Purification and Characterization of Dimethylsulfoniopropionate Lyase from an Alcaligenes-Like Dimethyl Sulfide-Producing Marine Isolate

Dimethyl sulfide (DMS) is quantitatively the most important biogenic sulfur compound emitted from oceans and salt marshes. It is formed primarily by the action of dimethylsulfoniopropionate (DMSP) lyase which cleaves DMSP, an algal osmolyte, to equimolar amounts of DMS and acrylate. This report is the first to describe the isolation and purification of DMSP lyase. The soluble enzyme was purified to electrophoretic homogeneity from a facultatively anaerobic gram-negative rod-shaped marine bacterium identified as an Alcaligenes species by the Vitek gram-negative identification method. The key to successful purification of the enzyme was its binding to, and hydrophobic chromatography on, a phenyl-Sepharose CL-4B column. DMSP lyase biosynthesis was induced by its substrate, DMSP; its product, acrylate; and also by acrylamide. The relative effectivenesses of the inducers were 100, 90, and 204%, respectively. DMSP lyase is a 48-kDa monomer with a Michaelis-Menten constant (K(infm)) for DMSP of 1.4 mM and a V(infmax) of 408 (mu)mol/min/mg of protein. It converted DMSP to DMS and acrylate stoichiometrically. The similar K(infm) values measured for pure DMSP lyase and the axenic culture, seawater, and surface marsh sediment suggest that the microbes in these ecosystems must have enzymes similar to the one purified from our marine isolate. Anoxic sediment populations, however, have a 40-fold-lower K(infm) for this enzyme (30 (mu)M), possibly giving them the capability to metabolize much lower levels of DMSP than the aerobes.     

DIMETHYLSULFONIOPROPIONATE CLEAVAGE BY MARINE PHYTOPLANKTON IN RESPONSE TO MECHANICAL,CHEMICAL, OR DARK STRESS1

Several bloom‐forming marine algae produce concentrated intracellular dimethylsulfoniopropionate (DMSP) and display high DMSP cleavage activity in vitro and during lysis after grazing or viral attack. Here we show evidence for cleavage of DMSP in response to environmental cues among different strains of the haptophyte Emiliania huxleyi (Lohmann) Hay et Mohler and the dinoflagellate Alexandrium spp. (Halim). Sparging or shaking live cells of either taxon increased dimethyl sulfide (DMS), especially in dinoflagellates, known to be very sensitive to shear stresses. Additions of polyamines, known triggers of exocytosis in some protists, also stimulated DMSP cleavage in a dose‐responsive manner. We observed DMS production by some algae after shifts in light regime. When most exponential‐phase E. huxleyi were transferred to continuous darkness, cells decreased in volume and DMSP content within 24 h; DMSP content per unit cell volume remained relatively steady. DMS accumulated as long as cells remained in the dark, but on returning to a light:dark cycle DMS accumulation ceased within 24 h. However, E. huxleyi strain CCMP 373, containing highly active in vitro DMSP lyase, produced only transient accumulations of DMS in the dark. This was apparently due to production and concomitant oxidation or uptake of DMS, because cells of this strain rapidly removed DMS added to cultures. Three strains of the dinoflagellate Alexandrium tamarense containing high in vitro DMSP lyase activity showed no DMS production in the dark, and all appeared to remove additions of DMS. Alexandrium tamarense strain CCMP 1771 also removed dimethyl disulfide, an inhibitor of bacterial DMS consumption. These data suggest that physical or chemical cues can trigger algal DMSP cleavage, but DMS production may be masked by subsequent oxidation and/or uptake.     

Release of Dimethylsulfide from Dimethylsulfoniopropionate by Plant-Associated Salt Marsh Fungi

