Publications

• Dibenzothiophene desulfurizing enzymes from moderately thermophilic bacterium Bacillus subtilis WU-S2B: purification, characterization and overexpression.

  Ohshiro T., Ishii Y., Matsubara T., Ueda K., Izumi Y., Kino K., Kirimura K.

  The moderately thermophilic bacterium Bacillus subtilis WU-S2B desulfurized dibenzothiophene (DBT) at 50 degrees C through the selective cleavage of carbon-sulfur bonds. In this study, three enzymes involved in the microbial DBT desulfurization were purified and characterized. The first two enzyme ... >> More

  The moderately thermophilic bacterium Bacillus subtilis WU-S2B desulfurized dibenzothiophene (DBT) at 50 degrees C through the selective cleavage of carbon-sulfur bonds. In this study, three enzymes involved in the microbial DBT desulfurization were purified and characterized. The first two enzymes, DBT monooxygenase (BdsC) and DBT sulfone monooxygenase (BdsA), were purified from the wild-type strain, and the last one, 2'-hydroxybiphenyl 2-sulfinic acid desulfinase (BdsB), was purified from the recombinant Escherichia coli overexpressing the gene, bdsB, with chaperonin genes, groEL/ES. The genes of BdsC and BdsA were also overexpressed. The molecular weights of BdsC and BdsA were determined to be 200 and 174 kDa, respectively, by gel filtration chromatography, suggesting that both enzymes had four identical subunits. BdsB had a monomeric structure of 40 kDa. The three enzymes were characterized and compared with the corresponding enzymes (DszC, DszA, and DszB) of mesophilic desulfurization bacteria. The specific activities of BdsC, BdsA, and BdsB were 84.2, 855, and 280 units/mg, respectively, and the latter two activities were higher than those of DszA and DszB. The heat stability and optimum temperature of BdsC, BdsA, and BdsB were higher than those of DszC, DszA, and DszB. Other enzymatic properties were investigated in detail. << Less

  J Biosci Bioeng 100:266-273(2005) [PubMed] [EuropePMC]

• Flavin-N5-oxide: A new, catalytic motif in flavoenzymology.

  Adak S., Begley T.P.

  Flavin-N5-oxide is a recently discovered intermediate used by EncM (1,3-diketone oxidation), DszA (sulfone monooxygenase) and RutA (amide monooxygenase). This review describes the mechanism of these enzymes and proposes criteria for the identification of additional Flavin-N5-oxide dependent enzyme ... >> More

  Flavin-N5-oxide is a recently discovered intermediate used by EncM (1,3-diketone oxidation), DszA (sulfone monooxygenase) and RutA (amide monooxygenase). This review describes the mechanism of these enzymes and proposes criteria for the identification of additional Flavin-N5-oxide dependent enzymes. << Less

  Arch Biochem Biophys 632:4-10(2017) [PubMed] [EuropePMC]

  This publication is cited by 6 other entries.

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

  Ohshiro T., Kojima T., Torii K., Kawasoe H., Izumi Y.

  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, D ... >> More

  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 degrees C and about 7.5. The activity of the enzyme was inhibited by Mn2+, Ni2+, 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. << Less

  J Biosci Bioeng 88:610-616(1999) [PubMed] [EuropePMC]

• Purification and characterization of dibenzothiophene sulfone monooxygenase and FMN-dependent NADH oxidoreductase from the thermophilic bacterium Paenibacillus sp. strain A11-2.

  Konishi J., Ishii Y., Onaka T., Ohta Y., Suzuki M., Maruhashi K.

  A dibenzothiophene (DBT) sulfone monooxygenase (TdsA), which catalyses the oxidative CS bond cleavage of DBT sulfone to produce 2-(2-hydroxyphenyl)benzenesulfinate (HPBS) was purified from the thermophilic DBT desulfurizing bacterium Paenibacillus sp. strain A11-2 by multistep chromatography. The ... >> More

