Fre Is the Major Flavin Reductase Supporting Bioluminescence from Vibrio harveyi Luciferase in Escherichia coli
Fre Is the Major Flavin Reductase Supporting Bioluminescence from Vibrio harveyi Luciferase in Escherichia coli
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DOI：
10.1074/jbc.m808977200
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发表时间：
2009-03-27
影响因子：
4.8
通讯作者：
Baldwin, Thomas O.
中科院分区：
文献类型：
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作者：
Campbell, Zachary T.;Baldwin, Thomas O.
与绝大多数黄素酶不同，细菌荧光素酶需要外源还原的黄素单核苷酸来进行生物发光活性。在生物发光细菌细胞中，物种特有的氧化还原酶被认为为荧光素酶活性提供了减少的黄素。在大肠杆菌中表达生物发光的还原黄素的来源尚不清楚。在大肠杆菌中有两种可能参与这一过程的候选蛋白质，一种是哈维氏弧菌FRP氧化还原酶的同源物NFSA，另一种是LuxG型氧化还原酶Fre。使用单基因敲除菌株，我们发现fre基因的缺失使大肠杆菌的光输出减少了两个数量级以上，但对荧光素酶的表达没有影响。以FMN或核黄素为底物，纯化的Fre在体外能够支持生物发光，其活性与内源哈维氏弧菌还原酶(FRP)相当。在下拉实验中，我们发现Fre和FRP都不能与荧光素酶共纯化。与以前的工作相比，我们没有发现荧光素酶和氧化还原酶之间形成稳定复合体的证据。我们的结论是，在大肠杆菌中，一种主要负责核黄素还原(FRE)的酶也可以用来支持高水平的生物发光。
Unlike the vast majority of flavoenzymes, bacterial luciferase requires an exogenous source of reduced flavin mononucleotide for bioluminescence activity. Within bioluminescent bacterial cells, species-specific oxidoreductases are believed to provide reduced flavin for luciferase activity. The source of reduced flavin in Escherichia coli-expressing bioluminescence is not known. There are two candidate proteins potentially involved in this process in E. coli, a homolog of the Vibrio harveyi Frp oxidoreductase, NfsA, and a luxG type oxidoreductase, Fre. Using single gene knock-out strains, we show that deletion of fre decreased light output by greater than two orders of magnitude, yet had no effect on luciferase expression in E. coli. Purified Fre is capable of supporting bioluminescence in vitro with activity comparable to that with the endogenous V. harveyi reductase (Frp), using either FMN or riboflavin as substrate. In a pull-down experiment, we found that neither Fre nor Frp co-purify with luciferase. In contrast to prior work, we find no evidence for stable complex formation between luciferase and oxidoreductase. We conclude that in E. coli, an enzyme primarily responsible for riboflavin reduction (Fre) can also be utilized to support high levels of bioluminescence.