No abstract

Flavin monoxygenases (FMOs) are enzymes of increasing biotechnological (e.g., crude oil biodesulfurization) and pharmacological (e.g., drug metabolism) interest that perform the oxidation of soft nucleophiles and play key roles in the excretion of xenobiotics or in sulfur amino acid metabolism. DszC is a key FMO involved in sulfur oxidation of dibenzothiophenes (DBTs) through the 4S metabolic pathway of some bacteria. This pathway can be a cheaper and greener alternative for sulfur removal, as DBTs are the major source of crude oil sulfur. Here, we investigate the reaction mechanism of DszC with quantum mechanics/molecular mechanics methods (SCS-MP2/def2-TZVPP:ff10//B3LYP/6-31G(d):ff10). We observe that the reaction mechanism of DBT oxidation occurs in three stages: (1) spin-forbidden formation of a C4a-hydroperoxyflavin (C4aOOH) intermediate; (2) oxidation of DBT to DBTO, upon nucleophilic attack of the DBT-sulfur on the distal oxygen of C4aOOH; and (3) proton transfer from the N5H of the flavin group to the His92-imidazole via Ser163-hydroxyl, releasing a water molecule and oxidized flavin mononucleotide. The overall reaction is computed to be exergonic (−38.7 kcal·mol–1), and the rate-limiting step is the oxidation of DBT to DBTO (ΔG ‡ = 19.7 kcal·mol–1, consistent with the experimental turnover of 1.6 min–1). We observe that oxygen activation is a nearly spontaneous process that occurs through a proton-coupled electron transfer to produce a hydroperoxyl radical, followed by a triplet-singlet spin-forbidden inversion to form the C4aOOH intermediate. In agreement with other studies, His391 is a key acid to activate O2 and form the covalent bond. Further clarifying previous mutagenesis results, we also propose that His92 and Tyr96 are key residues for the mechanism: His92 acts as acid to deprotonate N5H in flavin via Ser163; and Tyr96 enhances the oxidation of DBT-sulfur by anchoring the proximal oxygen of C4aOOH, and acts as acid to form the water byproduct and regenerate the flavin cofactor. These are important results to clarify the chemistry of flavin monoxygenases and to open doors for the rational design of DszC mutants with improved catalytic activity.

Dissimilatory sulfur metabolism was recently shown to be much more widespread among bacteria and archaea than previously believed. One of the key pathways involved is the dsr pathway that is responsible for sulfite reduction in sulfate-, sulfur-, thiosulfate-, and sulfite-reducing organisms, sulfur disproportionators and organosulfonate degraders, or for the production of sulfite in many photo- and chemotrophic sulfur-oxidizing prokaryotes. The key enzyme is DsrAB, the dissimilatory sulfite reductase, but a range of other Dsr proteins is involved, with different gene sets being present in organisms with a reductive or oxidative metabolism. The dsrD gene codes for a small protein of unknown function and has been widely used as a functional marker for reductive or disproportionating sulfur metabolism, although in some cases this has been disputed. Here, we present in vivo and in vitro studies showing that DsrD is a physiological partner of DsrAB and acts as an activator of its sulfite reduction activity. DsrD is expressed in respiratory but not in fermentative conditions and a ΔdsrD deletion strain could be obtained, indicating that its function is not essential. This strain grew less efficiently during sulfate and sulfite reduction. Organisms with the earliest forms of dsrAB lack the dsrD gene, revealing that its activating role arose later in evolution relative to dsrAB.

The development of biocatalytic desulfurization strategies of petroleum and its derivatives could result in more economic alternatives than the widely used chemical desulfurization. The organism Rhodococcus erythropolis IGTS8 has been shown to metabolize organic sulfur compounds through a mechanism known as 4S pathway, which involves four enzymes (DszA, DszB, DszC, and DszD) and has been explored in biodesulfurization. Here we have applied QM/MM methods to study the catalytic mechanism of the enzyme DszD, a NADH-FMN oxidoreductase that occupies a central place on the 4S pathway by catalyzing the formation of the FMNH2 that is used by the two monooxynases in the cycle: DszA and DszC. In addition, to clarify the catalytic mechanism of this enzyme, this study analyzed in detail the role played by the active site Thr residue and of Asn and Ala enzyme mutants. The results help to explain previous experimental evidence and suggest new strategies for improving biodesulfurization through an increase in the activity of DszD.