Investigating Amino Acid Residues in The Active Site of Dibenzothiophene Monooxygenase (DszC)

Abstract

Fossil fuel combustion releases sulfur dioxide into the atmosphere, which is a harmful air pollutant and precursor of sulfate aerosols that contribute to acid rain. It is necessary to reduce atmospheric sulfur levels to mitigate these effects. A current method, hydrodesulfurization, is costly and inefficient at removing sulfur from complex aromatic compounds. Biodesulfurization is a cost‐effective alternative that utilizes bacteria to remove sulfur. Rhodococcus erythropolis uses a four enzyme pathway to remove sulfur from dibenzothiophene. The first enzyme of the pathway, dibenzothiophene monooxygenase (DszC), requires molecular oxygen, reduced flavin, and the formation of an intermediate, C4a‐hydroperoxyflavin, to sequentially oxidize dibenzothiophene to dibenzothiophene sulfone. This study aims to elucidate the roles of DszC active site residues in catalysis and binding and to enable rational engineering of DszC for future application in fuel refinement. Active site residue mutants were created using polymerase chain reaction, followed by plasmid DNA isolation and sequence confirmation. Steady state activity assays of DszC, quantitated by reverse‐phase HPLC, compared the amount of product formed by the mutants relative to wild type‐DszC. The ability of the mutants to form the intermediate was studied using stopped‐flow spectrophotometry. In this study, seven active site residue mutants were created and the percent of product formation by each mutant relative to wild type‐DszC was: His92Asn (12%), His92Gln (20%), Tyr96Phe (12%), His388Ala (38%), His388Asn (39%), His391Val (0%) and Asp392Ala (3%). These results indicate that all of the residues analyzed are necessary for proper product formation. Additionally, the intermediate formation rate constant for Tyr96Phe (1.03 × 104 M−1 s−1) was similar to wild type‐DszC (1.69 × 104 M−1 s−1). These results indicate that Tyr96 is not critical for proper intermediate formation, but has another role in catalysis as evidenced by the low (12%) product formation. His92Asn and Asp392Ala were also analyzed using stopped‐flow spectrophotometry but showed no intermediate formation signifying that these residues are essential to proper intermediate formation. Overall, these results show the necessity of all the residues presented in product formation and the fundamental roles of residues His92 and Asp392 in intermediate formation.Support or Funding InformationNational Institute of Health (NIH SCORE 5SC2AI109500)Research Corporation (CCSA 22672)MBRS/RISE Program/National Institute of Health (Grant 2R25GM063787‐10)This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.