Purification and Characterization of Dimethylsulfide Monooxygenase fromHyphomicrobium sulfonivorans

Abstract

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. 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 FMNH 2 -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.Dimethylsulfide (DMS) is a volatile organosulfur compound, important in the biogeochemical cycling of sulfur and global climate regulation (4, 9). Bacterial metabolism of DMS is an important sink of the compound in nature and is thought to account for degradation of over 80% of the DMS produced in the marine environment. Although bacterial pathways of DMS degradation have been studied previously in Hyphomicrobium spp. and in Thiobacillus spp. (12, 36), they remain poorly characterized, and few enzymes of DMS metabolism have been purified (see reference 32). DMS monooxygenase was first reported from an assay of NADH-dependent oxygen uptake in the presence of DMS by cell extracts of Hyphomicrobium S (12), an activity also demonstrated in cell extracts of other Hyphomicrobium, Thiobacillus, and Arthrobacter isolates (6,7,34), with specific activities around 30 nmol NADH oxidized min Ϫ1 mg protein Ϫ1 . The enzyme has not previously been purified or characterized.The aims of this study were to purify and characterize the DMS monooxygenase enzyme from a member of the genus Hyphomicrobium. Since Hyphomicrobium S is no longer available, studies were undertaken using the type strain of H. sulfonivorans. The strain was originally isolated from garden soil and grows on DMS, as well as the related compounds dimethyl sulfoxide (DMSO) and dimethylsulfone (DMSO 2 ). During growth on DMSO 2 , H. sulfonivorans first reduces DMSO 2 to DMSO by a dimethylsulfone reductase, and subsequently a DMSO reductase converts DMSO to DMS, which is further oxidized to methanethiol and formaldehyde by a DMS mono-

The abundant marine bacterium Pelagibacter simultaneously catabolizes dimethylsulfoniopropionate to the gases dimethyl sulfide and methanethiol

“…3 and Fig. S1 were obtained from the original publications 3,31,32 , searches of NCBI, and E.C. number searches based on figures from 1,33 , and references therein (all starting sequence data is included in Table S4).…”

Heterologous production of the lantibiotic Ala(0)actagardine in Escherichia coli

“…Sequence analysis suggests that GarO is a luciferase-like monooxygenase that possesses a typical binding domain for flavin mononucleotide (FMN). Previous studies on luciferase-like enzymes 20, 21 have shown that reduced FMN reacts with molecular oxygen, forming a flavin-hydroperoxide, which is followed by transfer of an oxygen atom to the substrate. NAD(P)H then commonly reduces the oxidized flavin back to its reduced form.…”

Methylotrophy in a Lake: from Metagenomics to Single-Organism Physiology

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