JOURNAL OF BACTERIOLOGY, Feb. 2010, p. 964–974 Vol. 192, No. 40021-9193/10/$12.00 doi:10.1128/JB.01281-09Copyright © 2010, American Society for Microbiology. All Rights Reserved.

The Nonphosphorylative Entner-Doudoroff Pathway in theThermoacidophilic Euryarchaeon Picrophilus torridus

Involves a Novel 2-Keto-3-Deoxygluconate-Specific Aldolase�

Matthias Reher,1 Tobias Fuhrer,2 Michael Bott,3 and Peter Schonheit1*Institut fur Allgemeine Mikrobiologie, Christian-Albrechts-Universitat Kiel, Am Botanischen Garten 1-9, D-24118 Kiel, Germany1;

Institute for Molecular Systems Biology, ETH Zurich, 8093 Zurich, Switzerland2; and Institut fur Biotechnologie I,Forschungszentrum Julich, D-52425 Julich, Germany3

Received 25 September 2009/Accepted 5 December 2009

The pathway of glucose degradation in the thermoacidophilic euryarchaeon Picrophilus torridus has beenstudied by in vivo labeling experiments and enzyme analyses. After growth of P. torridus in the presence of[1-13C]- and [3-13C]glucose, the label was found only in the C-1 and C-3 positions, respectively, of theproteinogenic amino acid alanine, indicating the exclusive operation of an Entner-Doudoroff (ED)-type path-way in vivo. Cell extracts of P. torridus contained all enzyme activities of a nonphosphorylative ED pathway,which were not induced by glucose. Two key enzymes, gluconate dehydratase (GAD) and a novel 2-keto-3-deoxygluconate (KDG)-specific aldolase (KDGA), were characterized. GAD is a homooctamer of 44-kDasubunits, encoded by Pto0485. KDG aldolase, KDGA, is a homotetramer of 32-kDa subunits. This enzyme washighly specific for KDG with up to 2,000-fold-higher catalytic efficiency compared to 2-keto-3-deoxy-6-phos-phogluconate (KDPG) and thus differs from the bifunctional KDG/KDPG aldolase, KD(P)GA of crenarchaeacatalyzing the conversion of both KDG and KDPG with a preference for KDPG. The KDGA-encoding gene,kdgA, was identified by matrix-assisted laser desorption ionization–time of flight (MALDI-TOF) mass spec-trometry (MS) as Pto1279, and the correct translation start codon, an ATG 24 bp upstream of the annotatedstart codon of Pto1279, was determined by N-terminal amino acid analysis. The kdgA gene was functionallyoverexpressed in Escherichia coli. Phylogenetic analysis revealed that KDGA is only distantly related toKD(P)GA, both enzymes forming separate families within the dihydrodipicolinate synthase superfamily. Fromthe data we conclude that P. torridus degrades glucose via a strictly nonphosphorylative ED pathway with anovel KDG-specific aldolase, thus excluding the operation of the branched ED pathway involving a bifunctionalKD(P)GA as a key enzyme.

Comparative analyses of sugar-degrading pathways in mem-bers of the domain Archaea revealed that all species analyzedso far degrade glucose and glucose polymers to pyruvate viamodification of the classical Embden-Meyerhof (EM) andEntner-Doudoroff (ED) pathways found in bacteria and eu-karya. Modified EM pathways were reported for hyperther-mophilic archaea, including, e.g., the strictly fermentativeThermococcales and Desulfurococcales, the sulfur-reducingThermoproteus tenax, and the microaerophilic Pyrobaculumaerophilum. These pathways differ from the classical EMpathway by the presence of several novel enzymes and en-zyme families, catalyzing, e.g., the phosphorylation of glu-cose and fructose-6-phosphate, isomerization of glucose-6-phosphate, and oxidation of glyceraldehyde-3-phosphate (18,22, 25).

Modified ED pathways have been proposed for aerobicarchaea, including halophiles, and thermoacidophilic cren-archaea, such as Sulfolobus species, and the euryarchaeaThermoplasma acidophilum and Picrophilus torridus. The an-

aerobic Thermoproteus tenax, which degrades glucose predom-inantly via a modified EM pathway, also utilizes—to a minorextent (�20%)—a modified ED pathway for glucose degrada-tion. The following ED pathway modifications have been re-ported in archaea (25). A semiphosphorylative ED pathway wasreported in halophilic archaea. Accordingly, glucose is convertedto 2-keto-3-deoxy-6-gluconate (KDG) via glucose dehydrogenaseand gluconate dehydratase. KDG is then phosphorylated byKDG kinase to 2-keto-3-deoxy-6-phosphogluconate (KDPG),which is split by KDPG aldolase to pyruvate and glyceralde-hyde-3-phosphate (GAP). GAP is further converted to formanother pyruvate via common reactions of the EM pathway,i.e., phosphorylative GAP dehydrogenase, phosphoglyceratekinase, phosphoglycerate mutase, enolase, and pyruvate ki-nase. The net ATP yield of this pathway is 1 ATP/mol glucose.

