Flavin reductase

If the luciferase sample solution contains a flavin-reductase, luciferase activity can be measured by the addition of FMN and NADH, instead of FMNH2. In this case, the turnover of luciferase takes place repeatedly using the FMNH2 that is enzymatically generated thus, the luminescence reaction continues until aldehyde or NADH is exhausted. A crude luciferase extracted from luminous bacteria usually contains a flavin-reductase. 

Eschenbrenner M, E Coves, M Fontecave (1995) The flavin reductase activity of the flavoprotein component of sulfite reductase bom Escherichia coli. J Biol Chem 270 20550-20555. 

Russ R, J Rau, A Stolz (2000) The function of cytoplasmic flavin reductases in the reduction of azo dyes by bacteria. Appl Environ Microbiol 66 1429-1434. 

Matsubara T, T Ohshiro, Y Nishina, Y Izumi (2001) Purification, characterization, and overexpression of flavin reductase involved in dibenzothiophene desulfurization by Rhodococcus erythropolis D-1. Appl Environ Microbiol 67 1179-1184. 

There is some evidence that the iron-sulfur protein, FhuF, participates in the mobilization of iron from hydroxamate siderophores in E. coli (Muller et ah, 1998 Hantke, K. unpublished observations). However, a reductase activity of FhuF has not been demonstrated. Many siderophore-iron reductases have been shown to be active in vitro and some have been purified. The characterization of these reductases has revealed them to be flavin reductases which obtain the electrons for flavin reduction from NAD(P)H, and whose main functions are in areas other than reduction of ferric iron (e.g. flavin reductase Fre, sulfite reductase). To date, no specialized siderophore-iron reductases have been identified. It has been suggested that the reduced flavins from flavin oxidoreductases are the electron donors for ferric iron reduction (Fontecave et ah, 1994). Recently it has been shown, after a fruitless search for a reducing enzyme, that reduction of Co3+ in cobalamin is achieved by reduced flavin. Also in this case it was suggested that cobalamins and corrinoids are reduced in vivo by flavins which may be generated by the flavin... 

The flavin reductase has been purified by several researchers. This enzyme from R. erythropolis IGTS8 was partially purified by Ohshiro et al., and reported to have an optimum pH and temperature of 6.0 and 35°C, respectively [153], The DszD enzyme from IGTS8 was also purified [53] and reported to be of 25 kDa size however, no kinetic details related to DszD were reported. This enzyme couples with FMN with NADH to produce reduced flavin required for DszC and DszA catalyzed reactions. 

The Keasling group [175] demonstrated that the flavin reductase from Vibrio harveyi, when expressed in E. coli along with the dsz cassette, enhanced biodesulfurization rate by about six-fold. However, it should be noted that the overexpression of the flavin oxidoreductase results only in increase in DBT removal rate, but not HBP production rate. This is expected since the flavin reductase is only required for the first two steps of desulfurization. Another interesting result from this study was the reduced rate of HBP production when the flavin reductase was co-expressed. This was probably due... 

Table 8. Comparison of flavin reductase from various organisms capable of supporting biodesulfurization...

The thermophilic enzyme DszD from Paenibacillus All-2 has been cloned into E. coli and characterized [172], The sequence of this enzyme showed 30% similarity to the major flavin reductase of Vibrio fischeri. The optimum activity was reported to be at 45°C in resting cell cultures and 55°C in cell-free extracts. 

Another thermophilic flavin reductase, flavoenzyme Bfl, from Bacillus sp. DSM411 was reported to have high thermostability [167], compared to the DszD from R. erythropolis D-l or the P. Polymyxa A-l. It had an optimum temperature of 70°C and optimum pH of 6.0. It retained 90% of its activity at 70°C after 30 min of incubation and was stable in pH range 2-12. 

Xi, L. Squires, C. H. Monticello, D. J., and Childs, J. D., A flavin reductase stimulates DszA and DszC proteins of Rhodococcus erythropohs IGTS8 in vitro. Biochemical and Biophysical Research Communications, 1997. 230(1) pp. 73-75. 

Ohshiro, T. Yamada, H. Shimoda, T., et al., Thermostable Flavin Reductase That Couples With Dibenzothiophene Monooxygenase, From Thermophilic Bacillus Sp DSM411 Purification, Characterization, and Gene Cloning. Bioscience Biotechnology and Biochemistry, 2004. 68(8) pp. 1712-1721. 

Ohshiro, T. Aoi, Y. Torii, K., and Izumi, Y., Flavin Reductase Coupling With Two Monooxygenases Involved in Dibenzothiophene Desulfurization Purification and Characterization From a Non-Desulfurizing Bacterium, Paenibacillus Polymyxa A-l. Applied Microbiology, and Biotechnology, 2002. 59(6) pp. 649-657. 

Matsubara, T. Ohshiro, T. Nishina, Y., and Izumi, Y., Purification, Characterization, and Overexpression of Flavin Reductase Involved in Dibenzothiophene Desulfurization by Rhodococcus Erythropolis D-l. Applied and Environmental, Microbiology, 2001. 67(3) pp. 1179-1184. 

Squires, C. H. Childs, J. D., and Gray, K. A., Rhodococcus flavin reductase complementing DszA and DszC activity. Patent No. W09817787. 1998, April 30. 

Based on the previous publications, azo dye can be reduced by azoreductase-catalyzed reduction under anaerobic conditions. But still there is a speculation whether bacterial flavin reductases are responsible for the azo reductase activity observed with bacterial cell extracts. In a published report, it is reported that flavin reductases are indeed able to act as azo reductases [24]. Bacteria produce extracellular oxidative enzymes, which are relatively nonspecific enzymes catalyzing the oxidation of a variety of dyes. It was reported that so many diverse groups of bacteria play a role in decolorization. It has been also reported that mixed microbial community could reduce various azo dyes, and members of the y-proteabacteria and sulfate reducing bacteria (SRB) were found to be prominent members of mixed bacterial population by using molecular methods to determine the microbial population dynamics [1],... 

Reduced flavins (FADH2, FMNH2, and riboflavin) generated by flavin-dependent reductases have been hypothesized to reduce azo dyes in a nonspecific chemical reaction, and flavin reductases have been revealed to be indeed anaerobic azoreductases. Other reduced enzyme cofactors, for example, NADH, NADH, NADPH, and an NADPH-generating system, have also been reported to reduce azo dyes. Except for enzyme cofactors, different artificial redox mediating compounds, especially such as quinines, are important redox mediators of azo dye anaerobic reduction (Table 1). 

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