Picrophilus Torridus Matthias Reher1, Michael Bott2 & Peter Schonheit¨ 1

Characterization of glycerate kinase (2-phosphoglycerate forming), a key enzyme of the nonphosphorylative Entner^Doudoro¡ pathway,from the thermoacidophilic euryarchaeon Picrophilus torridus Matthias Reher1, Michael Bott2 & Peter Schonheit¨ 1

1Institut fur ¨ Allgemeine Mikrobiologie, Christian-Albrechts-Universitat¨ Kiel, Kiel, Germany; and 2Institut fur ¨ Biotechnologie I, Julich, ¨ Germany

Correspondence: Peter Schonheit,¨ Institut Abstract für Allgemeine Mikrobiologie, Christian- Albrechts-Universitat¨ Kiel, Am Botanischen Picrophilus torridus has been shown to degrade glucose via a nonphosphorylative Garten 1-9, D-24118 Kiel, Germany. Tel.: 149 Entner–Doudoroff (ED) pathway. Here we report the characterization of a key 431 880 4328/4330; fax: 149 431 880 2194; enzyme of this pathway, glycerate kinase (2-phosphoglycerate forming). The e-mail: [email protected] enzyme was purified 5100-fold to homogeneity. The 95 kDa homodimeric protein catalyzed the ATP-dependent phosphorylation of glycerate specifically to Received 15 March 2006; accepted 24 March 2-phosphoglycerate. The enzyme showed highest activity at 60 1C and pH 7.3, 2006. with ATP as phosphoryl donor and Mg21 as divalent cation. By MALDI-TOF First published online May 2006. analysis, ORF Pto1442 was identified in the genome of P. torridus as the encoding gene, designated gck. Homologs with high sequence identity were identified in the doi:10.1111/j.1574-6968.2006.00264.x genomes of the archaea Thermoplasma and Sulfolobus spp. and Thermoproteus

Editor: Dieter Jahn tenax, for which the operation of nonphosphorylative ED pathways, involving 2-phosphoglycerate forming glycerate kinases, has been proposed. Keywords Picrophilus torridus; nonphosphorylative Entner–Doudoroff pathway; glycerate kinase (2-phosphoglycerate forming).

Introduction glycerate forming glycerate kinase (GCK), a key enzyme of this nonphosphorylative ED pathway has not been charac- The thermoacidophilic euryarchaeon Picrophilus torridus terized in P. torridus and Thermoplasma acidophilum, and grows optimally at about 60 1C and at pH values between 0 the encoding genes have not been identified. In the cre- and 2, thus representing the most acidophilic organism narchaeota, Sulfolobus spp. and Thermoproteus tenax, glu- described so far (Schleper et al., 1995; Futterer¨ et al., 2004). cose degradation has been proposed to proceed via a Picrophilus torridus utilizes various sugars, including glucose branched ED pathway, involving both a nonphosphorylative as growth substrates (Serour & Antranikian, 2002). Recent branch and a part-phosphorylative branch (see Siebers & analysis of the glucose degradation pathway revealed that in Schonheit,¨ 2005). As described for P. torridus and Thermo- P. torridus a nonphosphorylative version of the Entner– plasma acidophilum, a 2-phosphoglycerate forming glycerate Doudoroff (ED) pathway is operative (Reher & Schonheit,¨ kinase has been implicated in the nonphosphorylative 2006). Accordingly, glucose is oxidized to gluconate via branch of the ED pathways in Sulfolobus and Thermoproteus glucose dehydrogenase, subsequent dehydration via gluco- (see Siebers & Schonheit,¨ 2005); however, activities of this nate dehydratase yields 2 keto-3-deoxy-gluconate, which is enzyme have not been reported so far in both organisms. cleaved to pyruvate and glyceraldehyde. Glyceraldehyde is Here, we present the first report on the purification and then oxidized to glycerate via a NADP1 reducing glycer- characterization of a 2-phosphoglycerate forming glycerate aldehyde dehydrogenase. A glycerate kinase phosphorylates kinase (gck) in archaea, from P. torridus, and the identifica- glycerate specifically to 2-phosphoglycerate, which is then tion of its encoding gene, by MALDI-TOF analysis. Homo- converted to phosphoenolypyruvate by enolase and further logs with high sequence identity were identified in the to pyruvate by pyruvate kinase (Fig. 1). A similar pathway genomes of Thermoplasma spp. and Sulfolobus species as has been reported earlier for closely related Thermoplasma well as in Thermoproteus tenax, suggesting the presence of acidophilum (Budgen & Danson, 1986). So far, 2-phospho- functional 2-phosphoglycerate forming glycerate kinases in

