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Sulfoacetaldehyde bisulfite adduct is a substrate for enzymes presumed to act on sulfoacetaldehyde
Journal of Microbiological Methods 53 (2003) 423 – 425 www.elsevier.com/locate/jmicmeth
Note
Sulfoacetaldehyde bisulfite adduct is a substrate for enzymes presumed to act on sulfoacetaldehyde Rachel F. Gritzer, Kenneth Moffitt, Walter Godchaux *, Edward R. Leadbetter Department of Molecular and Cell Biology, The University of Connecticut, Storrs, CT 06269-2131, USA Received 26 September 2002; received in revised form 22 November 2002; accepted 22 November 2002
Abstract Sulfoacetaldehyde, an intermediate of interest to those studying microbial metabolism of sulfonates, is commonly synthesized as the bisulfite adduct. A published method presumed to convert this to the free aldehyde (and cited several times elsewhere in the literature) has been shown to be ineffective; this had not been realized by its users because the enzymes under study recognize the adduct as a substrate. D 2002 Elsevier Science B.V. All rights reserved. Keywords: Sulfoacetaldehyde; Sulfonate metabolism
Recent interest in microbial degradation of sulfonates (reviewed in Cook et al., 1999; Kertesz, 2000) has generated the need to synthesize proposed intermediates in this process. In particular, certain lowmolecular-weight sulfonates may be converted to 2sulfoacetaldehyde, for example, by oxidation of isethionate (2-hydroxy-ethanesulfonate) or transamination of taurine (2-aminoethanesulfonate). In some species, the sulfoactaldehyde appears to be degraded by a sulfolyase (E.C. 4.4.1.12) with acetate and sulfite as products (cf. Kondo and Ishimoto, 1975; Denger et al., 2001). Sulfoacetaldehyde is synthesized by displacement of bromine from 2-bromoacetaldehyde dimethyl acetal by bisulfite (Kondo et al., 1971; White, 1988). Because the bisulfite is present in excess, the product
* Corresponding author. Tel.: +1-860-486-1931. E-mail address: [email protected] (W. Godchaux).
is obtained as the bisulfite adduct (disodium salt, SABA), in which the bisulfite moiety has added across the aldehyde double bond. Two means have been reported for conversion of this to the free aldehyde. In one (Kondo et al., 1973), excess BaCl2 is added, the resulting BaSO3 precipitate is removed, and residual Ba2 + is removed by adding excess Na2SO4. Unfortunately, this leaves a preparation contaminated with sulfate ion, which compromises the interpretation of some of our studies of the utilization of sulfonates as alternate electron acceptors by sulfatereducing bacteria (Lie et al., 1996). As we note below, this contamination can be avoided, but only by a complicated procedure. A simpler method of conversion had been reported (White, 1988) and involves the acidification of the SABA and volatilization of the adduct sulfite as SO2 by bubbling N2 through the solution. We report here that this method actually failed to remove the adduct sulfite, and that this failure was not perceived by this
0167-7012/02/$ - see front matter D 2002 Elsevier Science B.V. All rights reserved. doi:10.1016/S0167-7012(02)00254-3
424
R.F. Gritzer et al. / Journal of Microbiological Methods 53 (2003) 423–425
and at least one other research group, probably because SABA itself serves as a substrate for certain enzymes that act upon sulfoacetaldehyde. 2-Bromoacetaldehyde dimethyl acetal was obtained from Aldrich (Milwaukee, WI, USA) and SABA (disodium salt) was synthesized and crystallized as described by Kondo et al. (1971). A sample was converted to the free acid (by passage through a column of Dowex-50Wx8, hydrogen form) for electrospray mass spectrometry (ESMS; Micromass Quattro II, negative-ion mode) which gave peaks at m/z 205 (M –H) (20%), 123 (sulfoacetaldehyde anion) (100%) and 141 (sulfoacetaldehyde anion plus H2O) (30%). The SABA (disodium salt) was stable for months in filter-sterilized, aqueous solution in the cold. A portion of the SABA was converted to the free aldehyde using the Ba2 + method; instead of addition of sulfate ion, the preparation was passed through a column of the Dowex-50, hydrogen form, to remove all metal ions and yield the free acid. ESMS of the free acid gave peaks at m/z 123 (M –H) (100%) and 141 (M – H + H2O) (40%). The remainder of the material was neutralized with NaOH and stored in filter-sterilized