Arsenate Reductase, Mycothiol, and Mycoredoxin Concert Thiol/Disulfide Exchange
Arsenate Reductase, Mycothiol, and Mycoredoxin Concert Thiol/Disulfide Exchange
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DOI：
10.1074/jbc.m900877200
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发表时间：
2009-05-29
影响因子：
4.8
通讯作者：
Messens, Joris
中科院分区：
文献类型：
--
作者：
Ordonez, Efren;Van Belle, Karolien;Messens, Joris
我们确定了第一种在硫醇/二硫化物氧化还原级联反应中使用真菌硫醇和真菌氧化还原蛋白的酶。这两种酶是谷氨酸棒杆菌的两种砷酸盐还原酶（Cg ArsC 1和Cg ArsC 2），它们在抵御砷酸盐中起关键作用。体内敲除表明，Cg_ArsC1和Cg_ArsC2的基因和真菌硫醇生物合成途径的酶的基因赋予砷酸盐抗性。通过稳态反应动力学、亚砷酸盐分析和理论反应活性分析，揭示了C.谷氨酸。Cg_ArsCs中的活性位硫醇盐促进砷酸盐和真菌硫醇之间的加合物形成。真菌氧化还原蛋白是一种氧化还原酶，其功能以前从未显示过，还原巯基-砷键并形成亚砷酸盐和真菌硫醇-真菌氧化还原蛋白混合二硫化物。第二分子的真菌硫醇与真菌氧还蛋白结合形成真菌氧化还原酶，而真菌氧化还原酶又被NADPH依赖性真菌氧化还原酶还原。Cg_ArsCs显示出类似于5 M-1 s（-1）的低特异性常数，通常用于三种不同分子上的亲核试剂的硫醇/二硫键级联。随着这一新型电子传递途径的体外重建，我们为放线菌氧化还原机制的研究铺平了道路。
We identified the first enzymes that use mycothiol and mycoredoxin in a thiol/disulfide redox cascade. The enzymes are two arsenate reductases from Corynebacterium glutamicum (Cg ArsC1 and Cg ArsC2), which play a key role in the defense against arsenate. In vivo knockouts showed that the genes for Cg_ArsC1 and Cg_ArsC2 and those of the enzymes of the mycothiol biosynthesis pathway confer arsenate resistance. With steady-state kinetics, arsenite analysis, and theoretical reactivity analysis, we unraveled the catalytic mechanism for the reduction of arsenate to arsenite in C. glutamicum. The active site thiolate in Cg_ArsCs facilitates adduct formation between arsenate and mycothiol. Mycoredoxin, a redox enzyme for which the function was never shown before, reduces the thiol-arseno bond and forms arsenite and a mycothiol-mycoredoxin mixed disulfide. A second molecule of mycothiol recycles mycoredoxin and forms mycothione that, in its turn, is reduced by the NADPH-dependent mycothione reductase. Cg_ArsCs show a low specificity constant of similar to 5 M-1 s(-1), typically for a thiol/disulfide cascade with nucleophiles on three different molecules. With the in vitro reconstitution of this novel electron transfer pathway, we have paved the way for the study of redox mechanisms in actinobacteria.