BackgroundDibenzothiophene monooxygenase (DszC), a two‐component class D flavin monooxygenase (FMO) that catalyzes the first step of the Rhodococcus erythropolis biodesulfurization pathway, has potential for development as a biocatalyst. DszC catalyzes the sequential oxidation of dibenzothiophene to dibenzothiophene sulfoxide and dibenzothiophene sulfone, activating the molecule for desulfurization by DszA and DszB in a three‐step metabolic pathway. Here we examine catalysis by DszC and the functional roles of four proposed active site residues (H92, S163, H391 and V261) by site directed mutagenesis, steady state and transient kinetics, spectrophotometry and x‐ray crystallography.MethodsPlasmids carrying the N‐terminally six‐histidine‐tagged expression constructs for wild type and mutant DszC (H92A, S163A, H391A, V261F) were generated by subcloning and whole plasmid PCR. Protein was expressed in BL21(DE3) E. coli cells and purified by affinity chromatography. Steady state activity was monitored at varying pH, temperature, and substrate concentrations. A spectrophotometric assay was used to quantitate flavin binding, and preliminary stopped‐flow spectrophotometric experiments were conducted to observe catalytic intermediates. In addition, crystallization experiments focusing on the substrate‐bound form of the enzyme are being pursued.ResultsAll of our active site mutants of DszC appear to disrupt catalysis. Mutation of H391 and S163 to alanine results in lower enzymatic activity and weaker flavin binding, with observed dissociation constants for reduced flavin of 72.3, 105.0, and >155.1 μM for the wild type, H391A, and S163A, respectively. This is in agreement with the prediction that both residues play important roles in flavin binding. Wild type DszC catalyzes formation of the C4a‐(hydro)peroxyflavin intermediate in an O2‐dependent manner, but formation of this intermediate by the H391A and S163A mutants was not observed. Co‐crystallization experiments of wild type DszC and the V261F mutant with substrate and/or flavin have yielded initial crystals that are currently being optimized to improve diffraction.Conclusions and future directionsH391 and S163 play important roles in catalysis by DszC that likely involve formation or breakdown of the C4a‐(hydro)peroxyflavin intermediate. These roles, and those of the other active site residues under study here, will be further clarified by additional transient‐state kinetics studies and co‐crystallization experiments.Support or Funding InformationThis work was supported by funds provided by the CSUN Department of Chemistry and Biochemistry, the CSUN College of Science and Math, the Research Corporation (CCSA Grant Number 22672) and by the NIH (Grant R25GM063787‐12).