The range of types of microbes with dimethylsulfoniopropionate (DMSP) lyase capability (enzymatic release of dimethylsulfide [DMS] from DMSP) has recently been expanded from bacteria and eukaryotic algae to include fungi (a species of the genus Fusarium [M. K. Bacic and D. C. Yoch, Appl. Environ. Microbiol. 64:106–111, 1998]). Fungi (especially ascomycetes) are the predominant decomposers of shoots of smooth cordgrass, the principal grass of Atlantic salt marshes of the United States. Since the high rates of release of DMS from smooth cordgrass marshes have a temporal peak that coincides with peak shoot death, we hypothesized that cordgrass fungi were involved in this DMS release. We tested seven species of the known smooth cordgrass ascomycetes and discovered that six of them exhibited DMSP lyase activity. We also tested two species of ascomycetes from other DMSP-containing plants, and both were DMSP lyase competent. For comparison, we tested 11 species of ascomycetes and mitosporic fungi from halophytes that do not contain DMSP; of these 11, only 3 were positive for DMSP lyase. A third group tested, marine oomycotes (four species of the genera Halophytophthora and Pythium, mostly from mangroves), showed no DMSP lyase activity. Two of the strains of fungi found to be positive for DMSP lyase also exhibited uptake of DMS, an apparently rare combination of capabilities. In conclusion, a strong correlation exists between a fungal decomposer’s ability to catabolize DMSP via the DMSP lyase pathway and the host plant’s production of DMSP as a secondary product.     

Release of dimethylsulfide from dimethylsulfoniopropionate by plant-associated salt marsh fungi

The range of types of microbes with dimethylsulfoniopropionate (DMSP) lyase capability (enzymatic release of dimethylsulfide [DMS] from DMSP) has recently been expanded from bacteria and eukaryotic algae to include fungi (a species of the genus Fusarium [M. K. Bacic and D. C. Yoch, Appl. Environ. Microbiol. 64:106-111, 1998]). Fungi (especially ascomycetes) are the predominant decomposers of shoots of smooth cordgrass, the principal grass of Atlantic salt marshes of the United States. Since the high rates of release of DMS from smooth cordgrass marshes have a temporal peak that coincides with peak shoot death, we hypothesized that cordgrass fungi were involved in this DMS release. We tested seven species of the known smooth cordgrass ascomycetes and discovered that six of them exhibited DMSP lyase activity. We also tested two species of ascomycetes from other DMSP-containing plants, and both were DMSP lyase competent. For comparison, we tested 11 species of ascomycetes and mitosporic fungi from halophytes that do not contain DMSP; of these 11, only 3 were positive for DMSP lyase. A third group tested, marine oomycotes (four species of the genera Halophytophthora and Pythium, mostly from mangroves), showed no DMSP lyase activity. Two of the strains of fungi found to be positive for DMSP lyase also exhibited uptake of DMS, an apparently rare combination of capabilities. In conclusion, a strong correlation exists between a fungal decomposer's ability to catabolize DMSP via the DMSP lyase pathway and the host plant's production of DMSP as a secondary product.     

Molecular genetic analysis of a dimethylsulfoniopropionate lyase that liberates the climate-changing gas dimethylsulfide in several marine alpha-proteobacteria and Rhodobacter sphaeroides

The α-proteobacterium Sulfitobacter EE-36 makes the gas dimethylsulfide (DMS) from dimethylsulfoniopropionate (DMSP), an abundant antistress molecule made by many marine phytoplankton. We screened a cosmid library of Sulfitobacter for clones that conferred to other bacteria the ability to make DMS. One gene, termed dddL , was sufficient for this phenotype when cloned in pET21a and introduced into Escherichia coli . Close DddL homologues exist in the marine α-proteobacteria Fulvimarina , Loktanella Oceanicola and Stappia , all of which made DMS when grown on DMSP. There was also a dddL homologue in Rhodobacter sphaeroides strain 2.4.1, but not in strain ATCC 17025; significantly, the former, but not the latter, emits DMS when grown with DMSP. Escherichia coli containing the cloned, overexpressed dddL genes of R. sphaeroides 2.4.1 and Sulfitobacter could convert DMSP to acrylate plus DMS. This is the first identification of such a 'DMSP lyase'. Thus, DMS can be made either by this DddL lyase or by a DMSP acyl CoA transferase, specified by dddD , a gene that we had identified in several other marine bacteria.     

