GENE OVEREXPRESSION, PURIFICATION, AND IDENTIFICATION OF A DESULFURIZATION ENZYME FROM RHODOCOCCUS SP STRAIN IGTS8 AS A SULFIDE SULFOXIDE MONOOXYGENASE/

The oxidation of dibenzothiophene to dibenzothiophene sulfone has been linked to the enzyme encoded by the sox/dszC gene from Rhodococcus sp , strain IGTS8 (S. A. Denome, C. Oldfield, L. J. Nash, and K. D. Young , J. Bacteriol, 176:6707-6717, 1994; C. S. Piddington, B. R. Kovacevic h, and J. Rambosek, Appl. Environ. Microbiol, 61:468-475, 1995), Howev er, this enzyme has not been characterized, and the type of its cataly tic activity remains unclassified, In this work, the sox/dszC gene was overexpressed in Escherichia coli, a procedure for the purification o f the expressed enzyme was developed, and the properties of and the re actions catalyzed by the purified enzyme were characterized, This enzy me binds one flavin mononucleotide (K-d, 7 mu M) or reduced flavin mon onucleotide (FMNH(2)) (K-d < 10(-8) M) per 90,200-Da homodimer, and FM NH(2) is an essential cosubstrate for its activity, Patterns of produc t formation were examined under different FMNH, availabilitie(2), and results indicate that this enzyme catalyzes a stepwise conversion of d ibenzothiophene to the corresponding sulfoxide and subsequently to the sulfone, On the basis of isotope labeling patterns with (H2O)-O-18 an d O-18(2), dibenzothiophene sulfoxide and sulfone obtained their oxyge n atom(s) from molecular oxygen rather than water in their formation f rom dibenzothiophene, The enzyme also utilizes benzyl sulfide and benz yl sulfoxide as substrates. Hence, it is identified as a sulfide/sulfo xide monooxygenase, This monooxygenase is similar to the microsomal fl avin-containing monooxygenase but is unique among microbial flavomonoo xygenases In its ability to catalyze two consecutive monooxygenation r eactions.