Glucose adjusted to 1 M (NH4)2SO4 and applied to a Resource NAD(P)+ Phenyl (1 mL) column equilibrated with Tris-HCl pH 8, Glucose + Dehydrogenase NAD(P)H + H 1 M (NH4)2SO4. The protein was eluted with a decreasing gradient (30 mL) from 1 to 0 M (NH4)2SO4. The fraction Gluconate containing the highest GCK activity [0.8–0.7 M (NH4)2SO4] Gluconate were pooled and concentrated to 1000 mL by ultrafiltration H O Dehydratase 2 (cutoff: 10 kDa). The concentrated protein solution was KDG applied to a Superdex 200 HiLoad 16/60 column equili- KDG brated with 50 mM Tris-HCl pH 7.1, 150 mM NaCl. The À1 Aldolase protein was eluted at a flow rate of 1 mL min . The fractions with the highest GCK activity (70–75 mL) were Pyruvate Glyceraldehyde pooled, adjusted to pH 8 with 100 mM Tris-HCl pH 8 and NADP + Glyceraldehyde applied to a UNO-Q1 column equilibrated with the same Dehydrogenase NADPH + H + buffer. The protein was eluted with increasing gradient from Glycerate 0 to 0.7 M NaCl (30 mL). Eluted fraction (0.06 M NaCl) containing GCK activity indicated essentially pure as judged ATP Glycerate by sodium dodecyl sulphate-polyacrylamide gel electro- kinase ADP phoresis (SDS-PAGE) using 12% gels. 2- Phosphoglycerate Identification of the encoding gene Enolase H O 2 The encoding gene for GCK was identified by MALDI-TOF Phosphoenolpyruvate analyses. For peptide mass fingerprinting, the protein of interest was excised from Coomassie-stained gels and sub- Pyruvate ADP kinase jected to in-gel digestion with trypsin as described pre- ATP viously (Schaffer et al., 2001). Peptides were extracted by Pyruvate sequential addition of 12 mL water and 10 mL 0.1% (v/v) Fig. 1. Proposed nonphosphorylative Entner–Doudoroff pathway in the trifluoroacetic acid in 30% (v/v) acetonitrile. 0.5 mL of the thermoacidophilic euryarchaea P. torridus (Reher & Schonheit,¨ 2006) and resulting peptide solution was mixed on a stainless steel T. acidophilum (Budgen & Danson, 1986) involving 2-phosphoglycerate sample plate with 0.5 mL of a saturated a-cyano-4-hydroxy- forming glycerate kinase as a key enzyme. trans cinnamic acid solution in 50% (v/v) acetonitrile, 0.1% (v/v) trifluoroacetic acid. Samples were analysed manually the proposed nonphosphorylative branches of ED pathway with an Applied Biosystems Voyager STR MALDI-TOF mass in these organisms. spectrometer (Applied Biosystems, Weiterstadt, Germany) in positive reflector mode with 20 kV accelerating voltage, Materials and methods 63% grid voltage and the delay time set at 125 ns. External calibration was performed using calibration mixtures 1 and Purification of GCK from P. torridus 2 of the Sequazyme peptide mass standard kit (Applied Biosystems). Data acquisition and analysis was performed Picrophilus torridus was grown aerobically at 60 1C in a 8-l using the Voyager Control Panel software, version 5.0 and fermentor (FairmenTec, Germany) (stirred at 200 r.p.m.) the Voyager Data Explorer software, version 3.5 (Applied filled with 5000 mL medium (Serour & Antranikian, 2002), Biosystems). The generated mass lists were used to search a containing glucose (25 mM) and yeast extract (0.2%). For local digest database of 1535 P. torridus DSM9790 proteins GCK purification, fermentor-grown cells (30 g wet weight) (Futterer¨ et al., 2004) using ProteinProspector MS-Fit (Clau- were harvested in the late-exponential growth phase by ser et al., 1999) available at http://prospector.ucsf.edu/. centrifugation, suspended in 100 mM Tris-HCl pH 8, and disrupted by passage through a French pressure cell at Analytical assays 1.3 Â 108 Pa. Cell debris was removed by centrifugation for 90 min at 100 000 g. The supernatant was adjusted to pH of The apparent molecular mass of GCK was determined by