aqueous solution in the cold, but after several weeks, when a sample was re-converted to the free acid and analyzed, the spectrum had disappeared, that is, the free aldehyde is unstable. This method was too complex to use every time we needed fresh sulfoacetaldehyde, so we attempted to use a simpler one involving acidification of SABA and bubbling with N2 (White, 1988). ‘‘Conversion’’ reactions (as referred to in Table 1) contained 250 Amol Na2SO3 or SABA in 1.375 ml and were acidified by the addition of 0.125 ml 4 N HCl. Nitrogen was bubbled through the mixtures for 25 min and then the mixtures were neutralized by addition of 0.5 ml of 1 M NaHCO3. Aliquots were taken for assay within the linear response range (0– 1 A415, 0 – 125 nmol) of the Ellman reaction (Johnston et al., 1975) with a Na2SO3 standard. The Ellman reagent reacts not only with sulfite but also with aldehyde adducts of sulfite, though we found that the reaction took longer with the latter (15 min at 25 jC) than the former (5 min). For control samples, the HCl and NaHCO3 (in 1.5 ml of water) were mixed together before addition of Na2SO3 or SABA (0.5 ml of a 0.5 M solution) and the bubbling step was omitted. In some experiments, the Na2SO3, in
Table 1 Failure of acidification and bubbling to remove adduct bisulfite Sample
Treatment
Ellman reactivity remaining (Amol of Na2SO3)a
% Sulfite/ bisulfite removedb
Na2SO3 Na2SO3
none acidification/ bubbling acidification/ bubbling none acidification/ bubbling
230 1
– 100
230
0
340c 310
– 9
BABA SABA SABA a
Average of values for triplicate reactions that agreed to within
15%. b
The value with no treatment minus the value after acidification and bubbling, divided by the value with no treatment. c Our preparations of SABA consistently give about 35% more Ellman reactivity, by weight, than would be expected. The reason for this is not understood, but it does not result from contamination by free bisulfite derived from the synthesis procedure. On HPLC (Shodex Ionpak KC-810P, 0.1% H3PO4 mobile phase at 70 jC, conductimetric detection), the SABA gave a single peak. Injection of Na2SO3 gave a single peak well separated from that of SABA.
1.4 ml of water, was converted to the bisulfite adduct of butyraldehyde (BABA) by incubating with a 1.5fold molar excess of the aldehyde (from Aldrich; added neat) for 15 min at 25 jC before addition of HCl and normal completion of the procedure. Acidification and bubbling volatilized and removed the sulfite (as SO2) from Na2SO3, but not from SABA or from the bisulfite adduct of butyraldehyde (Table 1). Thus, it appears that, in general, adducts of aldehydes are not degraded by this method. Decreasing the concentrations of all components 10-fold did not alter these results, nor did doubling the amount of HCl relative to SABA or using a nonvolatile acid (H2SO4), or bubbling for longer times. Fortunately, given the failure of this simple conversion method, SABA itself can serve as a substrate for enzymes that are presumed to act upon sulfoacetaldehyde. Thus, White (1988) used the acidification – bubbling method and the ‘‘product’’ (which we now know was unaltered SABA) served as a precursor for coenzyme M in cell-free extracts of a methanogen. King et al. (1997) used this same conversion protocol to demonstrate sulfoacetaldehyde sulfolyase activity in an Acinetobacter isolate. This organism has a very active sulfite oxidase so it was not possible to measure sulfite formation in crude extracts but they observed
R.F. Gritzer et al. / Journal of Microbiological Methods 53 (2003) 423–425
‘‘sulfoacetaldehyde’’-dependent acetate production and demonstrated a sulfolyase band by nondenaturing-gel zymography (local detection of sulfite production). We have successfully reproduced both of these results in their Acinetobacter isolate using their methods (the gel staining procedure was from a companion paper, King and Quinn, 1997) but with no attempt by us to convert SABA to the free aldehyde. Cook’s group has also used SABA directly as a substrate for the sulfolyase (Denger et al., 2001). We will report, elsewhere, on other instances of this utilization of SABA. Possibly there is enough sulfite present in these organisms so that biogenic sulfoacetaldehyde actually exists as the adduct.
Acknowledgements We acknowledge support from NSF grant MCB/ 9905677 to E.R.L.
