Project description:Dimethylsulfide (DMS) is a volatile organosulfur compound which has been implicated in the biogeochemical cycling of sulfur and in climate control. Microbial degradation is a major sink for DMS. DMS metabolism in some bacteria involves its oxidation by a DMS monooxygenase in the first step of the degradation pathway; however, this enzyme has remained uncharacterized until now. We have purified a DMS monooxygenase from Hyphomicrobium sulfonivorans, which was previously isolated from garden soil. The enzyme is a member of the flavin-linked monooxygenases of the luciferase family and is most closely related to nitrilotriacetate monooxygenases. It consists of two subunits: DmoA, a 53-kDa FMNH₂-dependent monooxygenase, and DmoB, a 19-kDa NAD(P)H-dependent flavin oxidoreductase. Enzyme kinetics were investigated with a range of substrates and inhibitors. The enzyme had a K(m) of 17.2 (± 0.48) μM for DMS (k(cat) = 5.45 s⁻¹) and a V(max) of 1.25 (± 0.01) μmol NADH oxidized min⁻¹ (mg protein⁻¹). It was inhibited by umbelliferone, 8-anilinonaphthalenesulfonate, a range of metal-chelating agents, and Hg²(+), Cd²(+), and Pb²(+) ions. The purified enzyme had no activity with the substrates of related enzymes, including alkanesulfonates, aldehydes, nitrilotriacetate, or dibenzothiophenesulfone. The gene encoding the 53-kDa enzyme subunit has been cloned and matched to the enzyme subunit by mass spectrometry. DMS monooxygenase represents a new class of FMNH₂-dependent monooxygenases, based on its specificity for dimethylsulfide and the molecular phylogeny of its predicted amino acid sequence. The gene encoding the large subunit of DMS monooxygenase is colocated with genes encoding putative flavin reductases, homologues of enzymes of inorganic and organic sulfur compound metabolism, and enzymes involved in riboflavin synthesis.
Project description:DmoA is a monooxygenase which uses dioxygen (O2) and reduced flavin mononucleotide (FMNH2) to catalyze the oxidation of dimethylsulfide (DMS). Although it has been characterized, the structure of DmoA remains unknown. Here, the crystal structure of DmoA was determined to a resolution of 2.28 Å and was compared with those of its homologues LadA and BdsA. The results showed that their overall structures are similar: they all share a conserved TIM-barrel fold which is composed of eight α-helices and eight β-strands. In addition, they all have five additional insertions. Detailed comparison showed that the structures have notable differences despite their high sequence similarity. The substrate-binding pocket of DmoA is smaller compared with those of LadA and BdsA.