Biochemical,Kinetic, and Spectroscopic Characterization of Ruegeria pomeroyi DddW—A Mononuclear Iron-Dependent DMSP Lyase

The osmolyte dimethylsulfoniopropionate (DMSP) is a key nutrient in marine environments and its catabolism by bacteria through enzymes known as DMSP lyases generates dimethylsulfide (DMS), a gas of importance in climate regulation, the sulfur cycle, and signaling to higher organisms. Despite the environmental significance of DMSP lyases, little is known about how they function at the mechanistic level. In this study we biochemically characterize DddW, a DMSP lyase from the model roseobacter Ruegeria pomeroyi DSS-3. DddW is a 16.9 kDa enzyme that contains a C-terminal cupin domain and liberates acrylate, a proton, and DMS from the DMSP substrate. Our studies show that as-purified DddW is a metalloenzyme, like the DddQ and DddP DMSP lyases, but contains an iron cofactor. The metal cofactor is essential for DddW DMSP lyase activity since addition of the metal chelator EDTA abolishes its enzymatic activity, as do substitution mutations of key metal-binding residues in the cupin motif (His81, His83, Glu87, and His121). Measurements of metal binding affinity and catalytic activity indicate that Fe(II) is most likely the preferred catalytic metal ion with a nanomolar binding affinity. Stoichiometry studies suggest DddW requires one Fe(II) per monomer. Electronic absorption and electron paramagnetic resonance (EPR) studies show an interaction between NO and Fe(II)-DddW, with NO binding to the EPR silent Fe(II) site giving rise to an EPR active species (g = 4.29, 3.95, 2.00). The change in the rhombicity of the EPR signal is observed in the presence of DMSP, indicating that substrate binds to the iron site without displacing bound NO. This work provides insight into the mechanism of DMSP cleavage catalyzed by DddW.     

Structural and molecular basis for the novel catalytic mechanism and evolution of DddP,an abundant peptidase‐like bacterial Dimethylsulfoniopropionate lyase: a new enzyme from an old fold

The microbial cleavage of dimethylsulfoniopropionate (DMSP) generates volatile dimethyl sulfide (DMS) and is an important step in global sulfur and carbon cycles. DddP is a DMSP lyase in marine bacteria, and the deduced dddP gene product is abundant in marine metagenomic data sets. However, DddP belongs to the M24 peptidase family according to sequence alignment. Peptidases hydrolyze C‐N bonds, but DddP is deduced to cleave C‐S bonds. Mechanisms responsible for this striking functional shift are currently unknown. We determined the structures of DMSP lyase RlDddP (the DddP from Ruegeria lacuscaerulensis ITI_1157) bound to inhibitory 2‐(N‐morpholino) ethanesulfonic acid or PO₄^3? and of two mutants of RlDddP bound to acrylate. Based on structural, mutational and biochemical analyses, we characterized a new ion‐shift catalytic mechanism of RlDddP for DMSP cleavage. Furthermore, we suggested the structural mechanism leading to the loss of peptidase activity and the subsequent development of DMSP lyase activity in DddP. This study sheds light on the catalytic mechanism and the divergent evolution of DddP, leading to a better understanding of marine bacterial DMSP catabolism and global DMS production.     

Towards a systematic understanding of structure–function relationship of dimethylsulfoniopropionate‐catabolizing enzymes

Each year, several million tons of dimethylsulfoniopropionate (DMSP) are produced by marine phytoplankton and bacteria as an important osmolyte to regulate their cellular osmosis. Microbial breakdown of DMSP to the volatile gas dimethylsulfide (DMS) plays an important role in global biogeochemical cycles of the sulphur element between land and the sea. Understanding the enzymes involved in the transformation of DMSP and DMS holds the key to a better understanding of oceanic DMSP cycles. Recent work by Shao et al. (2019) has resolved the crystal structure of two important enzymes, DmdB and DmdC, involved in DMSP transformation through the demethylation pathway. Their work represents an important step towards a systematic understanding of the structure–function relationships of DMSP‐catabolizing enzymes in marine microbes.     

Metabolism of DMSP, DMS and DMSO by the cultivable bacterial community associated with the DMSP-producing dinoflagellate Scrippsiella trochoidea

