Arch Microbiol (2006) 186:61–67 DOI 10.1007/s00203-006-0123-7 ORIGINAL PAPER N-Acetyltaurine dissimilated via taurine by Delftia acidovorans NAT Jutta Mayer Æ Karin Denger Æ Theo H. M. Smits Æ Klaus Hollemeyer Æ Ulrich Groth Æ Alasdair M. Cook Received: 5 January 2006 / Revised: 24 April 2006 / Accepted: 11 May 2006 / Published online: 21 June 2006 Ó Springer-Verlag 2006 Abstract The naturally occurring sulfonate N-acetyl- Introduction taurine was synthesized chemically and its identity was confirmed. Aerobic enrichment cultures for bacteria N-Acetyltaurine (Fig. 1) is secreted by orb spiders in able to utilize N-acetyltaurine as sole source of fixed molar concentrations in the viscid droplets applied to nitrogen or as sole source of carbon were successful. their webs as one component of the complex mecha- One representative isolate, strain NAT, which was nism to catch prey (Vollrath et al. 1990; Higgins et al. identified as a strain of Delftia acidovorans, grew with 2001); the compound is thus widespread in Nature. The N-acetyltaurine as carbon source and excreted stoi- compound is usually the major, low molecular-weight chiometric amounts of sulfate and ammonium. Induc- species present on these webs, and it is one of the many ible enzyme activities were measured in crude extracts derivatives of taurine (2-aminoethanesulfonate) found of this organism to elucidate the degradative pathway. in Nature (Huxtable 1992). The bacterial transforma- Cleavage of N-acetyltaurine by a highly active amidase tions of taurine are very diverse (Cook and Denger yielded acetate and taurine. The latter was oxidatively 2006), but much less is known about the transformation deaminated by taurine dehydrogenase to ammonium of taurine derivatives and analogues (Cook and and sulfoacetaldehyde. This key intermediate of sul- Denger 2002; Weinitschke et al. 2006). fonate catabolism was desulfonated by the known Taurine (two routes), isethionate, sulfoacetate and reaction of sulfoacetaldehyde acetyltransferase to sul- ethane-1,2-disulfonate are all converted to sulfoacet- fite and acetyl phosphate, which was further degraded aldehyde, which is subject to phosphatolysis to acetyl to enter central metabolism. A degradative pathway phosphate and sulfite by sulfoacetaldehyde acetyl- including transport functions is proposed. transferase (Xsc) [EC 2.3.3.15]; three subgroups of sulfoacetaldehyde acetyltransferases are currently Keywords Sulfoacetaldehyde acetyltransferase Æ known (Denger et al. 2001; Cook and Denger 2002; Xsc Æ Amidase Æ Desulfonation Ruff et al. 2003; Bru¨ ggemann et al. 2004; Denger et al. 2004a). The only taurine derivative, whose complete degradative pathway has been elucidated, N-methyl- J. Mayer Æ K. Denger Æ T. H. M. Smits Æ A. M. Cook (&) taurine, is converted directly to sulfoacetaldehyde and Fachbereich Biologie der Universita¨t Konstanz, methylamine by (N-methyl)taurine dehydrogenase 78457 Konstanz, Germany (Tdh) [EC 1.4.2.-] in Alcaligenes spp. and Paracoccus e-mail: [email protected] spp. (Weinitschke et al. 2006). Taurocholate, a bile salt, U. Groth Æ J. Mayer is cleaved by bile salt hydrolases [EC 3.5.1.24] (e.g. Fachbereich Chemie der Universita¨t Konstanz, Kim et al. 2004) to taurine and cholate, whose degra- 78457 Konstanz, Germany dative path is unknown. N-acetyltaurine is known to be a substrate for a different amidase, hog-kidney K. Hollemeyer Institut fu¨ r Technische Biochemie, Universita¨t des N-acetyl-b-alanine deacetylase [EC 3.5.1.21] (Fujimoto Saarlandes, PO Box 151150, 66041 Saarbru¨ cken, Germany et al. 1968). 123 62 Arch Microbiol (2006) 186:61–67 –1 O Table 1 Enzyme activities (mkat (kg protein) ) of crude SO - extracts of Delftia acidovorans NAT under different growth N 3 conditions N-Acetyltaurine Enzyme Acetate Taurine NAT grown grown grown N-acetyltaurine 0.5 0.3 39 O amidase EC 3.5.1.- SO - N 3 Taurine dehydrogenase BLD 0.27 0.08 EC 1.4.2.- H O 2 Taurine:pyruvate aminotransferase BLD BLD ND Amidase EC 2.6.1.77 Sulfoacetaldehyde acetyltransferase BLD 2.5 1 O EC 2.3.3.15 - + SO Phosphate acetyltransferase 0.8 0.2 0.2 O- H N 3 3 EC 2.3.1.8 Acetate Taurine Sulfite dehydrogenase 1.3 34 78 EC 1.8.2.1 H2O Isocitrate lyase 1.3 1.7 1.7 Tdh HSCoA EC 4.1.3.1 + + NH4 NH4 NAT N-acetyltaurine, ND not determined, BLD below the limit - of detection. The limits of detection for Tdh, Tpa and Xsc were SO3 O 0.02, 0.5 and 0.3 mkat (kg protein)–1, respectively Sulfo- acetaldehyde Pi of desulfonation by Xsc is the need to remove sulfite, Xsc usually as sulfate, so both a sulfite dehydrogenase [EC Sdh - SO 2- 2- HSO3 4 SO4 1.8.2.1] and a sulfate exporter are needed. A putative Pi HSCoA 2- protein, OrfX, which belongs to the large group of SCoA PO3 O putative membrane proteins with domains of unknown Pta O function (DUF81), has been proposed for this