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

Abstract

The dibenzothiophene (DBT)-desulfurizing bacterium, Rhodococcus erythropolis D-1, removes sulfur from DBT to form 2-hydroxybiphenyl using four enzymes, DszC, DszA, DszB, and flavin reductase. In this study, we purified and characterized the flavin reductase from R. erythropolis D-1 grown in a medium containing DBT as the sole source of sulfur. It is conceivable that the enzyme is essential for two monooxygenase (DszC and DszA) reactions in vivo. The purified flavin reductase contains no chromogenic cofactors and was found to have a molecular mass of 86 kDa and four identical 22-kDa subunits. The enzyme catalyzed NADH-dependent reduction of flavin mononucleotide (FMN), and the K(m) values for NADH and FMN were 208 and 10.8 microM, respectively. Flavin adenine dinucleotide was a poor substrate, and NADPH was inert. The enzyme did not catalyze reduction of any nitroaromatic compound. The optimal temperature and optimal pH for enzyme activity were 35 degrees C and 6.0, respectively, and the enzyme retained 30% of its activity after heat treatment at 80 degrees C for 30 min. The N-terminal amino acid sequence of the purified flavin reductase was identical to that of DszD of R. erythropolis IGTS8 (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 reductase gene was amplified with primers designed by using dszD of R. erythropolis IGTS8, and the enzyme was overexpressed in Escherichia coli. The specific activity in crude extracts of the overexpressed strain was about 275-fold that of the wild-type strain.

FIG. 1

DBT desulfurization pathway of R. erythropolis D-1.

FIG. 2

SDS-PAGE of flavin reductase from R. erythropolis D-1. Lanes 1 and 8, marker proteins; lane 2, cell extract of R. erythropolis D-1; lane 3, pooled fractions containing DszD after Q-Sepharose chromatography; lane 4, pooled fractions containing DszD after Butyl-Toyopearl chromatography; lane 5, pooled fractions containing DszD after Phenyl-Toyopearl chromatography; lane 6, fractions after membrane treatment; lane 7, purified DszD enzyme after FMN agarose chromatography.

FIG. 3

Effects of temperature on flavin reductase stability. After purified enzyme was preincubated at different temperatures for 30 min, enzyme reactions were carried out as described in Materials and Methods. The total reaction volume was 0.5 ml, and 0.55 μg of purified enzyme from the wild-type strain was added to each reaction mixture. The enzyme activity was determined by measuring the decrease in absorbance at 340 nm.

FIG. 4

Effect of NEM on flavin reductase activity. Purified enzyme (5.5 μg) from the wild-type strain was preincubated in a total volume of 0.25 ml at 0°C with 1 mM NEM (■), 1 mM NEM and 200 μM FMN (○), or 1 mM NEM and 500 μM NADH (▴). At different times, 25-μl aliquots were withdrawn, and enzyme reactions were carried out by using 0.4 mM NADH and 0.2 mM FMN as described in Materials and Methods. The enzyme activity was determined by measuring the decrease in absorbance at 340 nm.

FIG. 5

Effect of FMN concentration on enzyme activity. Enzyme reactions were carried out as described in Materials and Methods by using reaction mixtures containing 20 mM potassium phosphate buffer (pH 7.0), 0.4 mM NADH, 0.55 μg of purified enzyme from the wild-type strain, and different concentrations of FMN in a total volume of 0.5 ml. The enzyme activity was determined by measuring the decrease in absorbance at 340 nm.