Purification and characterization of dibenzothiophene (DBT) sulfone monooxygenase, an enzyme involved in DBT desulfurization, from Rhodococcus erythropolis D-1

Abstract

Dibenzothiophene (DBT), a model of organic sulfur compound in petroleum, is microbially desulfurized to 2-hydroxybiphenyl by Rhodococcus erythropolis D-1. Three desulfurization (Dsz) enzymes—DszC, A, and B—and flavin reductase are involved in sulfur-specific DBT desulfurization. In this study, DszA was purified, characterized, and crystallized from R. erythropolis D-1. DszA, DBT sulfone monooxygenase, is the second enzyme in microbial DBT desulfurization metabolism and catalyzes the conversion of DBT sulfone to 2′-hydroxybiphenyl 2-sulfinic acid in the presence of flavin reductase with cleavage of the carbon-sulfur bond in the DBT skeleton. Using anion-exchange column chromatography, the four enzyme fractions responsible for DBT desulfurization were separated, and DszA was then purified to homogeneity. Polygonal crystals of DszA were observed within a week. DszA was found to have a molecular mass of 97 kDa and to consist of two subunits with identical masses of 50 kDa. The N-terminal amino acid sequence of the purified DszA completely coincided with the deduced amino acid sequence for dszA of R. erythropolis IGTS8 except for a methionine residue at the latter N-terminal. The optimal temperature and pH for DszA activity were 35°C and about 7.5. The activity of the enzyme was inhibited by Mn 2+, Ni 2+, 2,2′-bipyridine, and 8-quinolinol, suggesting that a metal might be involved in its activity. DszA acted on not only DBT sulfone but also on dibenz[ c,e][1,2]oxathiin 6-oxide and dibenz[ c,e][1,2]oxathiin 6,6-dioxide. Dihydroxybiphenyl was formed from the latter two substrates.