References Cook, A.M., Laue, H., Junker, F., 1999. Microbial desulfonation. FEMS Microbiol. Rev. 22, 399 – 419. Denger, K., Ruff, J., Rein, U., Cook, A.M., 2001. Sulphoacetaldehyde sulpho-lyase (EC 4.4.1.12) from Desulfonispora thiosulfa-
425
tigenes: purification, properties and primary sequence. Biochem. J. 357, 581 – 586. Johnston, J.B., Murray, K., Cain, R.B., 1975. Microbial metabolism of aryl sulphonates. A re-assessment of colorimetric methods for determination of sulphite and their use in measuring desulphonation of aryl and alkylbenzenesulphonate. Antonie van Leeuwenhoek 41, 493 – 511. Kertesz, M.A., 2000. Riding the sulfur cycle-metabolism of sulfonates and sulfate esters in Gram-negative bacteria. FEMS Microbiol. Rev. 24, 135 – 175. King, J.E., Quinn, J.P., 1997. Metabolism of sulfoacetate by environmental Aureobacterium sp. and Comamonas acidovorans isolates. Microbiology 143, 3907 – 3912. King, J.E., Jaouhari, R., Quinn, J.P., 1997. The role of sulfoacetaldehyde sulfo-lyase in the mineralization of isethionate by an environmental Acinetobacter isolate. Microbiology 143, 2339 – 2343. Kondo, H., Ishimoto, M., 1975. Purification and properties of sulfoacetaldehyde sulfo-lyase, a thiamine pyrophosphate-dependent enzyme forming sulfite and acetate. J. Biochem. 78, 317 – 325. Kondo, H., Anada, H., Ohsawa, K., Ishimoto, M., 1971. Formation of sulfoacetaldehyde from taurine in bacterial extracts. J. Biochem. 69, 621 – 623. Kondo, H., Kagotani, K., Oshima, M., Ishimoto, M., 1973. Purification and some properties of taurine dehydrogenase from a bacterium. J. Biochem. 73, 1269 – 1278. Lie, T.J., Pitta, T., Leadbetter, E.R., Godchaux III, W., Leadbetter, J.R., 1996. Sulfonates: novel electron acceptors in anaerobic respiration. Arch. Microbiol. 166, 204 – 210. White, R.H., 1988. Characterization of the enzymatic conversion of sulfoacetaldehyde and L-cysteine into coenzyme M (2-mercaptoethanesulfonic acid). Biochemistry 27, 7458 – 7462.
Note
Sulfoacetaldehyde bisulfite adduct is a substrate for enzymes presumed to act on sulfoacetaldehyde Rachel F. Gritzer, Kenneth Moffitt, Walter Godchaux *, Edward R. Leadbetter Department of Molecular and Cell Biology, The University of Connecticut, Storrs, CT 06269-2131, USA Received 26 September 2002; received in revised form 22 November 2002; accepted 22 November 2002
Abstract Sulfoacetaldehyde, an intermediate of interest to those studying microbial metabolism of sulfonates, is commonly synthesized as the bisulfite adduct. A published method presumed to convert this to the free aldehyde (and cited several times elsewhere in the literature) has been shown to be ineffective; this had not been realized by its users because the enzymes under study recognize the adduct as a substrate. D 2002 Elsevier Science B.V. All rights reserved. Keywords: Sulfoacetaldehyde; Sulfonate metabolism
Recent interest in microbial degradation of sulfonates (reviewed in Cook et al., 1999; Kertesz, 2000) has generated the need to synthesize proposed intermediates in this process. In particular, certain lowmolecular-weight sulfonates may be converted to 2sulfoacetaldehyde, for example, by oxidation of isethionate (2-hydroxy-ethanesulfonate) or transamination of taurine (2-aminoethanesulfonate). In some species, the sulfoactaldehyde appears to be degraded by a sulfolyase (E.C. 4.4.1.12) with acetate and sulfite as products (cf. Kondo and Ishimoto, 1975; Denger et al., 2001). Sulfoacetaldehyde is synthesized by displacement of bromine from 2-bromoacetaldehyde dimethyl acetal by bisulfite (Kondo et al., 1971; White, 1988). Because the bisulfite is present in excess, the product
* Corresponding author. Tel.: +1-860-486-1931. E-mail address: [email protected] (W. Godchaux).
