2-(2'-Hydroxyphenyl)benzene sulfinate desulfinase from the thermophilic desulfurizing bacterium Paenibacillus sp. strain A11-2: purification and characterization.
2-(2'-Hydroxyphenyl)benzene sulfinate (HPBSi) desulfinase (TdsB), which catalyzes the final step of desulfurization of dibenzothiophene (DBT), was purified from a thermophilic DBT- and benzothiophene (BT)-desulfurizing bacterium: Paenibacillus sp. strain A11-2. The molecular mass of the purified enzyme was 31 kDa and 39 kDa by gel filtration and sodium dodecyl sulfate polyacrylamide gel electrophoresis, respectively, suggesting a monomeric structure. The optimal temperature and pH for the reaction involving TdsB was 55 degrees C and the enzyme was more resistant to heat treatment than DszB, a counterpart purified from Rhodococcus erythropolis. The optimum pH for TdsB activity was pH 8. TdsB converted HPBSi to 2-hydroxybiphenyl (2-HBP) and sulfite stoichiometrically. The Km and kcat values for HPBSi were 0.33 mM and 0.32 s(-1), respectively. TdsB was inactivated by SH reagents such as p-chloromercuribenzoic acid and 5,5'-dithio-bis-2-nitrobenzoic acid, but was not inhibited by chelating reagents such as EDTA and o-phenanthroline. TdsB was also inhibited by o-hydroxystyrene, the final desulfurized product of BT. However, 2-HBP and its derivatives showed only a weak inhibitory effect. TdsB desulfurized 2-(2'-hydroxyphenyl)ethen-1-sulfinate to yield o-hydroxystyrene, but DszB could not. A site-directed mutagenesis study revealed the cysteine residue at position 17 to be essential to the catalytic activity of TdsB.
Konishi
J
J
Bio-Refining Process Laboratory, Japan Cooperation Center, Petroleum, 1900 Sodeshi-cho, 424-0037, Shimizu, Shizuoka, Japan. konishi@brpl.jccp.or.jp
Maruhashi
K
K
D001406:Bacillaceae / Q000201:enzymology* / Q000235:genetics; D006358:Hot Temperature; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D010088:Oxidoreductases / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D050862:Oxidoreductases Acting on Sulfur Group Donors; D013379:Substrate Specificity; D013455:Sulfur / Q000378:metabolism*; D013876:Thiophenes / Q000378:metabolism; D001426:Bacterial Proteins
D010088:Oxidoreductases; D050862:Oxidoreductases Acting on Sulfur Group Donors; D013455:Sulfur; D013876:Thiophenes; C113832:2-(2-hydroxybiphenyl)-benzenesulfinate desulfinase; D001426:Bacterial Proteins; C016366:dibenzothiophene