  A dibenzothiophene (DBT) sulfone monooxygenase (TdsA), which catalyses the oxidative CS bond cleavage of DBT sulfone to produce 2-(2-hydroxyphenyl)benzenesulfinate (HPBS) was purified from the thermophilic DBT desulfurizing bacterium Paenibacillus sp. strain A11-2 by multistep chromatography. The molecular mass of the purified enzyme was determined to be 120 kDa by gel filtration and the subunit molecular mass was calculated to be 48 kDa by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) indicating a dimeric structure. The N-terminal amino acid sequence of the purified TdsA was determined to be MRQMHLAGFFAAGNTHH, which revealed no significant similarity to any other known amino acid sequences. The purified TdsA absolutely required an oxidoreductase for its activity. This oxidoreductase (TdsD) was also purified to homogeneity, and its molecular size was calculated to be 50 kDa and 25 kDa by gel filtration and SDS-PAGE, respectively. TdsD was completely FMN-dependent, and FAD could not act as a cofactor. The N-terminal amino acid sequence of the purified TdsD was determined to be TSQTAEQSIAPIVAQYRHPEQPISALFVNR, which showed significant similarity to kinesin-like protein (44% identity). The optimal temperatures for the activity of TdsA and TdsD were 45 degrees C and 55 degrees C, respectively. Both enzymes showed optimal activity at pH 5.5. TdsA was slightly inhibited by sulfate, but not by 2-hydroxybiphenyl (2-HBP), which is another end product of DBT. TdsA showed higher activity toward bulkier substrates than its mesophilic counterpart, DszA. These properties suggest the applicability of biodesulfurization to the processing of actual petroleum fractions. << Less

  J Biosci Bioeng 90:607-613(2000) [PubMed] [EuropePMC]

• Molecular mechanisms of biocatalytic desulfurization of fossil fuels.

  Gray K.A., Pogrebinsky O.S., Mrachko G.T., Xi L., Monticello D.J., Squires C.H.

  The development of biocatalytic desulfurization of petroleum fractions may allow its use in place of conventional hydrodesulfurization (HDS). Dibenzothiophene (DBT) is representative of a broad range of sulfur heterocycles found in petroleum that are recalcitrant to desulfurization via HDS. Rhodoc ... >> More

  The development of biocatalytic desulfurization of petroleum fractions may allow its use in place of conventional hydrodesulfurization (HDS). Dibenzothiophene (DBT) is representative of a broad range of sulfur heterocycles found in petroleum that are recalcitrant to desulfurization via HDS. Rhodococcus sp. strain IGTS8 has the ability to convert DBT to 2-hydroxybiphenyl (HBP) with the release of inorganic sulfur. The conversion of DBT to HBP is catalyzed by a multienzyme pathway consisting of two monooxygenases and a desulfinase. The final reaction catalyzed by the desulfinase appears to be the rate limiting step in the pathway. Each of the enzymes has been purified to homogeneity and their kinetic and physical properties studied. Neither monooxygenase has a tightly bound cofactor and each requires an NADH-FMN oxidoreductase for activity. An NADH-FMN oxidoreductase has been purified from Rhodococcus and is a protein of approximately 25,000 molecular weight with no apparent sequence homology to any other protein in the databases. We describe a unique sulfur acquisition system that Rhodococcus uses to obtain sulfur from very stable heterocyclic molecules. << Less

  Nat. Biotechnol. 14:1705-1709(1996) [PubMed] [EuropePMC]

  This publication is cited by 3 other entries.

• Dibenzothiophene Catabolism Proceeds via a Flavin-N5-oxide Intermediate.

  Adak S., Begley T.P.

  The dibenzothiophene catabolic pathway converts dibenzothiophene to 2-hydroxybiphenyl and sulfite. The third step of the pathway, involving the conversion of dibenzothiophene sulfone to 2-(2-hydroxyphenyl)-benzenesulfinic acid, is catalyzed by a unique flavoenzyme DszA. Mechanistic studies on this ... >> More

  The dibenzothiophene catabolic pathway converts dibenzothiophene to 2-hydroxybiphenyl and sulfite. The third step of the pathway, involving the conversion of dibenzothiophene sulfone to 2-(2-hydroxyphenyl)-benzenesulfinic acid, is catalyzed by a unique flavoenzyme DszA. Mechanistic studies on this reaction suggest that the C2 hydroperoxide of dibenzothiophene sulfone reacts with flavin to form a flavin-N5-oxide. The intermediacy of the flavin-N5-oxide was confirmed by LC-MS analysis, a co-elution experiment with chemically synthesized FMN-N5-oxide and (18)O2 labeling studies. << Less

  J Am Chem Soc 138:6424-6426(2016) [PubMed] [EuropePMC]

  This publication is cited by 2 other entries.