Purification, characterization, and overexpression of flavin reductase involved in dibenzothiophene desulfurization by Rhodococcus erythropolis D-1

The dibenzothiophene (DBT)-desulfurizing bacterium, Rhodococcus erythropoli s D-l, removes sulfur from DBT to form 2-hydroxybiphenyl using four enzymes , DszC, DszA, DszB, and flavin reductase, In this study, we purified and ch aracterized the flavin reductase from R. erythropolis D-l grown in a medium containing DBT as the sole source of sulfur, It is conceivable that the en zyme is essential for two monooxygenase (DszC and DszA) reactions in vivo. The purified flavin reductase contains no chromogenic cofactors and was fou nd to have a molecular mass of 86 kDa and four identical 22-kDa subunits, T he enzyme catalyzed NADH-dependent reduction of flavin mononucleotide (FMN) , and the K-m values for NADH and FMN were 208 and 10.8 muM, respectively. Flavin adenine dinucleotide was a poor substrate, and NADPH was inert. The enzyme did not catalyze reduction of any nitroaromatic compound. The optima l temperature and optimal pH for enzyme activity were 35 degreesC and 6.0, respectively, and the enzyme retained 30% of its activity after heat treatm ent at 80 degreesC for 30 min. The N-terminal amino acid sequence of the pu rified flavin reductase was identical to that of DszD of R, erythropolis IG TS8 (K. A. Gray, O, S, Pogrebinsky, G. T. Mrachko, L. Xi, D. J. Monticello, and C. H. Squires, Nat, Biotechnol, 14:1705-1709, 1996). The flavin reduct ase gene was amplified with primers designed by using dszD of R, erythropol is IGTS8, and the enzyme was overexpressed in Escherichia coli. The specifi c activity in crude extracts of the overexpressed strain was about 275-fold that of the wild-type strain.