Bacterial species associated with the dimethylsulfoniopropionate (DMSP)-producing phytoplankton Scrippsiella trochoidea were cultured and identified, with the aim of establishing their ability to metabolise DMSP, dimethylsulfide (DMS) and dimethylsulfoxide (DMSO). Results demonstrate that of the cultivable bacteria only α-Proteobacteria were capable of producing DMS from DMSP. The concentration of DMSP was shown to affect the amount of DMS produced. Lower DMSP concentrations (1.5?μmol?dm^?3) were completely assimilated, whereas higher concentrations (10?μmol?dm^?3) resulted in increasing amounts of DMS being produced. By contrast to the restricted set of bacteria that metabolised DMSP,?~?70% of the bacterial isolates were able to ‘consume’ DMS. However, 98-100% of the DMS removed was accounted for as DMSO. Notably, a number of these bacteria would only oxidise DMS in the presence of glucose, including members of the γ-Proteobacteria and Bacteroidetes. The observations from this study, coupled with published field data, identify DMS oxidation to DMSO as a major transformation pathway for DMS, and we speculate that the fate of DMS and DMSP in the field are tightly coupled to the available carbon produced by phytoplankton.     

Evidence for Intracellular and Extracellular Dimethylsulfoniopropionate (DMSP) Lyases and DMSP Uptake Sites in Two Species of Marine Bacteria

The kinetics of dimethylsulfoniopropionate (DMSP) uptake and dimethylsulfide (DMS) production from DMSP in two bacterial species, Alcaligenes sp. strain M3A, an isolate from estuarine surface sediments, and Pseudomonas doudoroffii, from seawater, were investigated. In Alcaligenes cells induced for DMSP lyase (DL) activity, DMS production occurred without DMSP uptake. In DL-induced suspensions of P. doudoroffii, uptake of DMSP preceded the production of DMS, indicating an intracellular location of DL; intracellular DMSP levels reached ca. 7 mM. DMSP uptake rates in noninduced cells showed saturation at three concentrations (K(inft) [transport] values, 3.4, 127, and 500 (mu)M). In DL-induced cells of P. doudoroffii, DMSP uptake rates increased ca. threefold (V(infmax), 0.022 versus 0.065 (mu)mol of DMSP taken up min(sup-1) mg of cell protein(sup-1)), suggesting that the uptake binding proteins were inducible. DMSP uptake and DL activity in P. doudoroffii were both inhibited by CN(sup-), 2,4-dinitrophenol, and membrane-impermeable thiol-binding reagents, further indicating active uptake of DMSP by cell surface components. The respiratory inhibitors had limited or no effect on DL activity by the Alcaligenes sp. Of the structural analogs of DMSP tested for their effect on DMSP metabolism, glycine betaine (GBT), but not methyl-3-mercaptopropionic acid (MMPA), inhibited DMSP uptake by P. doudoroffii, suggesting that GBT shares a binding protein with DMSP and that MMPA is taken up at a separate site. Two models of DMSP uptake, induction, and DL location found in marine bacteria are presented.     

In Vivo Characterization of Dimethylsulfoniopropionate Lyase in the Fungus Fusarium lateritium

A fungus, Fusarium lateritium, with dimethylsulfoniopropionate (DMSP) lyase activity was isolated from both seawater and a salt marsh due to its ability to grow on DMSP (with the evolution of dimethyl sulfide) as the sole source of carbon. This is the first reported case of DMSP lyase activity in a fungus. Several other common fungal genera tested did not have DMSP lyase activity. DMSP was taken up more rapidly by F. lateritium than it was utilized, leading to its intracellular accumulation. Inhibitor studies with nystatin and cyanide indicated that DMSP uptake was an energy-dependent process. The lyase was inducible by its substrate, DMSP (K_m, 1.2 mM), and by the substrate analogs choline and glycine betaine. During induction, DMSP lyase activity increased with time and then dropped rapidly. This loss of activity could be prevented by spiking the culture with fresh DMSP or choline. The V_max for DMSP lyase was 34.7 mU · mg of protein⁻¹. The inhibitory effects of nystatin, and p-chloromercuriphenylsulfonate on DMSP lyase activity suggested that the enzyme is cytosolic. Because plants like Spartina (a marsh grass) and marine algae contain high concentrations of DMSP, we speculate that DMSP-utilizing fungi may be involved in their decay.     