is obtained as the bisulfite adduct (disodium salt, SABA), in which the bisulfite moiety has added across the aldehyde double bond. Two means have been reported for conversion of this to the free aldehyde. In one (Kondo et al., 1973), excess BaCl2 is added, the resulting BaSO3 precipitate is removed, and residual Ba2 + is removed by adding excess Na2SO4. Unfortunately, this leaves a preparation contaminated with sulfate ion, which compromises the interpretation of some of our studies of the utilization of sulfonates as alternate electron acceptors by sulfatereducing bacteria (Lie et al., 1996). As we note below, this contamination can be avoided, but only by a complicated procedure. A simpler method of conversion had been reported (White, 1988) and involves the acidification of the SABA and volatilization of the adduct sulfite as SO2 by bubbling N2 through the solution. We report here that this method actually failed to remove the adduct sulfite, and that this failure was not perceived by this
0167-7012/02/$ - see front matter D 2002 Elsevier Science B.V. All rights reserved. doi:10.1016/S0167-7012(02)00254-3
424
R.F. Gritzer et al. / Journal of Microbiological Methods 53 (2003) 423–425
and at least one other research group, probably because SABA itself serves as a substrate for certain enzymes that act upon sulfoacetaldehyde. 2-Bromoacetaldehyde dimethyl acetal was obtained from Aldrich (Milwaukee, WI, USA) and SABA (disodium salt) was synthesized and crystallized as described by Kondo et al. (1971). A sample was converted to the free acid (by passage through a column of Dowex-50Wx8, hydrogen form) for electrospray mass spectrometry (ESMS; Micromass Quattro II, negative-ion mode) which gave peaks at m/z 205 (M –H) (20%), 123 (sulfoacetaldehyde anion) (100%) and 141 (sulfoacetaldehyde anion plus H2O) (30%). The SABA (disodium salt) was stable for months in filter-sterilized, aqueous solution in the cold. A portion of the SABA was converted to the free aldehyde using the Ba2 + method; instead of addition of sulfate ion, the preparation was passed through a column of the Dowex-50, hydrogen form, to remove all metal ions and yield the free acid. ESMS of the free acid gave peaks at m/z 123 (M –H) (100%) and 141 (M – H + H2O) (40%). The remainder of the material was neutralized with NaOH and stored in filter-sterilized aqueous solution in the cold, but after several weeks, when a sample was re-converted to the free acid and analyzed, the spectrum had disappeared, that is, the free aldehyde is unstable. This method was too complex to use every time we needed fresh sulfoacetaldehyde, so we attempted to use a simpler one involving acidification of SABA and bubbling with N2 (White, 1988). ‘‘Conversion’’ reactions (as referred to in Table 1) contained 250 Amol Na2SO3 or SABA in 1.375 ml and were acidified by the addition of 0.125 ml 4 N HCl. Nitrogen was bubbled through the mixtures for 25 min and then the mixtures were neutralized by addition of 0.5 ml of 1 M NaHCO3. Aliquots were taken for assay within the linear response range (0– 1 A415, 0 – 125 nmol) of the Ellman reaction (Johnston et al., 1975) with a Na2SO3 standard. The Ellman reagent reacts not only with sulfite but also with aldehyde adducts of sulfite, though we found that the reaction took longer with the latter (15 min at 25 jC) than the former (5 min). For control samples, the HCl and NaHCO3 (in 1.5 ml of water) were mixed together before addition of Na2SO3 or SABA (0.5 ml of a 0.5 M solution) and the bubbling step was omitted. In some experiments, the Na2SO3, in
Table 1 Failure of acidification and bubbling to remove adduct bisulfite Sample
Treatment
Ellman reactivity remaining (Amol of Na2SO3)a
% Sulfite/ bisulfite removedb
Na2SO3 Na2SO3
none acidification/ bubbling acidification/ bubbling none acidification/ bubbling
230 1
– 100
230
0
340c 310
– 9
BABA SABA SABA a
Average of values for triplicate reactions that agreed to within
15%. b
The value with no treatment minus the value after acidification and bubbling, divided by the value with no treatment. c Our preparations of SABA consistently give about 35% more Ellman reactivity, by weight, than would be expected. The reason for this is not understood, but it does not result from contamination by free bisulfite derived from the synthesis procedure. On HPLC (Shodex Ionpak KC-810P, 0.1% H3PO4 mobile phase at 70 jC, conductimetric detection), the SABA gave a single peak. Injection of Na2SO3 gave a single peak well separated from that of SABA.
