Biogenic Sulfur in the Environment - American Chemical Society

Chapter 14

Metabolism of Acrylate and 3-Mercaptopropionate

Downloaded by CORNELL UNIV on November 5, 2017 | http://pubs.acs.org Publication Date: April 27, 1989 | doi: 10.1021/bk-1989-0393.ch014

Decomposition Products of (Dimethylsulfonio)propionate in Anoxic Marine Sediments 1

2

Ronald P. Kiene and Barrie F. Taylor 1

Marine Institute, University of Georgia, Sapelo Island, GA 31327 Division of Marine and Atmospheric Chemistry, Rosenstiel School of Marine and Atmospheric Science, University of Miami, Miami, FL 33149-1098 2

The anaerobic metabolism of acrylate and 3-mercaptopropionate (3-MPA) was studied in slurries of coastal marine sediments. The fate of these compounds is important because they are derived from the algal osmolyte dimethylsulfoniopropionate (DMSP), which is a major organic sulfur compound in marine environments. Micromolar levels of acrylate were fermented rapidly in the slurries to a mixture of acetate and propionate (1:2 molar ratio). Sulfate-reducing bacteria subsequently removed the acetate and propionate. 3-MPA has only recently been detected in natural environments. In our experiments 3-MPA was formed by chemical addition of sulfide to acrylate and was then consumed by biological processes. 3-MPA is a known inhibitor of fatty acid oxidation in mammalian systems. In accord with this fact, high concentrations of 3-MPA caused acetate to accumulate in sediment slurries. At lower concentrations, however, 3-MPA was metabolized by anaerobic bacteria. We conclude that the degradation of DMSP may ultimately lead to the production of substrates which are readily metabolized by microbes in the sediments. Dimethylsulfoniopropionate (DMSP) is an organic sulfur compound that occurs at high concentrations in many marine algae and plants, where it fulfills an osmotic function (1.2). D M S P is a principal form of organic sulfur in productive marine environments and its microbial catabolism entails either enzymatic cleavage to dimethyl sulfide (DMS) and acrylate (3.4): (CH )2S CH CH2COO+

3

2

= (CH ) S 3

2

+ CH =CHCOO2

+ H+

or conversion to 3-mercaptopropionate ( 3 - M P A ) (5.6). 3 - M P A is a novel organosulfur compound, only recently detected i n natural environments, which 0097-6156/89/0393-0222$06.00/0 • 1989 American Chemical Society

Saltzman and Cooper; Biogenic Sulfur in the Environment ACS Symposium Series; American Chemical Society: Washington, DC, 1989.

14. KIENE AND TAYLOR


is formed by a chemical addition of sulfide to acrylate (5*2) and by biochemical demethylation of D M S P (16) (Figure 1). Both acrylate and 3 - M P A have been reported to inhibit the /9-oxidation of fatty acids (89). The mechanism of action of 3 - M P A resides in its ability to form coenzyme A derivatives (Figure 2) which inhibit enzymes involved in fatty acid oxidation (1Q). However, in spite of these reported effects, we observed the metabolism of both acrylate and 3 - M P A in anoxic slurries of coastal marine sediments.


