Investigating the Kinetics, Mechanism, and Reaction Pathway of a Biodesulfurizing Enzyme from Rhodococcus erythropolis, Dibenzothiophene Monooxygenase

Fossil fuel combustion is a major source of sulfur dioxide, dangerous to respiratory health and the environment. Sulfur removal from fossil fuels eases these effects, however, the current method, hydrodesulfurization, requires costly high temperatures and pressures. The bacterial species Rhodococcus erythropolis uses a four‐step enzymatic process, known as biodesulfurization, to remove sulfur in fuel compounds. The first enzyme in the pathway, dibenzothiophene monooxygenase (DszC), uses reduced flavin and molecular oxygen to generate a flavin intermediate that oxygenates its substrate, dibenzothiophene, to dibenzothiophene sulfoxide, then dibenzothiophene sulfone. Our objectives are to understand DszC's enzymatic mechanism and steps of the catalytic pathway to engineer DszC to accept various organosulfur compounds and increase catalytic efficiency. Towards our goals, we have purified active DszC; determined steady‐state kinetic parameters of DszC as a function of dibenzothiophene, dibenzothiophene sulfoxide, and flavin substrates; performed steady‐state experiments to describe DszC's enzymatic mechanism; and used transient‐state kinetics to ascertain the effects of dibenzothiophene and age times on flavin binding. Using affinity chromatography, we purified DszC at 0.5 gram/liter of culture in its native tetrameric state. Reverse‐phase HPLC experiments showed DszC had faster product turnover using dibenzothiophene sulfoxide as substrate versus dibenzothiophene (11.8 × 10 −3 s −1 vs. 4.84 × 10 −3 s −1 ). HPLC assays also demonstrated that having equimolar flavin and DszC resulted in a faster turnover rate than using excess flavin (4.84 × 10 −3 s −1 vs. 1.49 × 10 −3 s −1 ). Varying flavin and dibenzothiophene concentrations under steady‐state conditions indicated DszC likely forms a ternary complex with the flavin intermediate and dibenzothiophene to facilitate oxygenation. Using stopped‐flow spectrophotometry, we observed fast flavin binding (<1 second) and that having equimolar dibenzothiophene and DszC increased intermediate formation rates. Dibenzothiophene pre‐incubation with DszC, however, decreased intermediate formation rates, suggesting reduced flavin is the initial substrate to bind to DszC. These results provided us kinetic and mechanistic insights towards understanding the full catalytic pathway of DszC to support our goals of engineering a robust and efficient DszC enzyme for industrial applications. Support or Funding Information National Institute of Health (SCORE 5SC2AI109500) Research Corporation CCSA 22672 MARC/RISE Program (NIH Grant Number 2R25GM063787‐10) CSUPERB New Investigator Grant This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal .