title: Gene overexpression, purification, and identification of a desulfurization enzyme from Rhodococcus sp. strain IGTS8 as a sulfide/sulfoxide monooxygenase.
abstract: 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. Kovacevich, and J. Rambosek, Appl. Environ. Microbiol. 61:468-475, 1995). However, this enzyme has not been characterized, and the type of its catalytic activity remains unclassified. In this work, the sox/dszC gene was overexpressed in Escherichia coli, a procedure for the purification of the expressed enzyme was developed, and the properties of and the reactions catalyzed by the purified enzyme were characterized. This enzyme binds one flavin mononucleotide (Kd, 7 micrometers) or reduced flavin mononucleotide (FMNH2) (Kd < 10(-8) M) per 90,200-Da homodimer, and FMNH2 is an essential cosubstrate for its activity. Patterns of product formation were examined under different FMNH2 availabilities, and results indicate that this enzyme catalyzes a stepwise conversion of dibenzothiophene to the corresponding sulfoxide and subsequently to the sulfone. On the basis of isotope labeling patterns with H2(18)O and 18O2, dibenzothiophene sulfoxide and sulfone obtained their oxygen atom(s) from molecular oxygen rather than water in their formation from dibenzothiophene. The enzyme also utilizes benzyl sulfide and benzyl sulfoxide as substrates. Hence, it is identified as a sulfide/sulfoxide monooxygenase. This monooxygenase is similar to the microsomal flavin-containing monooxygenase but is unique among microbial flavomonooxygenases in its ability to catalyze two consecutive monooxygenation reactions.
authors.affiliation: Department of Biochemical and Biophysical Sciences, University of Houston, Texas 77204-5934, USA.
authors.forename: B
authors.initials: B
authors.lastname: Lei
authors.forename: S C
authors.initials: SC
authors.lastname: Tu
mesh_terms: D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism*; D001593:Benzyl Compounds / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics; D005486:Flavin Mononucleotide / Q000378:metabolism; D005420:Flavoproteins / Q000235:genetics / Q000378:metabolism*; D013058:Mass Spectrometry; D010088:Oxidoreductases / Q000235:genetics / Q000378:metabolism*; D010100:Oxygen / Q000378:metabolism; D010103:Oxygen Isotopes; D011994:Recombinant Proteins / Q000378:metabolism; D012240:Rhodococcus / Q000201:enzymology* / Q000235:genetics; D013379:Substrate Specificity; D013876:Thiophenes / Q000378:metabolism*; D014867:Water / Q000378:metabolism
chemical_list: D001426:Bacterial Proteins; D001593:Benzyl Compounds; D005420:Flavoproteins; D010103:Oxygen Isotopes; D011994:Recombinant Proteins; D013876:Thiophenes; D014867:Water; C058517:dibenzothiophene 5-oxide; D005486:Flavin Mononucleotide; D010088:Oxidoreductases; C090100:dibenzothiophene monooxygenase; C016367:benzyl sulfide; D010100:Oxygen; C016366:dibenzothiophene