Experimental Sediments were collected i n polybutyrate tubes from nearshore regions in Biscayne Bay, Florida. Sediment slurries were prepared by mixing the upper 15 cm of the cores with equal volumes of deoxygenated seawater under a stream of N . The slurries were sieved through 1.5 mm screens to remove large particles and then 50 ml portions were dispensed into serum bottles (60 ml volume). The bottles were sealed with butyl rubber stoppers, held i n place by aluminum crimps, and incubated under N in the dark at 23-26°C with rotary snaking. The preparation of the slurries promoted the release of 3 - M P A into the seawater and 24-48 hours preincubation was usually necessary to allow the endogenous levels to decrease. Samples for H P L C analysis were removed with disposable plastic syringes (18 gauge needles) and centnfiiged at 13,000 x g for about 1 min to remove sediment. 3 - M P A was determined as its isoindole derivative using aminoethanol and £-phthalaldehyde ( H ) . 0.5 ml samples were derivatised, immediately after centrifugation of the slurries, with 10 /il of Q-phthalaldehyde (20 m g / m l in methanol) and 10 pi of aminoethanol (20 /*l/ml of sodium borate buffer, p H 9.2). After allowing 1 minute for reaction 20 pi of the sample was introduced into the H P L C via a Rheodyne 7125 valve (Rheodyne, Cotati, C A ) with an injection loop. A C18 reverse-phase column (Waters Kadial-Pak, 5/mi particles, 10 cm by 1 cm i.d.) was used to separate isoindoles using a binary gradient of 0.05M sodium acetate buffer ( p H 5.7) (solvent A ) and methanol (solvent B ) . The gradient protocol was: isocratic at 10% B for 1 min; 10% to 25% B in 1 min; isocratic at 25% B for 4 min; 25% to 50% B i n 7 min; isocratic at 50% B for 6 min; 50% to 60% B in 2 min; 60% to 67.5% B in 4 min; 67.5% to 100% B in 1 min; isocratic at 100% for 3 min; 100% to 10% B in 2 min; isocratic at 10% B for 3 min. Solvent was pumped at 1 ml/min with an Autochrom M500 pump. The isoindoles were detected with a Hitachi Model F1000 fluorimeter using excitation and detection wavelengths of 340 and 450 nm respectively. Peak areas were integrated with a Chromatopac C - R 3 A Data Processor (Shimadzu, Kyoto, Japan). 3 - M P A eluted after about 15 min and its detection limit was about 1.0 n M . Acrylate, propionate and acetate were separated by H P L C on a Benson carbohydrate column (30 cm by 0.6 cm i.d.) (Chromtec, r ort Worth, F L ) . The H P L C system used a Waters U K 6 injector and a Waters Model 6000A pump (Waters Associates, M i l f o r d , M A ) with a Conductomonitor III Detector (Laboratory Data Control, Riviera Beach, F L ) and a Shimadzu Data Processor. The solvent was 0.15 m M H 2 S 0 4 at a flow rate of 0.5 m l / m i n . Typical retention times (min) were: acrylate, 13.3; acetate, 12.3; propionate, 14.5. The detection limits for acrylate, acetate and propionate were 10, 30 and 40 /*M respectively. 20 m M molybdate was used to i n h i b i t sulfate-reduction (12.13). Chloramphenicol (125 /ig/ml) and tetracycline (50 Mg/ml) were used as general inhibitors of microbial activity and slurries were pre-incubated for 2-24 hr with these antibiotics before beginning an experiment. 2

2


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BIOGENIC SULFUR IN T H E ENVIRONMENT

(CH ) SCH .CH .C00Dimethylsulfoniopropionate\ 3

2

2

2


III CH3.S.C%CKy.C0CT 3-methiolpropionaie

Crl^CH.COO" Acrylate

HS.CHg.C^.COCT 3-lfercaptopropionate

Figure 1. Routes for the production of acrylate and 3 - M P A from D M S P . I = enzymatic cleavage of D M S P ; II = Michael addition; i n = biochemical demethylations. HS.CH2.CH2.COOH 3-mercaptopropionate

I acyl-CoA

synthetase

HS.CH2.CH2.CO.SC0A 3 - m e r c a p t o p r o p i o n y l CoA 3—ketoacyl—CoA thiolase R.CO.S.CH2.CH2.CO.SC0A S—acyl—3—mercaptopropionyl

CoA

Figure 2. Metabolism of 3 - M P A in mitochondria. Abstracted from Cuebas et al. (1Q).


14.

KIENE AND TAYLOR


Organic solvents were purchased from Burdick and Jackson (Muskogee, WI). Acrylic acid was bought from Fluka Chemicals (Happaugue, N Y ) ; other chemicals and biochemicals were obtained from either the Aldrich Chemical Co. (Milwaukee, WI) or the Sigma Chemical Co. (St. Louis, M O ) .


Results Acrylate was readily consumed and without a lag when it was added at about 700 pM (Figure 3). This activity was blocked oy autoclavine and strongly inhibited by antibiotics. Acetate and propionate accumulated transiently i n samples which received acrylate (Figure 4 A & B ) . Molybdate had no effect on the rate of consumption of acrylate (Figure 3) but it promoted the accumulation of acetate and propionate (Figure 4). Sediments treated with molybdate alone showed a substantial, linear accumulation of acetate. When this endogenous rate of acetate formation (molybdate present) was subtracted from that observed with acrylate plus molybdate, the molar ratio of propionate to acetate formed from acrylate was about 2 to 1. In contrast to micromolar levels, millimolar concentrations of acrylate were only utilized after a lag of about 3 days (Figure 5A). Again autoclaving but not molybdate inhibited acrylate consumption. Acetate and propionate were produced from acrylate, and acetate concentrations continued to increase after propionate levels began to decrease (Figure 5B & 5C). Molybdate prevented the disappearance of ooth acetate and propionate. 3 - M r A was formed from 12 m M acrylate at similar rates i n either autoclaved or uninhibited sediments (Figure 6). However, 3-MPA persisted in autoclaved systems but not in the untreated slurries. When 3-MPA was added at millimolar levels we observed the accumulation of acetate (Figure 7). 1 m M 3 - M P A caused a small, transient accumulation of acetate whilst 10 m M 3 - M P A caused acetate to accumulate for a period of 5 days. The initial rate of acetate accumulation was faster with a higher level (20 m M ) of 3 - M P A but its total production after 5 days was only about half that seen with 10 m M 3-MPA. Acetate concentrations did not change significantly upon further incubation (data not shown). Discussion Acrylate and 3-MPA were readily metabolised by sediment microbes when they were present at micromolar concentrations; but were not so easily degraded at millimolar levels. Acrylate is metabolised by a variety of bacteria. Escherichia coli converts acrylyl-CoA to pyruvate via lactyl-CoA, and some Clostridia ferment acrylate, v i a C o A intermediates, to a mixture of acetate and propionate. O f particular relevance to the fate of D M S P was the isolation by Wagner and Stadtman (14) o f a species of C l o s t r i d i u m (probably Q. propionicunri from river mud that fermented D M S P as follows: 3(CH )2S CH CH2COO- + 2 H 0 = 3 ( C H ) S + 2 C H C H C O O +