8, 1.5 M (NH4)2SO4 and applied to a Phenyl-Sepharose gelﬁltration on a Superdex 200 HiLoad 16/60 column at column 26/10 column equilibrated with 100 mM Tris-HCl ambient temperature. The molecular mass of the sub- pH 8, 1.5 M (NH4)2SO4. Protein was desorbed by a linear unit was determined by SDS-PAGE in 12% polyacrylamide gradient from 1.5 to 0 M (NH4)2SO4. The fractions with the gels followed by staining with Coomassie Blue according highest GCK activity [0.7–0.5 M (NH4)2SO4] were pooled, to standard procedures. Protein concentration were

c 2006 Federation of European Microbiological Societies FEMS Microbiol Lett 259 (2006) 113–119 Published by Blackwell Publishing Ltd. All rights reserved Glycerate kinase (2-phosphoglycerate forming) from P. torridus 115 determined by the method of Bradford with bovine serum (0–20 mM). The substrate specificity was tested in Assay 1 albumin as standard. by exchanging DL-glycerate for D-glycerate (2.6 mM), L-glycerate (2.6 mM), gluconate (0–12 mM), glucuronate Determination of glycerate kinase (0–12 mM), glyceraldehyde (0–5 mM), dihydroxyacetone (2 phosphoglycerate forming) (0–5 mM), glyceraldehyde-3-phosphate (0–3 mM) and glucose (0–12 mM). The nucleotide specificities were examined Assay 1 using the Assay 2 by exchanging ATP (4 mM) for alternative phosphoryl donors (UTP, CTP, GTP, acetyl-phosphate) at The ATP- and glycerate-dependent formation of 2-phos- equimolar concentrations. Cation specificity was tested phoglycerate and ADP was determined at 50 1C by coupling using Assay 1 by exchanging Mg21 for alternative divalent ADP formation to the oxidation of NADH via pyruvate cations (Ni21,Mn21,Co21, each 10 mM). The assays, were kinase and lactate dehydrogenase. The reaction mixture carried out in the presence of 50 mMMg21 to ensure that the (1 mL) contained 100 mM Tris-HCl, pH 7.3, 2.6 mM DL- auxiliary enzyme pyruvate kinase was not rate limiting. The glycerate, 10 mM MgCl , 0.3 mM NADH, 2 mM ATP, 5 mM 2 effect of K1 was tested at a concentration of 50 mM. The pH EGTA, 1 mM phosphoenolpyruvate, 1.4 U pyruvate kinase, dependence of glycerate kinase from P. torridus was mea- 2.8 U lactate dehydrogenase and enzyme. sured at 50 1C between pH 5.5 and pH 8.1 using different buffers (assay 1), Na-acetate 0.1 M (pH 5.5–6.0), BisTris Assay 2 0.1 M (pH 5.7–7.3), HEPES 0.1 M (pH 6.1–7.7) or Tris-HCl 0.1 M (pH 7.3–8.1). The temperature dependence was The ATP- and glycerate-dependent formation of 2-phos- determined between 42 and 62 1C using the assay 1. The phoglycerate and ADP was determined at 50 1C by coupling long term thermostability (0.024 mg protein in 20 mL Tris- 2-phosphoglycerate formation to the oxidation of NADH HCl, pH 8.0) was tested in sealed vials, which were in- via enolase, pyruvate kinase and lactate dehydrogenase. The cubated at 60, 70 and 80 1C up to 120 min. The vials were reaction mixture (1 mL) contained 100 mM Tris-HCl, pH cooled for 10 min and the remaining activity was tested. 7.3, 2.6 mM DL-glycerate, 10 mM MgCl2, 0.3 mM NADH, 2 mM ATP, 5 mM EGTA, 5 U enolase, 6.9 U pyruvate kinase, 7.9 U lactate dehydrogenase and enzyme. Results

