Dibenzothiophene (DBT) in fossil fuels can be efficiently biodesulfurized by a thermophilic bacterium Mycobacterium goodii X7B. Flavin reductase DszD, which catalyzes the reduction of oxidated flavin by NAD(P)H, is indispensable for the biodesulfurization process. In this work, a Flavin reductase DszD in M. goodii X7B was purified to homogeneity, and then its encoding gene dszD was amplified and expressed in Escherichia coli. DszD is a homodimer with each subunit binding one FMN as cofactor. The Km values for FMN and NADH of the purified recombinant DszD were determined to be 6.6 +/- 0.3 mu M and 77.9 +/- 5.4 mu M, respectively. The optimal temperature for DszD activity was 55 degrees C. DszD can use FMN or FAD as substrate to generate FMNH2 or FADH(2) as product. DszD was coexpressed with DBT monooxygenase DszC, the enzyme catalyzing the first step of the biodesulfurization process. It was indicated that the coexpressed DszD could effectively enhance the DszC catalyzed DBT desulfurization reaction. (c) 2012 Elsevier Ltd. All rights reserved.