Masters Thesis

Initial biochemical characterization of dibenzothiophene monooxygenase by steady state and transient state kinetics

Sulfur dioxide is generated by the combustion of organic sulfur compounds in fossil fuels. Anthropogenic emissions of sulfur dioxide are a major contributing factor to poor air quality associated with adverse environmental and health effects. The development of more efficient desulfurization technologies are needed to reduce the sulfur content of crude oils used as fuel. One approach involves the application of microbial desulfurization, such as the Rhodococcus erythropolis biodesulfurization pathway. This organism utilizes dibenzothiophene (DBT), which is a model organosulfur compound refractory to hydrodesulfurization, as its only sulfur source. The initiating step of this pathway is catalyzed by a Class D flavin monooxygenase, dibenzothiophene monooxygenase (DszC), which oxygenates DBT sequentially to dibenzothiophene sulfone (DBTO2). To investigate catalysis by DszC we used site directed mutagenesis coupled with steady state and transient state kinetics. We targeted four active site residues to determine the functional roles in the DszC reaction. Mutagenesis of H92, S163, and H391 to alanine destroys the steady state activity of DszC, while mutation of V261 to phenylalanine has minimal impact on catalysis. We quantified product formation with high-pressure liquid chromatography and found that DszC and the V261F mutant convert DBT to DBTO2 with an apparent turnover number (kcat) of 5.34 ± 0.026 x 10-4 s-1 and 4.03 ± 0.14 x 10-4 s-1, respectively. Spectrophotometric titrations were used to quantitate approximate dissociation constants (Kd) for wild-type DszC, S163A-DszC, and H391A-DszC. DszC appears to bind to reduced flavin with a dissociation constant of 53 ± 11 µM. Mutagenesis of the S163 increases the dissociation constant to 147 ± 49 µM. This is consistent with its predicted function of hydrogen binding to the N5 of FMN: removal of that side chain removes the hydrogen bonding interaction and weakens affinity. Conversely, the H391A mutant exhibits a dissociation constant of 64 ± 12 µM, similar to wild-type DszC, which suggests that the histidine side chain is not directly involved in FMN binding. Formation of the C4a-hydroperoxyflavin intermediate was monitored by stopped-flow spectrophotometry. DszC catalyzes the formation of the intermediate with a second order rate constant of 1.69 x 104 M-1 s-1. Although the S163A and H391A mutants can bind and oxidize reduced flavin, they cannot form or stabilize the intermediate. This is consistent with our steady state kinetic studies and the proposed roles of both residues involving the stabilization and formation of the intermediate. In the absence of H391, the residue cannot provide a positive charge and/or act as a Lewis acid to facilitate the formation of the C4a-hydroperoxyflavin. The V261F mutant does not form the C4a-hydroperoxyflavin intermediate as well as wild-type DszC and oxidizes reduced FMN much slower. Despite that this valine is not predicted to directly bind to FMN, mutation to phenylalanine decreases the enzyme’s ability to form the C4a-hydroperoxyflavin intermediate. We have confirmed that DszC binds reduced flavin and forms the C4a-hydroperoxyflavin, as expected based on similar Class D monooxygenase systems. Further studies are required to obtain more mechanistic information about the DszC catalyzed reaction.

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