Purification of 2-phosphoglycerate forming GCK Identification of the reaction product of glycerate from P. torridus kinase (assay 3) Cell extracts of P. torridus, grown on glucose, contained To test whether glycerate kinase forms 3-phosphoglycerate GCK at an activity of 0.07 U mgÀ1 (50 1C). The enzyme was as product, the enzyme was incubated for 30 min at 50 1Cin purified 5100-fold to a specific activity of 360 U mgÀ1 with a a 1 mL reaction mixture containing 100 mM Tris-HCl, pH yield of 2%, using four chromatographic purification steps 7.3, 2.6 mM DL-glycerate, 10 mM MgCl , 2 mM ATP, 5 mM 2 (Table 1). SDS-PAGE of the purified enzyme revealed one EGTA. The reaction was terminated by placing the tube on subunit of 50 kDa (Fig. 2). The molecular mass of the native ice at 0 1C. The formation of 3-phosphoglycerate was enzyme, as estimated by gelfiltration on Superdex 200, was determined by following its conversion to glyceraldehyde- 95 kDa indicating a homodimeric structure. 3-phosphate by coupling this reaction to the oxidation of NADH via 3-phosphoglycerate kinase and glyceraldehyde- 3-phosphate dehydrogenase. The assay (1 mL) contained Catalytic properties of GCK from P. torridus 50 mM potassium phosphate, pH 8.0, 1 mM ATP, 0.2 mM GCK catalyzed the ATP-dependent phosphorylation of NADH and 100 mL of the incubated mixture. NADH oxida- glycerate to 2-phosphoglycerate and ADP. The rate tion was followed after the addition of 8 U glyceraldehyde-

3-phosphate dehydrogenase and 9 U 3-phosphoglycerate Table 1. Purification of glycerate kinase of P. torridus kinase. Total Total Specific protein activity activity Enrichment Yield Kinetic parameters, substrate specificity, pH Purification step (mg) (U) (U mgÀ1) factor (%) dependence, temperature dependence and Cell extract 2930 205 0.07 thermal stability Phenyl sepharose 70 81 1.2 70 40 Resource PHE 7 63 9 129 31 For the determination of apparent Km and Vmax values for glycerate, ATP and Mg21, the following concentrations were Gel filtration 0.94 36 38.3 547 17 21 Anion exchange 0.012 4.3 358 5114 2 used (Assay 2): DL-glycerate (0–2.6), ATP (0–4) and Mg

kDa 12 (100%) could partially be replaced by GTP, CTP and UTP as 250 phosphoryl donor, acetyl phosphate was not used (Table 2). 150 GCK required divalent cations for activity, Mg21, which was 100 most effective (100%; apparent Km, 2 mM), could be replac- 75 ed to some extent by Ni21 (25%), Mn21 (11%), and Co21 (11%). K1 (50 mM), reported to activate glycerate kinase 50 50 kDa from Hyphomicrobium (Yoshida et al., 1992), was without effect. The pH optimum of GCK was at pH 7.3 and 50% of 37 activity at pH 5.6 and at pH 8.6. GCK showed moderate thermophilic properties; the enzyme had a temperature optimum at 60 1C and did not lose activity upon incubation 25 at 60 1C for 2 h. The half time at 70 1C was about 5 min.