1.4 ml of water, was converted to the bisulfite adduct of butyraldehyde (BABA) by incubating with a 1.5fold molar excess of the aldehyde (from Aldrich; added neat) for 15 min at 25 jC before addition of HCl and normal completion of the procedure. Acidification and bubbling volatilized and removed the sulfite (as SO2) from Na2SO3, but not from SABA or from the bisulfite adduct of butyraldehyde (Table 1). Thus, it appears that, in general, adducts of aldehydes are not degraded by this method. Decreasing the concentrations of all components 10-fold did not alter these results, nor did doubling the amount of HCl relative to SABA or using a nonvolatile acid (H2SO4), or bubbling for longer times. Fortunately, given the failure of this simple conversion method, SABA itself can serve as a substrate for enzymes that are presumed to act upon sulfoacetaldehyde. Thus, White (1988) used the acidification – bubbling method and the ‘‘product’’ (which we now know was unaltered SABA) served as a precursor for coenzyme M in cell-free extracts of a methanogen. King et al. (1997) used this same conversion protocol to demonstrate sulfoacetaldehyde sulfolyase activity in an Acinetobacter isolate. This organism has a very active sulfite oxidase so it was not possible to measure sulfite formation in crude extracts but they observed
R.F. Gritzer et al. / Journal of Microbiological Methods 53 (2003) 423–425
‘‘sulfoacetaldehyde’’-dependent acetate production and demonstrated a sulfolyase band by nondenaturing-gel zymography (local detection of sulfite production). We have successfully reproduced both of these results in their Acinetobacter isolate using their methods (the gel staining procedure was from a companion paper, King and Quinn, 1997) but with no attempt by us to convert SABA to the free aldehyde. Cook’s group has also used SABA directly as a substrate for the sulfolyase (Denger et al., 2001). We will report, elsewhere, on other instances of this utilization of SABA. Possibly there is enough sulfite present in these organisms so that biogenic sulfoacetaldehyde actually exists as the adduct.
Acknowledgements We acknowledge support from NSF grant MCB/ 9905677 to E.R.L.
References Cook, A.M., Laue, H., Junker, F., 1999. Microbial desulfonation. FEMS Microbiol. Rev. 22, 399 – 419. Denger, K., Ruff, J., Rein, U., Cook, A.M., 2001. Sulphoacetaldehyde sulpho-lyase (EC 4.4.1.12) from Desulfonispora thiosulfa-
425
tigenes: purification, properties and primary sequence. Biochem. J. 357, 581 – 586. Johnston, J.B., Murray, K., Cain, R.B., 1975. Microbial metabolism of aryl sulphonates. A re-assessment of colorimetric methods for determination of sulphite and their use in measuring desulphonation of aryl and alkylbenzenesulphonate. Antonie van Leeuwenhoek 41, 493 – 511. Kertesz, M.A., 2000. Riding the sulfur cycle-metabolism of sulfonates and sulfate esters in Gram-negative bacteria. FEMS Microbiol. Rev. 24, 135 – 175. King, J.E., Quinn, J.P., 1997. Metabolism of sulfoacetate by environmental Aureobacterium sp. and Comamonas acidovorans isolates. Microbiology 143, 3907 – 3912. King, J.E., Jaouhari, R., Quinn, J.P., 1997. The role of sulfoacetaldehyde sulfo-lyase in the mineralization of isethionate by an environmental Acinetobacter isolate. Microbiology 143, 2339 – 2343. Kondo, H., Ishimoto, M., 1975. Purification and properties of sulfoacetaldehyde sulfo-lyase, a thiamine pyrophosphate-dependent enzyme forming sulfite and acetate. J. Biochem. 78, 317 – 325. Kondo, H., Anada, H., Ohsawa, K., Ishimoto, M., 1971. Formation of sulfoacetaldehyde from taurine in bacterial extracts. J. Biochem. 69, 621 – 623. Kondo, H., Kagotani, K., Oshima, M., Ishimoto, M., 1973. Purification and some properties of taurine dehydrogenase from a bacterium. J. Biochem. 73, 1269 – 1278. Lie, T.J., Pitta, T., Leadbetter, E.R., Godchaux III, W., Leadbetter, J.R., 1996. Sulfonates: novel electron acceptors in anaerobic respiration. Arch. Microbiol. 166, 204 – 210. White, R.H., 1988. Characterization of the enzymatic conversion of sulfoacetaldehyde and L-cysteine into coenzyme M (2-mercaptoethanesulfonic acid). Biochemistry 27, 7458 – 7462.