Crystal Structure and Desulfurization Mechanism of 2′-Hydroxybiphenyl-2-sulfinic Acid Desulfinase

Abstract

The desulfurization of dibenzothiophene in Rhodococcus erythropolis is catalyzed by two monooxygenases, DszA and DszC, and a desulfinase, DszB. In the last step of this pathway, DszB hydrolyzes 2-hydroxybiphenyl-2-sulfinic acid into 2-hydroxybiphenyl and sulfite. We report on the crystal structures of DszB and an inactive mutant of DszB in complex with substrates at resolutions of 1.8 Å or better. The overall fold of DszB is similar to those of periplasmic substrate-binding proteins. Sulfur oxides released into the atmosphere by combustion of fossil fuel cause serious air pollution, which leads to acid rain and destroys forests and soils. Sulfur oxides also raise public health concerns associated with cardiopulmonary diseases (1).To alleviate these problems, efforts are being made to limit the sulfur content in diesel and gasoline fuel. Inorganic or nonaromatic organic sulfur compounds can easily be removed from fossil fuels by the conventional metal-catalyzed hydrodesulfurization method (2), but it is difficult to remove polycyclic aromatic sulfur compounds such as benzothiophene and dibenzothiophene (DBT) 2 (3). In particular, DBT is considered as a model compound of polycyclic organic sulfur compounds in fossil fuels, and degradation of DBT by microbial activities has been studied with a keen interest in biodesulfurization (4, 5).Sulfur-specific DBT degradation pathway, which is capable of transforming DBT to sulfite and 2-hydroxybiphenyl (HBP), was identified in the soil bacterium Rhodococcus erythropolis (6, 7) and found to be catalyzed by the following three enzymes: DszA, DszB, and DszC (8, 9). DszA and DszC are flavin-dependent monooxygenases and responsible for oxidation of DBT to 2Ј-hydroxybiphenyl-2-sulfinic acid (HBPS). A 39-kDa protein DszB (EC 3.13.1.3) participates in the last desulfurization step and hydrolyzes the sulfinate group of HBPS (Scheme 1).The reaction catalyzed by DszB is the slowest in the pathway and thus was proposed as the rate-limiting step in desulfurization (10). DszB is inhibited by the reaction product of HBP but not by biphenyl, which indicates that the hydroxyl group is required for the inhibition. However, DszB can accept biphenyl-2-sulfinic acid (BPS) as a substrate, and the hydroxyl group of HBPS does not seem to be essential for the activity (11). DszB is also strongly inhibited by cysteine-modifying reagents, and mutation of the unique cysteine residue, Cys 27 , into serine abolished the activity (11).Besides biodesulfurization, knowledge of sulfur metabolism by soil bacteria is particularly interesting because of their competition for sulfur nutrient with plants. Although biochemical studies have shown that DszB is a unique desulfinase, its detailed molecular reaction mechanism remains largely unknown. In an attempt to expand our understanding of bacterial desulfurization in molecular detail, we determined crystal structures of DszB in complex with substrates HBPS and BPS. EXPERIMENTAL PROCEDURESMaterials-BPS was synthesized as reported previously (12). Sodium salt of ...

Improvement of 2′-Hydroxybiphenyl-2-sulfinate Desulfinase, an Enzyme Involved in the Dibenzothiophene Desulfurization Pathway, fromRhodococcus erythropolisKA2-5-1 by Site-Directed Mutagenesis

“…Figure 1 shows the 3-D structure of the DszHBPSi complex near the catalytic center. 13) Since there were several aromatic amino acid residues, Phe61, Tyr63, Trp145, Trp155, and Trp228, that might interact with the substrate, alanine was substituted for these amino acids. Only the mutant enzyme at Tyr63 did not lose activity, and others showed little activity (data not shown).…”

“…We have proposed an enzyme reaction mechanism for DszB, unique judging by the chemical reaction formula, based upon the 3-D structure, suggesting that DszB belongs to a new family of desulfinases. 13) The Y63F mutant enzyme had higher maximum activity than the wild-type enzyme. This substitution might enhance the hydrophobic bond between the substrate and the enzyme, leading to increased catalytic activity without a decrease in the affinity for the substrate.…”

“…26) On the other hand, there are only four reports describing the purification and characterization of DszB, 11,18,19,27) and the mutation of the structural gene of DszB has not yet been investigated. Our previous study revealed the 3-D structures of DszB and the DszB-HBPSi complex in detail, 13) and that is the first structural study. In addition, our previous enzymatical investigation showed that Cys residue must be the catalytic center of DszB, because the SH reagents inhibited the enzyme activity.…”

“…Recently, we elucidated the 3-D structure of DszB, which was the first Xray crystallographic study of the enzymes involved in DBT desulfurization. 12,13) In addition, only one cysteine residue in DszB was found to be a catalytic center from the results of enzymological study.…”

“…On the HBPSi. This figure was prepared using the previously reported PDB File, 2DE3, 13) as starting data.…”