫醚相互作用，从而促进其在酶的还原半反应中的还原，从而对TrxR 1的催化起重要作用。硒基硫醚与酪氨酸苯环的相互作用，影响营业额，底物特异性的开关，和亲电子试剂的敏感性的调制，为TrxR 1的机制提供了重要线索，TrxR 1是一种硒蛋白，对哺乳动物细胞的命运和功能起着重要作用。结果还表明，重组硒蛋白TrxR可以产生高的量和足够的纯度，使晶体结构的测定，这表明这些类型的蛋白质的额外的结构研究是可行的。
Selenoproteins contain a highly reactive 21st amino acid selenocysteine (Sec) encoded by recoding of a predefined UGA codon. Because of a lack of selenoprotein supply, high chemical reactivity of Sec, and intricate translation machineries, selenoprotein crystal structures are difficult to obtain. Structural prerequisites for Sec involvement in enzyme catalysis are therefore sparsely known. Here we present the crystal structure of catalytically active rat thioredoxin reductase 1 (TrxR1), revealing surprises at the C-terminal Sec-containing active site in view of previous literature. The oxidized enzyme presents a selenenyl-sulfide motif in trans-configuration, with the selenium atom of Sec-498 positioned beneath the side chain of Tyr-116, thereby located far from the redox active moieties proposed to be involved in electron transport to the Sec-containing active site. Upon reduction to a selenolthiol motif, the Sec residue moved toward solvent exposure, consistent with its presumed role in reduction of TrxR1 substrates or as target of electrophilic agents inhibiting the enzyme. A Y116I mutation lowered catalytic efficiency in reduction of thioredoxin, but surprisingly increased turnover using 5-hydroxy-1,4-naphthoquinone (juglone) as substrate. The same mutation also decreased sensitivity to inhibition by cisplatin. The results suggest that Tyr-116 plays an important role for catalysis of TrxR1 by interacting with the selenenylsulfide of oxidized TrxR1, thereby facilitating its reduction in the reductive half reaction of the enzyme. The interaction of a selenenylsulfide with the phenyl ring of a tyrosine, affecting turnover, switch of substrate specificity, and modulation of sensitivity to electrophilic agents, gives important clues into the mechanism of TrxR1, which is a selenoprotein that plays a major role for mammalian cell fate and function. The results also demonstrate that a recombinant selenoprotein TrxR can be produced in high amount and sufficient purity to enable crystal structure determination, which suggests that additional structural studies of these types of proteins are feasible.