Metabolism of Acrylate to β-Hydroxypropionate and Its Role in Dimethylsulfoniopropionate Lyase Induction by a Salt Marsh Sediment Bacterium, Alcaligenes faecalis M3A

Dimethylsulfoniopropionate (DMSP) is degraded to dimethylsulfide (DMS) and acrylate by the enzyme DMSP lyase. DMS or acrylate can serve as a carbon source for both free-living and endophytic bacteria in the marine environment. In this study, we report on the mechanism of DMSP-acrylate metabolism by Alcaligenes faecalis M3A. Suspensions of citrate-grown cells expressed a low level of DMSP lyase activity that could be induced to much higher levels in the presence of DMSP, acrylate, and its metabolic product, β-hydroxypropionate. DMSP was degraded outside the cell, resulting in an extracellular accumulation of acrylate, which in suspensions of citrate-grown cells was then metabolized at a low endogenous rate. The inducible nature of acrylate metabolism was evidenced by both an increase in the rate of its degradation over time and the ability of acrylate-grown cells to metabolize this molecule at about an eight times higher rate than citrate-grown cells. Therefore, acrylate induces both its production (from DMSP) and its degradation by an acrylase enzyme. ¹H and ¹³C nuclear magnetic resonance analyses were used to identify the products resulting from [1-¹³C]acrylate metabolism. The results indicated that A. faecalis first metabolized acrylate to β-hydroxypropionate outside the cell, which was followed by its intracellular accumulation and subsequent induction of DMSP lyase activity. In summary, the mechanism of DMSP degradation to acrylate and the subsequent degradation of acrylate to β-hydroxypropionate in the aerobic β-Proteobacterium A. faecalis has been described.     

The Structure of RdDddP from Roseobacter denitrificans Reveals That DMSP Lyases in the DddP-Family Are Metalloenzymes

Marine microbes degrade dimethylsulfoniopropionate (DMSP), which is produced in large quantities by marine algae and plants, with DMSP lyases into acrylate and the gas dimethyl sulfide (DMS). Approximately 10% of the DMS vents from the sea into the atmosphere and this emission returns sulfur, which arrives in the sea through rivers and runoff, back to terrestrial systems via clouds and rain. Despite their key role in this sulfur cycle DMSP lyases are poorly understood at the molecular level. Here we report the first X-ray crystal structure of the putative DMSP lyase RdDddP from Roseobacter denitrificans, which belongs to the abundant DddP family. This structure, determined to 2.15 Å resolution, shows that RdDddP is a homodimeric metalloprotein with a binuclear center of two metal ions located 2.7 Å apart in the active site of the enzyme. Consistent with the crystallographic data, inductively coupled plasma mass spectrometry (ICP-MS) and total reflection X-ray fluorescence (TRXF) revealed the bound metal species to be primarily iron. A 3D structure guided analysis of environmental DddP lyase sequences elucidated the critical residues for metal binding are invariant, suggesting all proteins in the DddP family are metalloenzymes.     

ISOLATION, PURIFICATION, AND CHARACTERIZATION OF DMSP LYASE (DIMETHYLPROPIOTHETIN DETHIOMETHYLASE (4.4.1.3)) FROM THE RED ALGA POLYSIPHONIA PANICULATA1

Polysiphonia paniculata Montagne is an intertidal red alga known to produce large amounts of the compound dimethylsulfoniopropionate (DMSP). Conversion of this substrate into dimethylsulfide is accomplished in P, paniculata by an enzyme called DMSP lyase (dimethylpropiothetin dethiomethyla.se (4.4.1.3)). DMSP lyase has been purified and characterized from P. paniculata. Enzymie activity is found in two different proteins: the larger with a molecular weight of 9.26 ± 10⁴ daltons and the smaller with a molecular weight of 3.65 ± 10⁴ daltons. Specific activity of the enzyme is 526 μmols min⁻¹mg⁻¹ for the smaller protein a nd 263 μmols min ⁻¹ mg⁻¹ for the la rger protein. The Michaelis-Menten constant (K_m) is 72.8 μM ± 17.15 and the v_max is 1.62 μmols min⁻¹± 0.928 for the 92.6-kDa protein. The p1 of the larger protein is 5.8 and 5.9 for the smaller protein. Interaction with cysteine protease inhibitors L-trans-epoxysuccinyl-leucylamido (4-guanidino)-butane, dithiobis-(2-nitrobenzoate), or N -ethylmaleimide inactivated enzyme activity. The presence of either magnesium or calcium with DMSP lyase enhanced activity al concentrations between 20 and 40 μM but had little effect above these levels. Addition of the divalent chelators ethylenebis(oxyethylenenitrilo) tetraacetic acid and ethylenediaminetetraacetate decreased activity of the enzyme, but activity was restored when either chelator was removed and magnesium or calcium was added to the enzyme .     