3

2

2

3

2

3

2

+ CH3COO- + C 0 + 3H+ 2

The organism also fermented acrylate to acetate and propionate: 3CH =CHCOO2

+ 2 H 0 = 2CH CH COO- + CH COO- + C 0 2

3

2

3

2

Presumably an enzyme similar to that described for marine algae converted D M S P to D M S and acrylate with subsequent fermentation of the latter compound (&4). Clearly, similar fermentative bacteria may be common i n Saltzman and Cooper; Biogenic Sulfur in the Environment ACS Symposium Series; American Chemical Society: Washington, DC, 1989.

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BIOGENIC SULFUR IN T H E ENVIRONMENT

HOURS

Figure 3 . Metabolism of 7 0 0 pM acrylate i n sediment slurries. Treatments: acrylate alone, O ; acrylate plus autoclaved slurry, A ; acrylate plus 2 0 m M molybdate, # ; acrylate plus antibiotics, A ; endogenous (no additions), M .

Figure 4 . Accumulation of acetate (panel A ) and propionate (panel B ) in sediment slurries. Treatments: 7 0 0 M M acrylate, O ; 7 0 0 / i M acrylate plus 2 0 m M molybdate, # ; 7 0 0 pM acrylate and autoclaved slurry, A ; * 0 m M molybdate alone, A ; endogenous (no additions), Q .



14.

KIENE AND TAYLOR

Metabolism ofAcrylate and 3-Mercaptopropionate

15

Figure 5. Metabolism of 10 m M acrylate in sediment slurries. Panel A shows acrylate concentrations; panel B shows acetate concentrations; panel C shows propionate concentrations. Treatments: uninhibited slurries, # ; autoclaved slurries, O ; 20 m M molybdate added, A ; endogenous (no acrylate added), A .

Figure 6. 3-Mercaptopropionate production and metabolism in sediment slurries. Treatments: 10 m M acrylate added, O ; 10 m M acrylate with autoclaved slurries, A ; endogenous (no acrylate added), A .


22


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BIOGENIC SULFUR I N T H E ENVIRONMENT

300

DAYS Figure 7. Effects of millimolar concentrations of 3-mercaptopropionate on acetate accumulation in sediment slurries. Symbols: no 3 - M P A , Q ; 1 m M 3-MPA, • ; l O m M 3 - M P A , A ; 2 0 m M 3 - M P A , A .



14.

