Can Selenoenzymes Resist Electrophilic Modification? Evidence from Thioredoxin Reductase and a Mutant Containing α-Methylselenocysteine.
Can Selenoenzymes Resist Electrophilic Modification? Evidence from Thioredoxin Reductase and a Mutant Containing α-Methylselenocysteine.
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DOI：
10.1021/acs.biochem.0c00608
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发表时间：
2020-09-15
期刊：
影响因子：
2.9
通讯作者：
Hondal RJ
中科院分区：
文献类型：
--
作者：
Ste Marie EJ;Wehrle RJ;Haupt DJ;Wood NB;van der Vliet A;Previs MJ;Masterson DS;Hondal RJ
硒半胱氨酸(Sec)是遗传密码中第21个蛋白生成氨基酸。将SEC掺入蛋白质是一个复杂且生物能量成本高昂的过程，这引发了一个问题：“为什么大自然选择了硒？”过去十年出现的一个答案是，SEC对活性氧物种(ROS)不可逆转的氧化失活具有抵抗力。在这里，我们探讨了这个概念是否可以扩展到包括对活性亲电物种(RES)的抵抗力的问题，因为氧和相关化合物只是RES的一个子集。为了验证这一假设，我们灭活了哺乳动物硫氧还蛋白还原酶(Sec-TrxR)，一个含有α-甲基硒半胱氨酸((αMe)Sec-TrxR)的突变体，以及一个半胱氨酸-原生TrxR(Cys-TrxR)与各种亲电体，包括丙烯醛、4-羟基壬烯醛和姜黄素。结果表明，丙烯醛灭活的Sec-TrxR和(αMe)Sec-TrxR突变体分别在2 mM的H_2O_2和5 mM的咪唑中恢复了25%和30%的活性。相比之下，Cys-TrxR在同样的条件下没有恢复活性。我们假设Sec-酶可以经历一个修复过程，通过β-syn Seenoxide消除，排出电泳层，使酶处于氧化的硫化硒状态。(αMe)Sec-TrxR是通过半合成将非天然氨基酸((αMe)Sec)掺入TrxR而产生的，并允许对我们的假设进行严格的测试。这种Sec-衍生物具有更高的抗氧化性和亲电性，因为它缺乏主干Cα-H，后者可以防止通过形成脱氢丙氨酸而损失硒。这是这种独特的氨基酸首次被结合到酶中，是最先进的蛋白质工程的例子。
Selenocysteine (Sec) is the 21st proteogenic amino acid in the genetic code. Incorporation of Sec into proteins is a complex and bioenergetically costly process that evokes the question: “Why did nature choose selenium?” An answer that has emerged over the past decade is that Sec confers resistance to irreversible oxidative inactivation by reactive oxygen species (ROS). Here, we explore the question of whether this concept can be broadened to include resistance to reactive electrophilic species (RES) since oxygen and related compounds are merely a subset of RES. To test this hypothesis we inactivated mammalian thioredoxin reductase (Sec-TrxR), a mutant containing alpha-methylselenocysteine ((αMe)Sec-TrxR), and a cysteine-ortholog TrxR (Cys-TrxR) with various electrophiles including acrolein, 4-hydroxynonenal, and curcumin. Our results show that the acrolein-inactivated Sec-TrxR and the (αMe)Sec-TrxR mutant could regain 25% and 30% activity respectively, when incubated with 2 mM H2O2 and 5 mM imidazole. In contrast, the Cys-TrxR did not regain activity under the same conditions. We posit that Sec-enzymes can undergo a repair process via β-syn selenoxide elimination that ejects the electrophile, leaving the enzyme in the oxidized selenosulfide state. (αMe)Sec-TrxR was created by incorporating the non-natural amino acid ((αMe)Sec into TrxR by semisynthesis and allowed for rigorous testing of our hypothesis. This Sec-derivative enables higher resistance to both oxidative and electrophilic inactivation because it lacks a backbone Cα-H, which prevents loss of selenium through formation of dehydroalanine. This is the first time this unique amino acid has been incorporated into an enzyme and is example of state-of-the-art protein engineering.
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影响因子：
3.7
作者：
Anestål K;Prast-Nielsen S;Cenas N;Arnér ES
通讯作者：
Arnér ES
影响因子：
4.1
作者：
Cai, Jian;Bhatnagar, Aruni;Pierce, William M., Jr.
通讯作者：
Pierce, William M., Jr.
DOI：
10.1073/pnas.2134510100
发表时间：
2003-10-28
影响因子：
11.1
作者：
Gromer, S;Johansson, L;Arnér, ESJ
通讯作者：
Arnér, ESJ
影响因子：
1.8
作者：
Albericio, F;Cases, M;Kates, SA
通讯作者：
Kates, SA
影响因子：
--
作者：
Davies SS;Zhang LS
通讯作者：
Zhang LS