Purification and characterization of the monooxygenase catalyzing sulfur-atom specific oxidation of dibenzothiophene and benzothiophene from the thermophilic bacterium Paenibacillus sp. strain A11-2.
A benzothiophene (BT) and dibenzothiophene (DBT) monooxygenase (TdsC), which catalyzes the oxidation of the sulfur atoms in BT and DBT molecules, was purified from Paenibacillus sp. strain A11-2. The molecular mass of the purified enzyme and its subunit were determined to be 200 kDa and 43 kDa by gel filtration and sodium dodecyl sulfate polyacrylamide gel electrophoresis, respectively, indicating a tetrameric structure. The N-terminal amino acid sequence of the purified TdsC completely matched the amino acid sequence deduced from the nucleotide sequence of the tdsC gene reported previously [Ishii et al. (2000) Biophys Biochem Res Commun 270:81-88]. The optimal temperature and pH for the TdsC reaction were 65 degrees C and pH 9, respectively. TdsC required NADH, FMN and TdsD, a NADH-dependent FMN oxidoreductase, for its activity, as was observed for TdsA. FAD, lumiflavin and/or NADPH had some effect on the maintenance of TdsC activity. A comparison of the substrate specificity of TdsC and DszC, the homologous monooxygenase purified from Rhodococcus erythropolis strain KA2-5-1, demonstrated a contrasting pattern towards alkylated DBTs and BTs.
Konishi
J
J
Bio-Refining Process Laboratory, Japan Cooperation Center, Petroleum, 1900 Sodeshi-cho, Shimizu, Shizuoka, 424-0037, Japan. konishi@brpl.jccp.or.jp
Ishii
Y
Y
Onaka
T
T
Maruhashi
K
K
D000478:Alkylation; D001426:Bacterial Proteins; D004791:Enzyme Inhibitors / Q000494:pharmacology; D016937:Gram-Positive Endospore-Forming Rods / Q000201:enzymology*; D006863:Hydrogen-Ion Concentration; D010088:Oxidoreductases / Q000302:isolation & purification* / Q000378:metabolism; D050862:Oxidoreductases Acting on Sulfur Group Donors; D013379:Substrate Specificity; D013455:Sulfur / Q000378:metabolism*; D013696:Temperature; D013876:Thiophenes / Q000378:metabolism*
D001426:Bacterial Proteins; D004791:Enzyme Inhibitors; D013876:Thiophenes; C088015:benzothiophene; D013455:Sulfur; D010088:Oxidoreductases; D050862:Oxidoreductases Acting on Sulfur Group Donors; C407144:TdsC protein, Paenibacillus; C016366:dibenzothiophene