Structural insights into the stabilization of active, tetrameric DszC by its C-terminus

Abstract

Dibenzothiophene (DBT) is a typical sulfur-containing compound found in fossil fuels. This compound and its derivatives are resistant to the hydrodesulfurization method often used in industry, but they are susceptible to enzymatic desulfurization via the 4S pathway, which is a well-studied biochemical pathway consisting of four enzymes. DBT monooxygenase (DszC) from Rhodococcus erythropolis is involved in the first step of the 4S pathway. We determined the crystal structure of DszC, which reveals that, in contrast to several homologous proteins, the C-terminus (410-417) of DszC participates in the stabilization of the substrate-binding pocket. Analytical ultracentrifugation analysis and enzymatic assays confirmed that the C-terminus is important for the stabilization of the active conformation of the substrate-binding pocket and the tetrameric state. Therefore, the C-terminus of DszC plays a significant role in the catalytic activity of this enzyme.

Initial investigations of C4a-(hydro)peroxyflavin intermediate formation by dibenzothiophene monooxygenase

“…Wild-type and mutant DszC expressed at high levels and were purified to apparent homogeneity using nickel affinity chromatography (Supplemental Figure 3) at yields of 60-70 mg of purified protein per liter of culture. Native PAGE (Supplemental Figure 4) and size exclusion chromatography results are consistent with DszC existing as a tetramer in solution, as observed by others [8,11,12,13]. …”

“…DszC activity is determined by monitoring conversion of DBT (or DBTO) to DBTO 2 , using HPLC to quantitate reaction rates [5,8,12]. Fre and excess NADH are included in our assays to provide a sufficient amount of FMNH − for catalysis and catalase is included to minimize any nonenzymatic DBT oxidation due to reaction with free H 2 O 2 generated by autocatalytic oxidation of flavin.…”

“…The FMNH − required for catalysis is produced by DszD [9], the flavin reductase component of the two-component monooxygenase system [10]. Five X-ray crystal structures have shown DszC to be a tetrameric protein with sequence and structural similarity to the acyl-CoA dehydrogenases [8,11,12,13]. Based on its sequence homology and catalytic requirements, DszC is a Class D monooxygenase [14,15], along with p-hydroxyphenylacetate hydroxylase (HpaH) [16], chlorophenol-4-monooxygenase [17], nitrososynthase [18], and others [19,20,21,22].…”

“…Based on its sequence homology and catalytic requirements, DszC is a Class D monooxygenase [14,15], along with p-hydroxyphenylacetate hydroxylase (HpaH) [16], chlorophenol-4-monooxygenase [17], nitrososynthase [18], and others [19,20,21,22]. DszC interacts with flavin as do other Class D monooxygenases [8,11,12,13], and two loops adjacent to the active site, both of which adopt distinct “open” and “closed” conformations, may control access to the active site pocket [8,13]. Mutagenesis of individual active site residues, including H92, S163, H388, H391 and D392 (Supplemental Figure 2), abolishes enzymatic activity [8,12].…”

“…DszC interacts with flavin as do other Class D monooxygenases [8,11,12,13], and two loops adjacent to the active site, both of which adopt distinct “open” and “closed” conformations, may control access to the active site pocket [8,13]. Mutagenesis of individual active site residues, including H92, S163, H388, H391 and D392 (Supplemental Figure 2), abolishes enzymatic activity [8,12]. The specific catalytic roles of each of these residues are currently unknown.…”