Purification and characterization of the aromatic desulfinase, 2-(2'-hydroxyphenyl)benzenesulfinate desulfinase.
2-(2(')-Hydroxyphenyl)benzenesulfinate desulfinase (HPBS desulfinase) catalyzes the cleavage of the carbon-sulfur bond of 2-(2(')-hydroxyphenyl)benzenesulfinate (HPBS) to form hydroxybiphenyl and sulfite. This is the final step in the desulfurization of dibenzothiophene, the organosulfur compound used to study biodesulfurization of petroleum middle distillate. HPBS desulfinase was purified 1600-fold from Rhodococcus IGTS8. The purification was monitored using a spectrofluorimetric assay and SDS-PAGE. The pI of HPBS desulfinase is 5.6, the temperature optimum is 35 degrees C, and the pH optimum is 7.0. HPBS desulfinase has a K(m) of 0.90+/-0.15 microM and a k(cat) of 1.3+/-0.07 min(-1). Several analogs were tested for their ability to act as substrates or inhibitors of HPBS desulfinase. No alternative substrates and very few inhibitors were identified. HPBS desulfinase activity decreases in the presence of Cu(2+) and Zn(2+), while no metals significantly enhance enzyme activity. HPBS desulfinase is susceptible to tyrosine, tryptophan, and cysteine specific modification agents.
Watkins
L M
LM
Department of Chemistry and Biochemistry, Southwest Texas State University, 601 University Dr., San Marcos, TX 78666, USA. LW09@swt.edu
Rodriguez
R
R
Schneider
D
D
Broderick
R
R
Cruz
M
M
Chambers
R
R
Ruckman
E
E
Cody
M
M
Mrachko
G T
GT
D002478:Cells, Cultured; D004789:Enzyme Activation; D004791:Enzyme Inhibitors / Q000737:chemistry; D004795:Enzyme Stability; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D010088:Oxidoreductases / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000302:isolation & purification* / Q000378:metabolism; D011786:Quality Control; D012240:Rhodococcus / Q000737:chemistry* / Q000145:classification / Q000201:enzymology / Q000302:isolation & purification; D013045:Species Specificity; D013696:Temperature; D001426:Bacterial Proteins; D050862:Oxidoreductases Acting on Sulfur Group Donors
D004791:Enzyme Inhibitors; D010088:Oxidoreductases; C113832:2-(2-hydroxybiphenyl)-benzenesulfinate desulfinase; D001426:Bacterial Proteins; D050862:Oxidoreductases Acting on Sulfur Group Donors