KIENE AND TAYLOR


coastal marine sediments since micromolar levels of acrylate was rapidly metabolized in our experiments to acetate and propionate. Fermentation rates for aciylate exceeded the oxidation rates for acetate and propionate, thereby allowing these products to accumulate transiently (Figure 4 & 5). Millimolar concentrations of acrylate generated about equimolar amounts of acetate and propionate (Figure 3). A t the lower substrate level, and with molybdate present to block further metabolism, the molar ratio of acetate to propionate was about 1:2 after correcting for acetate production from endogenous substrates (Figure 4A). The recovery of fermentation products was about 108% after three days. These results are in agreement with the stoichiometry reported by Wagner and Stadtman (14) for acrylate fermentation. The inhibition by molybdate of propionate and acetate utilization implicates sulfate-reducing bacteria i n their catabolism. Acetate is an important substrate for sulfate-reducing bacteria i n marine sediments (15-17) but acrylate has not previously been recognized as a precursor of acetate i n marine sediments. The propionate formed from acrylate may also be a precusor of acetate because it is converted to acetate by Desulfobulbus species, which have been identified as the dominant propionate-oxidising sulfatereducers in marine and estuarine sediments (18.19). This is suggested i n our experiments by the continued production of acetate after propionate began to decrease (Figure 5), and by the blockage with molybdate of propionate consumption. Thus D M S P may be a precursor for several volatile fatty acids in marine sediments. The importance of D M S P as a source of metabolisable substrates in natural environments is not known. The chemical addition of sulfide to acrylate to yield 3 - M P A occurred in the slurries. However, consumption of acrylate v i a the addition reaction was probably secondary to its fermentation to acetate and propionate. Kiene and Taylor (£) showed that 10 fM acrylate gave very little accumulation of 3 - M P A i n sediments, probably because botn acrylate and 3 - M P A were rapidly metabolised. Millimolar concentrations of 3 - M P A caused acetate accumulation in the sediment slurries (Figure 7). This might be expected because 3 - M P A inhibits fatty acid oxidation in rat heart mitochondria (2). In mitochondria, 3 - M P A is converted into 3-mercaptopropionyl-CoA and S-acyl-3-mercaptopropionyl-CoA (Figure 2; 1Q) which respectively inhibit the medium and long-chain acyl-CoA dehydrogenases of ^-oxidation (g). Clearly different enzymes will be affected in the inhibition of acetate oxidation but 3 - M P A may prove useful as a selective inhibitor of fatty acid catabolism i n environmental studies. Thiols such as 3M P A bind significantly to sediment particles. In sterile slurries about half of the added 3 - M P A leaves s o l u t i o n w i t h i n 24 hours (&). The transient accumulation of acetate with 1 m M 3 - M P A may be due to eventual lowering of the dissolved concentration to non-inhibitory levels during the incubation. A t lower concentrations 3 - M P A was consumed biologically (Figure 4; £). Its lack of use i n autoclaved slurries suggests biotransformation and tnis raises biochemical questions about its metabolism. We are currently studying the metabolism o f 3 - M P A by bacterial cultures and in sediments. Acknowledgments We thank K e n Mopper and A . Vairavamurthy for helpful discussions, Marcia House for help with organic acid analysis, and the National Science Foundation for financial support (Grant No. OCE-8516020). Contribution No. 622 from the University of Georgia Marine Institute.


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Literature Cited 1. Vairavamurthy, A.; Andreae, M.O.; Iverson,R.L.Limnol.Oceanogr. 1985, 30, 59-70. 2. White, R.H. J.Mar.Res. 1982, 40, 529-36. 3. Cantoni, G.L.; Anderson, D.G. J. Biol. Chem. 1956, 222, 171-7. 4. Kadota, H.; Ishida, Y. Am. Rev. Microbiol. 1972, 26, 127-38. 5. Mopper, K.; Taylor, B. F. In Organic Marine Chemistry; Sohn, M., Ed.;


ACS Symposium Series No. 305; American Chemical Society: Washington, D.C., 1986. 6. Kiene, R. P.; Taylor, B.F. Nature 1988,332,148-50. 7. Vairavamurthy, A.; Mopper, K. Nature 1987,329,623-5. 8. Thijsse, G. J. E . Biochim. Biophys. Acta 1964, 84, 195-7. 9. Sabbagh, E.; Cuebas, D.; Schulz, H . J. Biol. Chem. 1985, 260, 7337-42 10. Cuebas, D.; Beckmann, J. D.; Frerman, F. E.; Schulz, H . J. Biol. Chem. 260, 7330-6. 11. Mopper, K.; Delmas, D. Analyt. Chem. 1984, 56, 2557-60. 12. Oremland, R. S.; Taylor, B. F. Geochim. Cosmochem. Acta 1978, 42, 209-14. 13. Taylor, B. F.; Oremland, R. S. Curr. Microbiol. 1979,3,101-3. 14. Wagner, C.; Stadtman, E. R. Arch. Biochem. Biophys. 1962,98,331-6. 15. Banat, I. M.; Lindstrom, E . B.; Nedwell, D. B.; Balba, M. T. Appl. Environ. Microbiol. 1981, 42, 985-92. 16. Sorensen, J.; Christensen, D.; Jorgensen, B. B. Appl. Environ. Microbiol. 1981, 42, 5-11. 17. Winfrey, M. R.; Ward, D. M. Appl. Environ. Microbiol. 1983, 45, 193-9. 18. Laanbroek, H .J.;Pfennig, N. Arch. Microbiol. 1981, 128, 330-5 19. Widdel, F.; Pfennig, N. Arch. Microbiol. 1982,131,360-5. RECEIVED August 22, 1988


Biogenic Sulfur in the Environment - American Chemical Society

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