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.     

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.     

Phylogenetic analysis of culturable dimethyl sulfide-producing bacteria from a spartina-dominated salt marsh and estuarine water

Dimethylsulfoniopropionate (DMSP), an abundant osmoprotectant found in marine algae and salt marsh cordgrass, can be metabolized to dimethyl sulfide (DMS) and acrylate by microbes having the enzyme DMSP lyase. A suite of DMS-producing bacteria isolated from a salt marsh and adjacent estuarine water on DMSP agar plates differed markedly from the pelagic strains currently in culture. While many of the salt marsh and estuarine isolates produced DMS and methanethiol from methionine and dimethyl sulfoxide, none appeared to be capable of producing both methanethiol and DMS from DMSP. DMSP, and its degradation products acrylate and beta-hydroxypropionate but not methyl-3-mecaptopropionate or 3-mercaptopropionate, served as a carbon source for the growth of all the alpha- and beta- but only some of the gamma-proteobacterium isolates. Phylogenetic analysis of 16S rRNA gene sequences showed that all of the isolates were in the group Proteobacteria, with most of them belonging to the alpha and gamma subclasses. Only one isolate was identified as a beta-proteobacterium, and it had >98% 16S rRNA sequence homology with a terrestrial species of Alcaligenes faecalis. Although bacterial population analysis based on culturability has its limitations, bacteria from the alpha and gamma subclasses of the Proteobacteria were the dominant DMS producers isolated from salt marsh sediments and estuaries, with the gamma subclass representing 80% of the isolates. The alpha-proteobacterium isolates were all in the Roseobacter subgroup, while many of the gamma-proteobacteria were closely related to the pseudomonads; others were phylogenetically related to Marinomonas, Psychrobacter, or Vibrio species. These data suggest that DMSP cleavage to DMS and acrylate is a characteristic widely distributed among different phylotypes in the salt marsh-estuarine ecosystem.     

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.     

Comparative Physiology of Dimethyl Sulfide Production by Dimethylsulfoniopropionate Lyase in Pseudomonas doudoroffii and Alcaligenes sp. Strain M3A

Dimethylsulfoniopropionate (DMSP) lyase enzymatically cleaves DMSP, an algal metabolite, to produce acrylate, a proton, and dimethyl sulfide (DMS), the most abundant volatile sulfur compound emitted from oceans. The physiology of DMS production by DMSP lyase was studied in vivo in an Alcaligenes-like organism, strain M3A, a salt marsh bacterial isolate, and in a marine strain, Pseudomonas doudoroffii. Enzymes from both strains were induced at optimum rates by 1 mM DMSP and vigorous aeration. P. doudoroffii was very sensitive to continued aeration and lost activity rapidly; the enzyme was more stable when aeration ceased. In addition to DMSP, acrylate and several of its analogs acted as inducers of DMSP lyase in Alcaligenes sp. strain M3A but not in P. doudoroffii. Turnover of DMSP by P. doudoroffii was enhanced by 3.5% NaCl or seawater, whereas the Alcaligenes sp. strain M3A enzyme was not salt dependent and salt did not greatly affect its activity. The pH profile showed two peaks of DMSP lyase activity (6.5 and 8.8) for Alcaligenes sp. strain M3A and a single peak at pH 8 for P. doudoroffii. Enzyme activity in both organisms was inhibited by methyl-3-mercaptopropionate and homocysteine. Cyanide, azide and p-chloromercuribenzoate inhibited only the P. doudoroffii DMSP lyase. The apparent K(infm) values for DMSP for cell cultures of Alcaligenes sp. strain M3A and P. doudoroffii were ca. 2 mM and <20 (mu)M, respectively. The differences in the physiology of DMSP metabolism in these two bacterial isolates may enable them to exist in diverse ecological niches.     

Autumnal biomass and potential productivity of salt marsh fungi from 29 degrees to 43 degrees north latitude along the United States Atlantic Coast

It has been established that substantial amounts of fungal mass accumulate in standing decaying smooth cordgrass (Spartina alterniflora) marshes in the southeastern United States (e.g., in standing decaying leaf blades with a total fungal organic mass that accounts for about 20% of the decay system organic mass), but it has been hypothesized that in marshes farther north this is not true. We obtained samples of autumnal standing decaying smooth cordgrass from sites in Florida to Maine over a 3-year period. The variation in latitude could not explain any of the variation in the living fungal standing crop (as determined by ergosterol content) or in the instantaneous rates of fungal growth (as determined by acetate incorporation into ergosterol at a standard temperature, 20 degrees C), which led to the conclusion that the potential levels of fungal production per unit of naturally decaying grass are not different in northern and southern marshes. Twenty-one percent of the variation in the size of the living fungal standing crop could be explained by variation in the C/N ratio (the higher the C/N ratio the smaller the fungal crop), but the C/P ratio was not related to the size of the fungal crop. Instantaneous rates of fungal growth were negatively related to the size of the living fungal crop (r = -0.35), but these rates were not correlated with C/nutrient ratios. The same two predominant species of ascomycetes (one Phaeosphaeria species and one Mycosphaerella species) were found ejecting ascospores from standing decaying smooth cordgrass blades at all of the sites examined from Florida to Maine.     