20 Identification of the gene encoding GCK in P. torridus and of putative homologs Fig. 2. Purified 2-phosphoglycerate forming glycerate kinase from P.torridus as analyzed by SDS-PAGE. Lanes: 1, molecular mass standards; The identity of the purified GCK from P. torridus protein 2, purified enzyme from P. torridus. was determined by peptide mass fingerprinting after tryptic digestion of the protein (MALDI-TOF). Analysis of the dependence (at 50 1C) on glycerate and ATP followed peptide masses with MS-Fit using a local digest database of

Michaelis–Menten kinetics with apparent Km values of 0.34 1535 P. torridus proteins showed that 18 peptides matched and 0.5 mM, respectively. Apparent Vmax values were to the protein derived from gene Pto1442 (46% sequence 435 U mgÀ1. The specific formation of 2-phosphoglycerate coverage). Thus, this ORF annotated as putative glycerate as product of P. torridus GCK was demonstrated by coupling kinase, represents the gene in P. torridus coding for 2- its formation to NADH oxidation via enolase, pyruvate phosphoglycerate forming glycerate kinase, designated gck. kinase and lactate dehydrogenase (Assay 1). The formation Pto1442 consists of 1248 bp coding for a polypeptide of 415 of 3-phosphoglycerate as product could not be detected amino acids with a calculated molecular mass of 46.6 kDa. using Assay 3. The enzyme was specific for glycerate as phosphoryl acceptor, showing highest activity with D-glyce- Discussion rate (100%), and lower activity (34%) with L-glycerate. ATP In this paper, we report the first characterization of 2-phosphoglycerate forming GCK, a key enzyme of the Table 2. Biochemical and kinetic properties of 2-phosphoglycerate nonphosphorylative ED pathway in archaea, from the ther- forming glycerate kinases from P. torridus and Hyphomicrobium methylovorum GM2 (Yoshida et al., 1992) moacidophile P. torridus. The enzyme is a 95 kDa homodimer and showed moderate thermophilic properties around P. torridus H. methylovorum GM2 60 1C, which is in accordance with the optimal growth Apparent molecular mass temperature (about 60 1C) of Picrophilus. The pH-optimum Native enzyme (kDa) 95 41 from GCK, at 7.3, is significantly higher than the internal pH Subunit (kDa) 50 52 (pH 4.6) in this organism (van de Vossenberg et al., 1998). Calculated (kDa) 46.5 – The gene encoding glycerate kinase (assigned as gck) was Oligomeric structure a2 a pH optimum 7.3 8 identified in P. torridus (Pto1442) by MALDI-TOF analysis. Temperature optimum ( 1C) 60 50 Homologs with high sequence identity were found in À1 Vmax (U mg ) Thermoplasma acidophilum, Sulfolobus solfataricus, Sulfolo- Glycerate 435 181 bus acidocaldarius and Thermoproteus tenax. Since for these ATP 432 – archaea nonphosphorylative ED pathways have been pro- K (mM) m posed for glucose degradation, it is likely that the identified Glycerate 0.34 0.13 ATP 0.51 0.13 homologous ORFs code for functional 2-phosphoglycerate Mg21 2.1 – forming glycerate kinases in this organisms. This has to be Phosphoryl donor specificity (%) verified by detailed biochemical studies of the corresponding ATP 100 100 recombinant proteins. GTP 37 59 So far, 2-phosphoglycerate (2-PG) forming glycerate kin- CTP 24 59 ases have been described in several bacteria and eukarya. UTP 39 64 These include methanol-utilizing bacteria, e.g. Hyphomicro- –, not tested. bium, Methylobacterium (Yoshida et al., 1992; Chistoserdova

Fig. 3. Multiple sequence alignment of amino acid sequences of characterized and putative glycerate kinases from archaea form Thermotoga maritima, Methylobacterium extorquens and Homo sapiens. The alignment was generated with ClustalX using the gonnet matrix. The highly conserved residues of the presumed active site at the C-terminal domain (Glu312, Arg325, Asp341, Asn407) and from the Rossmann-like domain (Lys47, Asp189), based on crystal structure of the Thermotoga maritima protein are indicated by arrows. NCBI accession numbers: Picrophilus torridus DSM 9790 AAT44027.1; Ferroplasma acidarmanus Fer1 EAM93653.1; Thermoplasma acidophilum NP_393931.1; Thermoplasma volcanium GSS1 BAB59939.1; Sulfolobus solfataricus CAB57636.1; Sulfolobus acidocaldarius AAY79538.1; Thermoproteus tenax CAF18535.1; Thermotoga maritima Q9x1s1; Methylobacterium extorquens AAB66496.1; Pyrococcus furiosus AAL80148.1; Homo sapiens AAP41923.1.

