Structural and Mechanistic Insights Into Dimethylsulfoxide Formation Through Dimethylsulfide Oxidation

Abstract

Dimethylsulfide (DMS) and dimethylsulfoxide (DMSO) are widespread in marine environment, and are important participants in the global sulfur cycle. Microbiol oxidation of DMS to DMSO represents a major sink of DMS in marine surface waters. The SAR11 clade and the marine Roseobacter clade (MRC) are the most abundant heterotrophic bacteria in the ocean surface seawater. It has been reported that trimethylamine monooxygenase (Tmm, EC 1.14.13.148) from both MRC and SAR11 bacteria likely oxidizes DMS to generate DMSO. However, the structural basis of DMS oxidation has not been explained. Here, we characterized a Tmm homolog from the SAR11 bacterium Pelagibacter sp. HTCC7211 (Tmm₇₂₁₁). Tmm₇₂₁₁ exhibits DMS oxidation activity in vitro. We further solved the crystal structures of Tmm₇₂₁₁ and Tmm₇₂₁₁ soaked with DMS, and proposed the catalytic mechanism of Tmm₇₂₁₁, which comprises a reductive half-reaction and an oxidative half-reaction. FAD and NADPH molecules are essential for the catalysis of Tmm₇₂₁₁. In the reductive half-reaction, FAD is reduced by NADPH. In the oxidative half-reaction, the reduced FAD reacts with O₂ to form the C4a-(hydro)peroxyflavin. The binding of DMS may repel the nicotinamide ring of NADP⁺, and make NADP⁺ generate a conformational change, shutting off the substrate entrance and exposing the active C4a-(hydro)peroxyflavin to DMS to complete the oxidation of DMS. The proposed catalytic mechanism of Tmm₇₂₁₁ may be widely adopted by MRC and SAR11 bacteria. This study provides important insight into the conversion of DMS into DMSO in marine bacteria, leading to a better understanding of the global sulfur cycle.

Keywords: DMS; DMSO; SAR11; catalytic mechanism; flavin-containing monooxygenase.

FIGURE 1

Characterization of Tmm₇₂₁₁. (A) SDS-PAGE analysis of the purified Tmm₇₂₁₁ protein. (B) Absorbance spectra of Tmm₇₂₁₁. Black line, the absorbance spectrum of the purified Tmm₇₂₁₁. Red line, the absorbance spectrum of the purified Tmm₇₂₁₁ with the addition of equimolar amount of NADPH (0.1 mM) under aerobic conditions. (C) HPLC assay of the enzymatic activity of the recombinant Tmm₇₂₁₁ on DMS. The peaks of DMSO and NADP⁺ monitored at 210 nm were indicated with black and red arrows, respectively. The reaction system without Tmm₇₂₁₁ was used as the control. The DMSO (1.25 mM) and NADP⁺ (0.4 mM) standards were used as positive controls. (D) Effect of temperature on the enzymatic activity of Tmm₇₂₁₁. (E) Effect of pH on the enzymatic activity of Tmm₇₂₁₁. The optimal pH was examined at 25°C using Bis-Tris buffer for pH 6–7, Tris buffer for pH 7–9 and glycine buffer for pH 9–10. The standard errors are from three independent experiments.

FIGURE 2

Overall structure of Tmm₇₂₁₁. (A) Two monomers of Tmm₇₂₁₁ arranged as a dimer in an asymmetric unit. The monomers are colored in magenta and orange, respectively. (B) The overall structure of Tmm₇₂₁₁ monomer. Tmm₇₂₁₁ contains an NADPH binding domain (colored in orange) and a FAD binding domain (colored in magenta) connected through two hinge regions (colored in cyan). The NADP⁺ molecule and the FAD molecule are shown as sticks colored in green and yellow, respectively. (C) Gel filtration analysis of Tmm₇₂₁₁. Inset, semilog plot of the molecular mass of all standards used vs. their K_av values (black squares). The red arrow indicates the position of the K_av value of Tmm₇₂₁₁ (0.48) interpolated in the regression line. Tmm₇₂₁₁ monomer has a calculated molecular mass of 52 kDa. The apparent molecular mass of Tmm₇₂₁₁ is 95 kDa, indicating that Tmm₇₂₁₁ is a dimer in solution.

FIGURE 3

The binding of the NADP⁺ molecule and the FAD molecule in Tmm₇₂₁₁. The NADP⁺ molecule and the FAD molecule are shown as sticks colored in green and yellow, respectively. (A) Electrostatic surface of Tmm₇₂₁₁. The NADP⁺ molecule is partially visible through the surface. (B) Electrostatic surface of Tmm₇₂₁₁ after the transparency of surface was set to 40%. (C) Interactions between NADP⁺ and Tmm₇₂₁₁ residues. (D) Interactions between FAD and Tmm₇₂₁₁ residues. Water molecules are shown in red dots. The possible hydrogen bonds are represented by dashed lines. (E) The enzymatic activities of WT Tmm₇₂₁₁ and its mutants. The activity of WT Tmm₇₂₁₁ is taken as 100%. The standard errors are from three independent experiments. (F) CD spectra of WT Tmm₇₂₁₁ and its mutants.

FIGURE 4

Analysis of the conformational change of NADP⁺. (A) Structure of Tmm₇₂₁₁. (B) Structure of Tmm₇₂₁₁-5-min. (C) Structure of Tmm₇₂₁₁-20-min. The NADP⁺ molecule and the FAD molecule are shown as sticks colored in green and yellow, respectively. The F_o-F_c densities for NADP⁺ are contoured in blue meshes at 3.0σ. (D) Kinetic analysis of WT Tmm₇₂₁₁ toward NADPH. (E) Kinetic analysis of the mutant Asp314Ala toward NADPH.

FIGURE 5

A proposed catalytic cycle of Tmm₇₂₁₁ oxidizing DMS to generate DMSO. In the reductive half-reaction, FAD is reduced by NADPH. In the oxidative half-reaction, the reduced FAD reacts with O₂, and a C4a-(hydro)peroxyflavin (FAD intermediate) is formed. The nicotinamide ring of NADP⁺ protects the FAD intermediate from solvent attack. When DMS enters the catalytic pocket, NADP⁺ generates a conformational change to form a hydrogen bond with Asp314, shutting off the substrate entrance and exposing the FAD intermediate to DMS. After the reaction, DMSO, NADP⁺ and a water molecule are released and the oxidized FAD is regenerated.

FIGURE 6

Structural comparisons between Tmm₇₂₁₁ and three other reported bacterial FMOs. (A) Superimposition of the structures of Tmm₇₂₁₁, NiFMO (PDB code: 6HNS), mFMO (PDB code: 2VQ7) and RnTmm (PDB code: 5IPY). Structures of Tmm₇₂₁₁, NiFMO, mFMO and RnTmm are colored in yellow, purple, salmon and cyan, respectively. (B) The locations of the NADP⁺ and FAD in the four structures. The NADP⁺ and FAD molecules are shown as sticks.