Autumnal Biomass and Potential Productivity of Salt Marsh Fungi from 29° to 43° North Latitude along the United States Atlantic Coast

It has been established that substantial amounts of fungal mass accumulate in standing decaying smooth cordgrass (Spartina alterniflora) marshes in the southeastern United States (e.g., in standing decaying leaf blades with a total fungal organic mass that accounts for about 20% of the decay system organic mass), but it has been hypothesized that in marshes farther north this is not true. We obtained samples of autumnal standing decaying smooth cordgrass from sites in Florida to Maine over a 3-year period. The variation in latitude could not explain any of the variation in the living fungal standing crop (as determined by ergosterol content) or in the instantaneous rates of fungal growth (as determined by acetate incorporation into ergosterol at a standard temperature, 20°C), which led to the conclusion that the potential levels of fungal production per unit of naturally decaying grass are not different in northern and southern marshes. Twenty-one percent of the variation in the size of the living fungal standing crop could be explained by variation in the C/N ratio (the higher the C/N ratio the smaller the fungal crop), but the C/P ratio was not related to the size of the fungal crop. Instantaneous rates of fungal growth were negatively related to the size of the living fungal crop (r = −0.35), but these rates were not correlated with C/nutrient ratios. The same two predominant species of ascomycetes (one Phaeosphaeria species and one Mycosphaerella species) were found ejecting ascospores from standing decaying smooth cordgrass blades at all of the sites examined from Florida to Maine.     

Metabolism of acrylate to beta-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, beta-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. (1)H and (13)C nuclear magnetic resonance analyses were used to identify the products resulting from [1-(13)C]acrylate metabolism. The results indicated that A. faecalis first metabolized acrylate to beta-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 beta-hydroxypropionate in the aerobic beta-Proteobacterium A. faecalis has been described.     

Dimethylsulfoniopropionate lyase from the marine macroalga Ulva curvata: purification and characterization of the enzyme

This is a report on the purification and characterization of an algal dimethylsulfoniopropionate (DMSP) lyase. This enzyme, also found in bacteria, is responsible for producing most of the dimethylsulfide (DMS) in marine environments. It was purified from the green macroalga, Ulva curvata (Kützing) De Toni. Initial in-vivo experiments showed that DMSP lyase activity from endogenous DMSP in Ulva increased for 24 h and then decreased as the culture aged and endogenous DMSP levels were depleted. When amended with exogenous DMSP, rates of DMSP lyase activity remained high even when the culture was 5 d old. Following disruption of the DMSP-depleted U. curvata cells by grinding, a soluble DMSP lyase was purified. This enzyme is a monomer of 78 kDa which has a K _m for DMSP of 0.52 mM. Soluble DMSP lyase had an optimum pH of 8 and an optimum osmotic strength of 75 mM NaCl. Following disruption of the algae by either grinding with sand or blending, and washing out the soluble enzyme, the green tissue, when treated with the non-ionic detergent, Triton X-100, solubilized additional DMSP lyase activity. Three hydrophobic variant forms of Ulva DMSP lyase were isolated and partially characterized from the detergent-solubilized activity. While the molecular and kinetic properties of the algal enzyme are different from the bacterial enzymes we purified earlier, both the soluble and membrane-bound forms did, nevertheless, cross-react with antibodies raised against the bacterial (Alcaligenes strain M3A) DMSP lyase.Abbreviations DMS dimethylsulfide - DMSP dimethylsulfoniopropionate This paper is dedicated to D.I. Arnon (1910–1995). We thank Dr. Richard Zingmark for helpful discussions on the speciation of the natural algal samples used in these experiments, and Robin Krest for collecting samples for us on numerous occasions. This work was supported, in part, by a grant from the University of South Carolina Venture Fund.     