I Fig. 4. Phylogenetic relationship of glycerate T. tenax S. acidocaldarius H. sapiens kinases with selected members of the proposed three glycerate kinase families from eukarya, S. solfataricus H. marismortui bacteria and archaea. Characterized enzymes are underlined. The numbers at the nodes are T. volcanium M. extorquens bootstrapping values according to neighbor-joining [generated using the neighbor-joining algo- rithm of ClustalX (Thompson et al., 1997)]. NCBI T. acidophilum 1000 660 954 accession numbers: I: Picrophilus torridus DSM 897 P. furiosus 1000 9790 AAT44027.1; Ferroplasma acidarmanus F. acidarmanus 999 Fer1 EAM93653.1; Thermoplasma acidophilum 1000 823 1000 NP_393931.1; Thermoplasma volcanium GSS1 T. maritima P. torridus BAB59939.1; Sulfolobus solfataricus CA- 1000 B57636.1;AAY79538.1; Thermoproteus tenax 649 CAF18535.1; Homo sapiens AAP41923.1; Haloar- K. pneumoniae cula marismortui ATCC 43049 AAV44739.1; 1000 Methylobacterium extorquens AAB66496.1; Pyro- 562 1000 coccus furiosus DSM 3638 AAL80148.1; Thermo- A. variabilis 1000 624 E. coli GK 1 toga maritima Q9x1s1; II: Klebsiella pneumoniae BAD14998; Escherichia coli K12 GK1 P77364; Escherichia coli K12 GK2 P23524; Bacillus lichen- E. coli GK 2 formis ATCC 14580 AAU21658.1; Bacillus cereus S. cerevisiae ATCC 10987 AAS39089.1; Neisseria meningitidis B. lichenformis serogroup A P57098; III: Arabidopsis thaliana III B. cereus II Q94414; Saccharomyces cerevisiae NP_011721.1; A. thaliana N. menigitidis Anabaena variabilis ATCC 29413 COG4240. The 0.1 scale bar corresponds to 0.1 substitutions per site. & Lidstrom, 1997), where the enzyme is involved in for- bacteria Thermotoga maritima and M. extorquens and of maldehyde assimilation via the serin pathway. In Escherichia human, as follows. Ferroplasma acidarmanus (52%), Ther- coli a 2-phosphoglycerate forming glycerate kinase has been moplasma acidophilum (49%), Thermoplasma volcanium implicated in glucarate and galacturate degradation (Hub- (47%), Thermotoga maritima (29%), S. solfataricus (27%), bard et al., 1998), in Flavobacterium in ethylene glycol Pyrococcus furiosus (27%), Haloarcula marismortui (25%), S. degradation (Willetts, 1978) and in mammals in serine acidocaldarius (24%), Thermoproteus tenax (21%), M. ex- metabolism (Hagopian et al., 2005). A 2-phosphoglycerate torquens (20%) and human (20%). An alignment of these forming glycerate kinase activity has also been reported for sequences, shown in Fig. 3, indicates several conserved the hyperthermophilic euryarchaeon Pyrococcus furiosus amino acids stretches. For example, based on crystal struc- (Schafer¨ & Schonheit,¨ 1992). From these glycerate kinases ture of the Thermotoga glycerate kinase, four residues of the only the enzyme from H. methylovorans GM2 has been presumed active site (E312, R325, D 359, W407, Thermotoga biochemically characterized in some detail (Table 2). The numbering), and two residues from a Rossman-like domain enzyme shows similar properties as the Picrophilus GCK, (K47, D189) were identified. These six amino acids residues however, they differ in oligomeric structure, being a mono- are highly conserved in all aligned sequences. mer in Hyphomicrobium, whereas the Picrophilus enzyme is 95 kDa homodimer (Table 4). Recently, the crystal struc- Phylogenetic affiliation tures of two putative glycerate kinases were solved, from The identification and characterization of the first archaeal Thermotoga maritima showing high sequence identity to 2- 2-phosphoglycerate forming GCK from P. torridus and thus, phosphoglycerate forming GCK from Methylobacterium the identification of putative archaeal homologs with high extorquens AM 1, and from Neisseria meningitidis. The sequence identity, allow phylogenetic studies. The phyloge- proteins from both organisms were found to represent two netic relationship of selected GCK sequences, including different structural folds (Cheek et al., 2005). those from archaea, from bacteria and eukarya is shown in Fig 4. In accordance with a recent phylogenetic study (Boldt Sequence alignment et al., 2005) the sequences were found to cluster in three The GCK sequence from P. torridus showed high sequence main groups. Group I include all sequences from archaea, identity to putative homologs of other archaea, of the from the bacteria M. extorquens and Thermotoga maritima,

