Unprecedented pathway of reducing equivalents in a diflavin-linked disulfide oxidoreductase
Unprecedented pathway of reducing equivalents in a diflavin-linked disulfide oxidoreductase
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DOI：
10.1073/pnas.1713698114
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发表时间：
2017-11-28
影响因子：
11.1
通讯作者：
Balsera, Monica
中科院分区：
文献类型：
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作者：
Buey, Ruben M.;Arellano, Juan B.;Balsera, Monica
黄蛋白参与各种各样的生理相关过程，通常涉及氧化还原反应。在这个蛋白质超家族中，有一组能够将还原等价物从FAD转移到氧化还原活性的二硫键上，从而进一步减少靶蛋白中的二硫键，从而调节其结构和功能。我们已经确定了一种以前未被描述的黄素酶，它是产氧光合原核生物所特有的，它基于被指定为NADPH依赖的硫氧还蛋白还原酶(NTR)的初级序列。然而，我们的实验数据表明，该蛋白质不像NTRs那样将还原等价物从黄素转移到二硫化物，而是以相反的方向发挥作用。紫球藻和聚球藻蛋白质的高分辨结构通过X射线结晶学得到的PCC6803显示，每个单体有两个并列的FAD分子，在NTRS采用的折叠的变体中，具有活性的二硫键进行氧化还原通讯。我们暂时将黄素蛋白命名为DDOR(双黄素连接的二硫键氧化还原酶)，并提出其活性与细菌膜上基于硫醇的还原等价物的转移有关。这些发现扩展了具有氧化还原酶活性的黄素酶的结构和机制，并为探索新的蛋白质工程方法铺平了道路，旨在为不同的生物技术应用设计氧化还原活性蛋白质。
Flavoproteins participate in a wide variety of physiologically relevant processes that typically involve redox reactions. Within this protein superfamily, there exists a group that is able to transfer reducing equivalents from FAD to a redox-active disulfide bridge, which further reduces disulfide bridges in target proteins to regulate their structure and function. We have identified a previously undescribed type of flavin enzyme that is exclusive to oxygenic photosynthetic prokaryotes and that is based on the primary sequence that had been assigned as an NADPH-dependent thioredoxin reductase (NTR). However, our experimental data show that the protein does not transfer reducing equivalents from flavins to disulfides as in NTRs but functions in the opposite direction. High-resolution structures of the protein from Gloeobacter violaceus and Synechocystis sp. PCC6803 obtained by X-ray crystallography showed two juxtaposed FADmolecules per monomer in redox communication with an active disulfide bridge in a variant of the fold adopted by NTRs. We have tentatively named the flavoprotein "DDOR" (diflavin-linked disulfide oxidoreductase) and propose that its activity is linked to a thiol-based transfer of reducing equivalents in bacterial membranes. These findings expand the structural and mechanistic repertoire of flavoenzymes with oxidoreductase activity and pave the way to explore new protein engineering approaches aimed at designing redox-active proteins for diverse biotechnological applications.