Abundance and Distribution of Dimethylsulfoniopropionate Degradation Genes and the Corresponding Bacterial Community Structure at Dimethyl Sulfide Hot Spots in the Tropical and Subtropical Pacific Ocean

Dimethylsulfoniopropionate (DMSP) is mainly produced by marine phytoplankton but is released into the microbial food web and degraded by marine bacteria to dimethyl sulfide (DMS) and other products. To reveal the abundance and distribution of bacterial DMSP degradation genes and the corresponding bacterial communities in relation to DMS and DMSP concentrations in seawater, we collected surface seawater samples from DMS hot spot sites during a cruise across the Pacific Ocean. We analyzed the genes encoding DMSP lyase (dddP) and DMSP demethylase (dmdA), which are responsible for the transformation of DMSP to DMS and DMSP assimilation, respectively. The averaged abundance (±standard deviation) of these DMSP degradation genes relative to that of the 16S rRNA genes was 33% ± 12%. The abundances of these genes showed large spatial variations. dddP genes showed more variation in abundances than dmdA genes. Multidimensional analysis based on the abundances of DMSP degradation genes and environmental factors revealed that the distribution pattern of these genes was influenced by chlorophyll a concentrations and temperatures. dddP genes, dmdA subclade C/2 genes, and dmdA subclade D genes exhibited significant correlations with the marine Roseobacter clade, SAR11 subgroup Ib, and SAR11 subgroup Ia, respectively. SAR11 subgroups Ia and Ib, which possessed dmdA genes, were suggested to be the main potential DMSP consumers. The Roseobacter clade members possessing dddP genes in oligotrophic subtropical regions were possible DMS producers. These results suggest that DMSP degradation genes are abundant and widely distributed in the surface seawater and that the marine bacteria possessing these genes influence the degradation of DMSP and regulate the emissions of DMS in subtropical gyres of the Pacific Ocean.     

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.     

Dimethylsulfoniopropionate metabolism by Pfiesteria-associated Roseobacter spp

The Roseobacter clade of marine bacteria is often found associated with dinoflagellates, one of the major producers of dimethylsulfoniopropionate (DMSP). In this study, we tested the hypothesis that Roseobacter species have developed a physiological relationship with DMSP-producing dinoflagellates mediated by the metabolism of DMSP. DMSP was measured in Pfiesteria and Pfiesteria-like (Cryptoperidiniopsis) dinoflagellates, and the identities and metabolic potentials of the associated Roseobacter species to degrade DMSP were determined. Both Pfiesteria piscicida and Pfiesteria shumwayae produce DMSP with an average intracellular concentration of 3.8 microM. Cultures of P. piscicida or Cryptoperidiniopsis sp. that included both the dinoflagellates and their associated bacteria rapidly catabolized 200 microM DMSP (within 30 h), and the rate of catabolism was much higher for P. piscicida cultures than for P. shumwayae cultures. The community of bacteria from P. piscicida and Cryptoperidiniopsis cultures degraded DMSP with the production of dimethylsulfide (DMS) and acrylate, followed by 3-methylmercaptopropionate (MMPA) and methanethiol (MeSH). Four DMSP-degrading bacteria were isolated from the P. piscicida cultures and found to be taxonomically related to Roseobacter species. All four isolates produced MMPA from DMSP. Two of the strains also produced MeSH and DMS, indicating that they are capable of utilizing both the lyase and demethylation pathways. The diverse metabolism of DMSP by the dinoflagellate-associated Roseobacter spp. offers evidence consistent with a hypothesis that these bacteria benefit from association with DMSP-producing dinoflagellates.     

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.     

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.     

Nuclear magnetic resonance analysis of [1-13C]dimethylsulfoniopropionate (DMSP) and [1-13C]acrylate metabolism by a DMSP lyase-producing marine isolate of the alpha-subclass of Proteobacteria.

The prominence of the alpha-subclass of Proteobacteria in the marine bacterioplankton community and their role in dimethylsulfide (DMS) production has prompted a detailed examination of dimethylsulfoniopropionate (DMSP) metabolism in a representative isolate of this phylotype, strain LFR. [1-(13)C]DMSP was synthesized, and its metabolism and that of its cleavage product, [1-(13)C]acrylate, were studied using nuclear magnetic resonance (NMR) spectroscopy. [1-(13)C]DMSP additions resulted in the intracellular accumulation and then disappearance of both [1-(13)C]DMSP and [1-(13)C]beta-hydroxypropionate ([1-(13)C]beta-HP), a degradation product. Acrylate, the immediate product of DMSP cleavage, apparently did not accumulate to high enough levels to be detected, suggesting that it was rapidly beta-hydroxylated upon formation. When [1-(13)C]acrylate was added to cell suspensions of strain LFR it was metabolized to [1-(13)C]beta-HP extracellularly, where it first accumulated and was then taken up in the cytosol where it subsequently disappeared, indicating that it was directly decarboxylated. These results were interpreted to mean that DMSP was taken up and metabolized by an intracellular DMSP lyase and acrylase, while added acrylate was beta-hydroxylated on (or near) the cell surface to beta-HP, which accumulated briefly and was then taken up by cells. Growth on acrylate (versus that on glucose) stimulated the rate of acrylate metabolism eightfold, indicating that it acted as an inducer of acrylase activity. DMSP, acrylate, and beta-HP all induced DMSP lyase activity. A putative model is presented that best fits the experimental data regarding the pathway of DMSP and acrylate metabolism in the alpha-proteobacterium, strain LFR.     

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.     

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.     

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.