c 2006 Federation of European Microbiological Societies FEMS Microbiol Lett 259 (2006) 113–119 Published by Blackwell Publishing Ltd. All rights reserved Glycerate kinase (2-phosphoglycerate forming) from P. torridus 119 and from human. All members of this group show a high sequence of Picrophilus torridus and its implications sequence identity (20–50%) to the P. torridus GCK (see for life around pH 0. Proc Natl Acad Sci USA 101: above), which is in accordance with a close phylogenetic 9091–9096. relationship. Within this group, glycerate kinase sequences Hagopian K, Ramsey JJ & Weindruch R (2005) Serine utilization from Thermoplasmales form a distinct subgroup. Group II in mouse liver: influence of caloric restriction and aging. FEBS contains only bacterial glycerate kinase sequences, including Lett 579: 2009–2013. those from Bacillus species (B. licheniformis, B. cereus), Hubbard BK, Koch M, Palmer DR, Babbitt PC & Gerlt JA sequences from the 2- and 3 phosphoglycerate forming (1998) Evolution of enzymatic activities in the enolase superfamily: characterization of the (D)-glucarate/galactarate glycerate kinases (GK1 and GK2) from E. coli (Hubbard catabolic pathway in Escherichia coli. Biochemistry 37: et al., 1998), and putative sequences from Neisseria meningi- 14369–14375. tidis and Klebsiella pneumoniae. Archaeal sequences were not Reher M & Schonheit¨ P (2006) Glyceraldehyde dehydrogenases found in this group. The distinct clustering of glycerate from the thermoacidophilic euryarchaeota Picrophilus torridus kinase sequences in group I and II is in accordance with a and Thermoplasma acidophilum, key enzymes of the non- low sequence identity ( o 10%) between members of these phosphorylative Entner-Doudoroff pathway, constitute a two groups. Further, the different structural folds of putative novel enzyme family within the aldehyde dehydrogenase glycerate kinase from Thermotoga maritima (group I) and superfamily. FEBS Lett 580: 1198–1204. from N. meningitidis (group II) (Cheek et al., 2005) support Schaffer S, Weil B, Nguyen VD, Dongmann G, Gunther K, the distinct clustering of both groups. Very recently (Boldt Nickolaus M, Hermann T & Bott M (2001) A high-resolution et al., 2005), a third distinct phylogenetic glycerate kinase reference map for cytoplasmic and membrane-associated group was identified following the first characterization of proteins of Corynebacterium glutamicum. Electrophoresis 22: 3-phosphoglycerate forming glycerate kinase from Arabi- 4404–4422. dopsis thaliana involved in photorespiration. This group Schafer¨ T & Schonheit¨ P (1992) Maltose fermentation to (group III) also contains sequences from cyanobacteria, e.g. acetate, CO2 and H2 in the anaerobic hyperthermophilic Anabena variabilis, and from Saccharomyces species, e.g. S. archaeon Pyrococcus furiosus. Evidence for the operation of a cerevisiae. All members of this group show a low sequence novel sugar fermentation pathway. Arch Microbiol 158: 188–202. identity (o 10%) to members of group I and II, respectively, Schleper C, Puehler G, Holz I, Gambacorta A, Janekovic D, supporting the proposed trichotomy of glycerate kinases Santarius U, Klenk HP & Zillig W (1995) Picrophilus gen. nov., tree topology. fam. nov.: a novel aerobic, heterotrophic, thermoacidophilic genus and family comprising archaea capable of growth References around pH 0. J Bacteriol 177: 7050–7059. 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