Quantification of total and particulate dimethylsulfoniopropionate (DMSP) in five Bermudian coral species across a depth gradient

The symbiotic dinoflagellate microalgae of corals (Symbiodinium spp.) contain high concentrations of dimethylsulfoniopropionate (DMSP), a multifunctional metabolite commonly found in many species of marine algae and dinoflagellates. A photoprotective antioxidant function for DMSP and its breakdown products has often been inferred in algae, but its role(s) in the coral–algal symbiosis remains elusive. To examine potential correlations between environmental and physiological parameters and DMSP, total DMSP (DMSP_t, from the host coral and zooxanthellae), particulate DMSP (DMSP_p, from the zooxanthellae only), coral surface area, and total protein, as well as zooxanthellae density, chlorophyll concentration, cell volume and genotype (i.e., clade) were measured in five coral species from the Diploria-Montastraea-Porites species complex in Bermuda along a depth gradient of 4, 12, 18, and 24 m. DMSP_t concentrations were consistently greater than DMSP_p concentrations in all species suggesting the possible translocation of DMSP from symbiont to host. D. labyrinthiformis was notably different from the other corals examined, showing DMSP_p and DMSP_t increases (per coral surface area or tissue biomass) with increasing water depth. However, overall, there were no consistent depth-related patterns in DMSP_p and DMSP_t concentrations. Further research, investigating dimethylsulfide (DMS), dimethylsulfoxide, and acrylate levels and DMSP-lyase activity in correlation with other biomarker endpoints that have been shown to be depth (i.e., temperature and light) responsive are needed to substantiate the significance of these findings.     

Metabolism of dimethylsulfoniopropionate and glycine betaine by a marine bacterium

Abstract The metabolism of the methylated osmolytes glycine betaine (GB) and dimethylsulfoniopropionate (DMSP) was studied in a bacterium (strain MD 14–50) isolated from a colony of the cyanobacterium Trichodesmium . MD 14–50 when grown on DMSP cleaved dimethylsulfide (DMS) from DMSP and oxidized acrylate. In contrast to DMSP, GB was metabolized by sequential N-demethylations. Low concentrations (100 μM) of DMSP or GB allowed the growth of MD 14–50 on glucose at higher salinities than in their absence. At elevated salinities, DMSP was accumulated intracellularly with less catabolism and DMS production. Thus, DMSP and GB were catabolized by different mechanisms but functioned interchangeably as osmolytes.     

Production of volatiles by the red seaweed Gelidium arbuscula (Rhodophyta): emission of ethylene and dimethyl sulfide

The effects of different light conditions and exogenous ethylene on the emission of volatile compounds from the alga Gelidium arbuscula Bory de Saint‐Vincent were studied. Special emphasis was placed on the possibility that the emission of ethylene and dimethyl sulfide (DMS) are related through the action of dimethylsulfoniopropionate (DMSP) lyase. The conversion of DMSP to DMS and acrylate, which is catalyzed by DMSP lyase, can indirectly support the synthesis of ethylene through the transformation of acrylate to ethylene. After mimicking the desiccation of G. arbuscula thalli experienced during low tides, the volatile compounds emitted were trapped in the headspace of 2 mL glass vials for 1 h. Two methods based on gas chromatography/mass spectrometry revealed that the range of organic volatile compounds released was affected by abiotic factors, such as the availability and spectral quality of light, salinity, and exogenous ethylene. Amines and methyl alkyl compounds were produced after exposure to white light and darkness but not after exposure to exogenous ethylene or red light. Volatiles potentially associated with the oxidation of fatty acids, such as alkenes and low‐molecular‐weight oxygenated compounds, accumu‐lated after exposure to exogenous ethylene and red light. Ethylene was produced in all treatments, especially after exposure to exogenous ethylene. Levels of DMS, the most abundant sulfur‐compound that was emitted in all of the conditions tested, did not increase after incubation with ethylene. Thus, although DMSP lyase is active in G. arbuscula, it is unlikely to contribute to ethylene synthesis. The generation of ethylene and DMS do not appear to be coordinated in G. arbuscula.