Differential transfers of reduced flavin cofactor and product by bacterial flavin reductase to luciferase

被引:37
作者
Jeffers, CE
Tun, SC [1 ]
机构
[1] Univ Houston, Dept Biol & Biochem, Houston, TX 77204 USA
[2] Univ Houston, Dept Chem, Houston, TX 77204 USA
关键词
D O I
10.1021/bi0024310
中图分类号
Q5 [生物化学]; Q7 [分子生物学];
学科分类号
071010 ; 081704 ;
摘要
It is believed that the reduced FMN substrate required by luciferase from luminous bacteria is provided in vivo by NAD(P)H-FMN oxidoreductases (flavin reductases). Our earlier kinetic study indicates a direct flavin cofactor transfer from Vibrio harveyi NADPH-preferring flavin reductase P (FRPH) to the luciferase (L-H) from the same bacterium in the in vitro coupled luminescence reaction. Kinetic studies were carried out in this work to characterize coupled luminescence reactions using FRPH and the Vibrio fischeri NAD(P)H-utilizing flavin reductase G (FRG(F)) in combination with L-H or luciferase from V. fischeri (L-F) Comparisons of K-m values of reductases for flavin and pyridine nucleotide substrates in single-enzyme and luciferase-coupled assays indicate a direct transfer of reduced flavin, in contrast to free diffusion, from reductase to luciferase by all enzyme couples tested. Kinetic mechanisms were determined for the FRG(F)-L-F and FRPH-L-F coupled reactions. For these two and the FRG(F)-L-H coupled reactions, patterns of FMN inhibition and effects of replacement of the FMN cofactor of FRPH and FRG(F) by 2-thioFMN were also characterized. Similar to the FRPH-L-H couple, direct cofactor transfer was detected for FRG(F)-L-F and FRPH-L-F. In contrast, despite the structural similarities between FRG(F) and FRPH and between L-F and L-H, direct flavin product transfer was observed for the FRG(F)-L-H couple. The mechanism of reduced flavin transfer appears to be delicately controlled by both flavin reductase and luciferase in the couple rather than unilaterally by either enzyme species.
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收藏
页码:1749 / 1754
页数:6
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