Crystal structures of TdsC, a dibenzothiophene monooxygenase from the thermophile Paenibacillus sp. A11-2, reveal potential for expanding its substrate selectivity

Abstract

Sulfur compounds in fossil fuels are a major source of environmental pollution, and microbial desulfurization has emerged as a promising technology for removing sulfur under mild conditions. The enzyme TdsC from the thermophile sp. A11-2 is a two-component flavin-dependent monooxygenase that catalyzes the oxygenation of dibenzothiophene (DBT) to its sulfoxide (DBTO) and sulfone (DBTO) during microbial desulfurization. The crystal structures of the apo and flavin mononucleotide (FMN)-bound forms of DszC, an ortholog of TdsC, were previously determined, although the structure of the ternary substrate-FMN-enzyme complex remains unknown. Herein, we report the crystal structures of the DBT-FMN-TdsC and DBTO-FMN-TdsC complexes. These ternary structures revealed many hydrophobic and hydrogen-bonding interactions with the substrate, and the position of the substrate could reasonably explain the two-step oxygenation of DBT by TdsC. We also determined the crystal structure of the indole-bound enzyme because TdsC, but not DszC, can also oxidize indole, and we observed that indole binding did not induce global conformational changes in TdsC with or without bound FMN. We also found that the two loop regions close to the FMN-binding site are disordered in apo-TdsC and become structured upon FMN binding. Alanine substitutions of Tyr-93 and His-388, which are located close to the substrate and FMN bound to TdsC, significantly decreased benzothiophene oxygenation activity, suggesting their involvement in supplying protons to the active site. Interestingly, these substitutions increased DBT oxygenation activity by TdsC, indicating that expanding the substrate-binding site can increase the oxygenation activity of TdsC on larger sulfur-containing substrates, a property that should prove useful for future microbial desulfurization applications.

Mechanistic Studies of a Flavin Monooxygenase: Sulfur Oxidation of Dibenzothiophenes by DszC

“…An X-ray structure of a DszC ortholog (TdsC) was very recently obtained with FMN and DBT present in the active site. Therefore, 33 we decided to characterize two reactant states, with DBT modeled from HPA in C 2 :HPAH structure (DBT HPAH ), and from the DBT in the TdsC structure (DBT TdsC ) (both shown in Figure 2). 48 From REACT to TS1, we noted that the hydrogen bond between O d and the N ε of His391 seemed stronger for the DBT TdsC pose (0.06 Å increase) than for DBT HPAH (0.32 Å increase), again suggesting that the DBT TdsC pose was better placed for the nucleophilic attack of S DBT to O d ; while the hydrogen bond between O p and the Tyr96-hydroxyl became shorter in about 0.58 and 0.31 Å, in agreement with the increase in electron density in O p (−0.08 to −0.24 au).…”

“…On a side note, a structure of an enzyme similar to DszC (TdsC from Paenibacillus sp., with 64% identity and identical active site residues) with DBT bound was very recently published (PDB ID: 5XDE). The pose of DBT in this structure showed some differences in relation to the X-ray structure of the parent enzyme HPAH from Acinetobacter baumanni used for the modeling here (see Figure ).…”

Structural and Biochemical Characterization of BdsA from Bacillus subtilis WU-S2B, a Key Enzyme in the “4S” Desulfurization Pathway

“…The structure of DszB was first reported in 2006, and the reaction mechanism was further illustrated ( Lee et al, 2006 ). The crystal structures of DszC and its complex with FMN have also been reported ( Liu et al, 2014 ; Zhang et al, 2014 ; Guan et al, 2015 ), and the homology TdsC has also been reported ( Hino et al, 2017 ). Recently, a liquid chromatography–mass spectrometry analysis showed that the C2 hydroperoxide of the DBT sulfone reacts with reduced flavin to form a flavin-N5-oxide intermediate in DszA, which is involved in subsequent protonation ( Adak and Begley, 2016 ).…”

Improved Efficiency of the Desulfurization of Oil Sulfur Compounds in Escherichia coli Using a Combination of Desensitization Engineering and DszC Overexpression

“…The K I values of the DszC mutants were slightly higher than that of the WT protein, indicating that their substrate inhibition was alleviated (Table 2). The DszC substrate binding pocket has been previously described in detail, [33][34][35][36]43 and the HBP binding site should not be the same as the substrate binding site because the inhibition of DszC by HBP is noncompetitive. 23 Although the true HBP binding site could not be verified in this study, the acquisition of the AKWC mutant made a significant contribution to the feedback inhibition desensitization of DszC.…”

“…XP, Rhodococcus erythropolis D-1, and Paenibacillus sp. A11-2 have been solved, and the binding sites of DBT/FMN have been identified. − Abinfuentes et al stated that there was a noncompetitive inhibition of DszC by HBP, showing that HBP has one or several binding sites on DszC that are different from the DBT substrate binding site. However, the binding site of HBP to DszC is still unknown, hindering efforts to modify DszC using rational design.…”