• Abstract and Figures
• Public Full-text

RESEARCH LETTER Assimilation of homotaurine-nitrogen by Burkholderia sp. and excretion of sulfopropanoate Jutta Mayer1, Karin Denger1, Katrin Kaspar2, Klaus Hollemeyer3, Theo H. M. Smits1, Thomas Huhn2 & Alasdair M. Cook1 1Department of Biology University of Konstanz, Konstanz, Germany; 2Department of Chemistry, University of Konstanz, Konstanz, Germany; and 3Institute of Biochemical Engineering, University of the Saarland, Saarbr ¨ucken,Germany Correspondence: Alasdair M. Cook, Abstract Department of Biology University of Konstanz, D-78457 Konstanz, Germany. Tel.: Homotaurine (3-aminopropanesulfonate), free or derivatized, is in widespread 149 7531 88 4247; fax: 149 7531 88 2966; pharmaceutical and laboratory use. Studies with enrichment cultures indicated E-mail: [email protected] that the compound is degradable as a sole source of carbon or as a sole source of nitrogen for bacterial growth. A pure culture of Burkholderia sp. was isolated which The sequence reported in this paper has been assimilated the amino group from homotaurine in a glucose–salts medium, and deposited in the GenBank database which released an organosulfonate, 3-sulfopropanoate, into the medium stoichio- (accession no. EU035638). metrically. The deamination involved an inducible 2-oxoglutarate-dependent aminotransferase to yield glutamate, and 3-sulfopropanal. Release of the amino Present address: Theo H.M. Smits, Agroscope Changins-Wadenswil¨ ACW, Swiss group was attributed to the measured NADP-coupled glutamate dehydrogenase. Federal Research Station, Schloss, Postfach 185, CH-8820 Wadenswil,¨ Switzerland. First published online December 2007. Editor: Christiane Dahl Keywords assimilation of homotaurine-nitrogen; Burkholderia sp.; excretion of sulfopropanoate; homotaurine:2-oxoglutarate aminotransferase. assimilation of taurine-nitrogen was seldom accompanied Introduction by cleavage of the carbon–sulfonate bond, and organosulfo- Taurine (2-aminoethanesulfonate) is a major organic solute nates were excreted quantitatively into the growth medium in mammals, and with its derivatives, it is found in all (Denger et al., 2004a, b; Styp von Rekowski et al., 2005; vertebrates and a wide range of marine invertebrates (Allen Weinitschke et al., 2005). & Garrett, 1971; Huxtable, 1992). Major roles have been The many functions of taurine in mammals led pharma- proposed for taurine in brain and muscle, and they have led cologists to examine the properties of homologues of to prolonged interest in the compound (e.g. Oja & Saransaari, taurine. Aminomethanesulfonate decays spontaneously in 2006). Corresponding to the widespread occurrence of mineral medium for bacterial growth (K. Denger, unpub- taurine and derivatives in eukaryotic organisms, the com- lished data), but homotaurine (3-aminopropanesulfonate, pound has been found to serve as a sole source of sulfur, APS) (Fig. 1) is stable and is a drug candidate (AlzhemedTM, nitrogen or carbon for a range of aerobic bacteria (Eichhorn CerebrilTM) for two major conditions (Alzheimer and et al., 1997, 2000; Cook & Denger, 2006; Mayer et al., 2006; stroke). The N-acetyl derivative of homotaurine Weinitschke et al., 2006; see also Lie et al., 1998). The (Camprals) is used to treat alcoholics (Scott et al., 2005). 78 3-Sulfopropanoate Homotaurine O− + H3N − SO3 − O SO3 I membrane V − H2O 3-Sulfopropanal O II IV +H N − − − 3 SO3 O SO3 O SO3 2-OG Glu acceptorox acceptorred + H2O + III NADP H H O NADPH 2 + NH4 Cell polymers Fig. 1. Presumed pathway of assimilatory deamination of homotaurine-nitrogen by Burkholderia sp. strain N-APS2. It is axiomatic that sulfonates require transport into the cell (Graham et al., 2002), and it was presumed that either an ATP-binding cassette transporter or a tripartite ATP-independent transporter (I) is involved in transporting homotaurine into the cell, analogous to the transport of other sulfonates (Kertesz, 2001; Denger et al., 2006; Gorzynska et al., 2006). Evidence is provided in the text for the presence of homotaurine:2-oxoglutarate aminotransferase (EC 2.6.1.-) (II) and glutamate dehydrogenase (EC 1.4.1.4) (III). The authors postulate a sulfopropanal dehydrogenase (EC 1.2.1.-) (IV) to generate the sulfopropanoate, which was detected in the medium (Fig. 2), and an exporter (V), because the cell membrane is impermeable to sulfonates. Glu, L-glutamate; 2-OG, 2- oxoglutarate. Other derivatives of homotaurine are the amphoteric the enrichment and isolation procedures were described surfactant 3-[(3-cholamidopropyl)-dimethylammonio]-1- previously (Mayer et al., 2006). The isolate Burkholderia sp. propanesulfonate (CHAPS) and the buffers cyclohexylami- strain N-APS2 was deposited with the German Culture nopropanesulfonate and 3-[N-morpholino]propanesulfonate Collection DSMZ, Braunschweig, Germany, under the (MOPS). Homotaurine and derivatives can, thus, be ex- accession number DSM 19529. pected in communal sewage works. Growth of Burkholderia sp. strain N-APS2 with 2 mM This study reports that homotaurine