function O Acetyl CoA Acetyl phosphate in b-Proteobacteria (all Burkholderiales) (Rein et al. 2005; Cook and Denger 2006). The acetate generated from N-acetyltaurine will need activation, effectively Krebs cycle, Glyoxylate cycle to acetyl-CoA, such that acetyl-CoA from both C2-moieties of N-acetyltaurine can be processed via Fig. 1 Proposed dissimilatory pathway for N-acetyltaurine in the Krebs cycle and, in b-Proteobacteria, the glyoxy- Delftia acidovorans NAT. A transport system for the charged N-acetyltaurine can be predicted (Cook and Denger 2002). The late shunt. Examination of these possibilities led to an amidase was found in the present paper, as were three typical hypothesis (Fig. 1), much of which was confirmed in enzymes of taurine catabolism (Cook and Denger 2006) taurine this study. dehydrogenase (Tdh), sulfoacetaldehyde acetyltransferase (Xsc) and phosphotransacetylase (Pta). A representative enzyme of the glyoxylate pathway, isocitrate lyase, was also detected (Table 1). Candidates for export functions are AmtB for Materials and methods ammonia (Denger et al. 2006; Gorzynska et al. 2006) and OrfX for sulfate (Rein et al. 2005) Materials A hypothetical degradative pathway for N-acetyl- The sodium salt of N-acetyltaurine was synthesized taurine has many components. A transporter for the on the gram scale in an SN2 reaction (e.g. Clayden substrate with its charged sulfonate group is essential et al. 2001) from taurine and acetic anhydride under (Fig. 1). Cleavage of N-acetyltaurine to acetamide and alkaline conditions in ethanol (Teraoka 1925). The sulfoacetaldehyde (not shown) or hydrolysis to taurine product was recrystallized in ethanol with a yield of and acetate (Fig. 1) can be hypothesized. Taurine dis- 7% (Lit.: 13%). It gave a sharp melting point at similation via Tdh [EC 1.4.2.-] (Bru¨ ggemann et al. 234°C (Lit.: 233–234°C). The product, a white pow- 2004) or via taurine:pyruvate aminotransferase and der, was highly soluble in water, as described previ- alanine dehydrogenase (e.g. Denger et al. 2004a) re- ously (Teraoka 1925), and neither acetate nor taurine quires an export function to remove excess ammonium was detected in the product. Analysis by 1H-NMR ion (e.g. Bru¨ ggemann et al. 2004), while the follow-up (400 MHz, DMSO) gave the following data: d [ppm] 123 Arch Microbiol (2006) 186:61–67 63 3 – 1.76 (3H; s; –CH3), 2.57 (2H; t; J = 7.4; –CH2–SO3), tions of acetate, taurine and the sulfite and sulfate 3.27 (2H; m; N–CH2–C) and 7.79 (1H; s; –NH–). ions. In order to quantify the amount of protein, Analysis by 13C-NMR (400 MHz, DMSO) gave the ammonia and sulfate after growth with different ini- following data: d [ppm] 22.8 (–CH3), 35.5 (N–CH2– tial concentrations of N-acetyltaurine (0–5 mM), the – C), 50.6 (–CH2–SO3) and 168.9 (C=O). Analysis by medium was adapted to a reduced concentration of matrix-assisted laser-desorption ionization time-of- ammonium chloride (0.5 mM). Cultures (50 ml) of flight mass spectrometry (MALDI-TOF-MS) in the cells grown with N-acetyltaurine, taurine or acetate as negative ion mode (m/z = 166[M–1]–) confirmed the sole carbon source were used for enzyme assays after identity of the product (M = 167). The preparation of harvesting by centrifugation (30,000 g, 15 min, 4°C), sulfoacetaldehyde, as the bisulfite addition complex, washing with 50 mM potassium phosphate buffer, pH was described elsewhere (Denger et al. 2001). Com- 7.2 and storage (–20°C). Cell suspensions were dis- mercial chemicals were of the highest purity avail- rupted by three to four passages through a chilled able, and they were purchased from Fluka, Merck, French pressure cell at 140 MPa. Debris was removed Roth, Serva or Sigma. by centrifugation (30,000 g,15min,4°C). Crude ex- tracts could be used immediately or stored frozen Enrichment cultures, isolations, growth media, without loss of activity. growth conditions and cell disruption Enzyme assays The salts medium for carbon-limited aerobic enrich- ment cultures (5 ml in 50 ml tubes) was a 50 mM N-Acetyltaurine amidase was assayed discontinuously potassium phosphate buffer, pH 7.2, which contained as the N-acetyltaurine-dependent formation of acetate 0.25 mM magnesium sulfate, 20 mM ammonium chlo- and taurine at 30°C. The reaction mixture (1 ml) con- ride and trace elements (Thurnheer et al. 1986). tained 50 lmol potassium phosphate buffer, pH 7.2, N-acetyltaurine (5 mM) was added as sole source of 25 lmol N-acetyltaurine and 0.1–0.9 mg protein with carbon or omitted in negative controls. The inocula which the reaction was started. Samples were taken at were from activated sludge from the waste-water intervals and the reaction stopped by the addition of treatment plant in Konstanz, Germany, from forest (a) NaHCO3 for the determination of taurine by soil, or from garden soil.