can be utilized by homotaurine as sole source of nitrogen (carbon source: bacteria as a sole source of carbon or of nitrogen for growth, 5 mM glucose) was followed in 100-mL cultures in 1-L as indicated in a review (Cook et al., 2006). The assimilation Erlenmeyer flasks shaken at 30 1C in a water bath. Samples of nitrogen was readily studied in pure culture. It involved a were taken at intervals to measure turbidity and to deter- homotaurine:2-oxoglutarate aminotransferase and the mine the concentrations of protein, homotaurine, 3-sulfo- excretion of 3-sulfopropanoate. propanoate, and ammonium and sulphate ions. Crude cell extracts for enzyme assays were prepared from Materials and methods cultures (50 mL) grown with homotaurine or ammonium chloride as sole nitrogen source. Harvesting and cell disrup- Enrichment cultures, isolations, growth media, tion with a French pressure cell were done as described growth conditions and cell extracts elsewhere (Denger et al., 2004a). Enrichment cultures (3 mL in 30-mL tubes) were grown at Analytical methods 30 1C in a 50 mM potassium phosphate-buffered salts med- ium, pH 7.2, which contained 0.25 mM magnesium sulfate Growth was followed as turbidity at 580 nm or quantified as and trace elements (Thurnheer et al., 1986). Carbon-limited protein in a Lowry-type reaction (Cook & Hutter,¨ 1981). enrichments contained 20 mM ammonium chloride and Sulfate was quantified turbidimetrically as a suspension of 7.5 mM homotaurine; nitrogen-limited medium contained BaSO4 (Sorbo,¨ 1987). Sulfite was determined photometri- 2 mM homotaurine and 5 mM glucose, 7 mM succinate and cally as the fuchsin-derivative (Denger et al., 2001). Ammo- 10 mM glycerol. Negative controls were done by omitting nium ion was assayed colorimetrically by the Berthelot homotaurine, positive controls by replacing the compound reaction (Gesellschaft Deutscher Chemiker, 1996). Homo- with glucose, succinate and glycerol (carbon-limited taurine and glutamate were determined after derivatization cultures) or with ammonium chloride (nitrogen-limited with 2,4-dinitrofluorobenzene (DNFB) (Sanger, 1945) and cultures). The inocula were from garden soil, forest soil or separation by HPLC. Reactions in samples (50 mL; activated sludge from the wastewater treatment plant in 0–100 nmol amine) from growth experiments or enzyme Konstanz, Germany. The preparation of inocula as well as assays were stopped by addition to 1 mL of 0.1 M NaHCO3, 79 and derivatization mixtures were shaken at 30 1C for 2 h Chemicals after addition of 0.2 mL 3% (v/v) DNFB in ethanol. Deriva- The sodium salt of 3-sulfopropanoate was synthesized in a tization was stopped by acidification with 50 mL of concen- modification of the published method (Kharasch & Brown, trated HCl, and after centrifugation (1 min, 11 000 g at room 1940). The key alteration was the addition of the radical starter temperature) the supernatant fluid was subject to analysis by azoisobutyronitrile [2,20-azobis(2-methylpropionitrile)] to reversed-phase HPLC (0–80% methanol gradient in 50 mM the mixture of sulfonyl chloride (0.25 mol) in an excess of potassium phosphate buffer, pH 2.2 over 17 min: the propanoic acid (0.5 mol) under reflux and illumination from detector was set at 360 nm). Aldehydes were deriva- a 200 W tungsten lamp. Residual propanoic acid was removed tized with 2-(diphenylacetyl)indane-1,3-dione-1-hydrazone under vacuum. Further unreacted material and putative by- (DIH; 0.3 mg mLÀ1) in acetonitrile (100 mL sample plus products were removed by extraction with 400 mL light 300 mL DIH reagent) and then separated by reversed-phase petroleum. The white crystals of 3-sulfopropanoic acid (yield HPLC (Cunningham et al., 1998). A gradient of acetonitrile 16%; lit.: 37%) were identified by nuclear magnetic resonance (30–80%) in 0.11 M NaClO was applied over 17 min and 4 (NMR) and shown to contain o 1% impurity with the the detection wavelength was 400 nm. A Eurospher 100–5 educts. Analysis by 1H-NMR (400 MHz, D6–DMSO) gave C18 column (125 mm  3 mm; Knauer, Berlin, Germany) 3 À1 the following data: d [ppm] = 2.49 (2H; t, J=8.2Hz, was used at a flow of 0.5 mL min . Confirmation of the 3 –CH2–COOH), 2.75 (2H; t, J = 8.0 Hz, CH2–SO3H), 12.78 formation of an o-aldehyde in enzyme reactions was 13 (2H; s, –COOH, –SO H). Analysis by C-NMR (100 MHz, obtained colorimetrically by reaction with o-aminobenzal- 3 D6–DMSO) gave the following data:[ppm] = 30.4 (–CH –), dehyde, which does not react with oxo-groups (Toyama 2 46.9 (–CH –SO H), 173.2 (–COOH). This product, however, et al., 1974). Matrix-assisted laser-desorption ionization 2 3 was oxygen-sensitive and had to be stored under nitrogen, so time-of-flight MS (MALDI-TOF-MS) in the negative-ion no melting point was determined. Neutralization of the free mode was used to confirm the identity of homotaurine (m/z acid with NaOH resulted in the disodium salt that was stable = 138 = [M-H]À1), assay its presence, and identify the inter- in air; this compound decomposed at about 298 1C.

Load more

  6

Copy Link