From initial enzyme studies of the thermoacidophilic ar-chaea Sulfolobus solfataricus, Thermoplasma acidophilum, andThermoproteus tenax, a nonphosphorylative ED pathway wasproposed (25). In this modification of the ED pathway, glucoseis converted to KDG via glucose dehydrogenase and gluconatedehydratase, as in the semiphosphorylative pathway, but thenthe steps differ as follows: KDG is cleaved into pyruvate andglyceraldehyde via 2-keto-3-deoxygluconate-specific aldolase(KDGA). The subsequent oxidation of glyceraldehyde to glyc-erate involves either NAD(P)�-dependent dehydrogenases or

* Corresponding author. Mailing address: Institut fur AllgemeineMikrobiologie, Christian-Albrechts-Universitat Kiel, Am BotanischenGarten 1-9, D-24118 Kiel, Germany. Phone: 49-431-880-4328. Fax:49-431-880-2194. E-mail: peter.schoenheit@ifam.uni-kiel.de.

� Published ahead of print on 18 December 2009.

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oxidoreductases. Glycerate is then phosphorylated by a specifickinase to 2-phosphoglycerate, which is finally converted topyruvate via enolase and pyruvate kinase. This modification ofthe ED pathway was called “nonphosphorylative” since it is notcoupled with net ATP synthesis.

However, recent comparative genomic studies and refinedenzyme analyses suggest that the crenarchaea Sulfolobus andThermoproteus utilize a so-called branched ED pathway, inwhich a semiphosphorylated route is simultaneously operativein addition to the nonphosphorylative route (25, 32). Accord-ingly, the semiphosphorylated route involves—via KDG ki-nase—the phosphorylation of KDG to KDPG, which is thencleaved to pyruvate and GAP by means of a bifunctional KDG/KDPG aldolase, KD(P)GA. GAP is then converted to an-other pyruvate via nonphosphorylative GAP dehydrogenase(GAPN), phosphoglycerate mutase, enolase, and pyruvate ki-nase. The net ATP yield of the branched ED pathway is zero.In support of this pathway, the genes encoding gluconate de-hydratase, bifunctional KD(P)GA, KDG kinase, and GAPNwere found to be clustered in Sulfolobus solfataricus (see Dis-cussion) and Thermoproteus tenax. The key enzyme of the pro-posed branched ED pathway is the bifunctional KD(P)GA,which catalyzes the cleavage of KDG to pyruvate and glycer-aldehyde and cleavage of KDPG to pyruvate and glyceralde-hyde-3-phosphate. This bifunctional aldolase, which has beencharacterized from S. solfataricus, was found to be identical toa previously described KDG aldolase of the same organism;however, its catalytic property to also utilize KDPG as a substratehas been recognized only recently. In fact, the bifunctionalKD(P)GA showed a higher catalytic efficiency for KDPG thanfor KDG (1, 14). Crystal structures of bifunctional KD(P)GAsof S. solfataricus and T. tenax have been reported (16, 27, 30; G.Taylor [United Kingdom], unpublished data).

The branched ED pathway in S. solfataricus has been re-ported to be promiscuous and therefore represents an equiv-alent degradation route for both glucose and its C-4 epimer,galactose. Accordingly, glucose dehydrogenase, gluconate de-hydratase, KDG kinase, and bifunctional KD(P)GA werefound to catalyze the conversion of both glucose and galactoseand the corresponding subsequent intermediates, i.e., gluco-nate/galactonate, KDG/KDGal (KDGal stands for 2-keto-3-deoxygalactonate), and KDPG/KDPGal (KDPGal stands for2-keto-3-deoxy-6-phosphogalactonate) (4, 12–14).

In contrast to crenarchaea, the modified ED pathway inthe thermoacidophilic euryarchaea Thermoplasma acidophilumand Picrophilus torridus has not been studied in detail. Enzymemeasurements in cell extracts and the characterization of fewenzymes suggest the operation of a nonphosphorylative EDpathway in these organisms (2, 3, 17, 19, 25). However, in vivoevidence for the operation of an ED-type pathway, e.g., by13C-labeling experiments with growing cultures, has not beenprovided yet. Furthermore, the KDG aldolase activity mea-sured in cell extracts of P. torridus and T. acidophilum has notbeen purified and characterized, in particular with respect tosubstrate specificity, and the genes encoding these enzymeshave not been identified. The biochemical analysis of this al-dolase is crucial to define the enzyme as a KDG-specific aldo-lase, indicative of a nonphosphorylative ED pathway, or asbifunctional KD(P)GA, indicative of the branched ED path-

way as proposed for the crenarchaea Sulfolobus and Thermo-proteus.

In this communication we studied the sugar-degrading path-way in P. torridus by in vivo labeling experiments with [13C]glu-cose, by enzyme measurements, and by characterization of twokey enzymes, gluconate dehydratase and KDG aldolase. Thedata indicate that P. torridus utilizes a strict nonphosphoryla-tive ED pathway, involving a novel KDG-specific aldolase as akey enzyme, and thus exclude the operation of a branched EDpathway, as in crenarchaea involving a bifunctional KD(P)GAas a key enzyme.

MATERIALS AND METHODS

Growth conditions. Picrophilus torridus (7, 21) was routinely grown aerobicallyat pH 0.9 and 60°C in 100-ml Erlenmeyer flasks filled with 20 ml mediumcontaining 25 mM glucose and 0.2% yeast extract (19, 24) and shaken at 150 rpm.For mass culturing, cells were grown in a 8-liter fermentor (FairmenTec, Ger-many) (stirred at 200 rpm) filled with 5 liters of medium. Sulfolobus acidocal-darius was grown at 70°C on a synthetic medium with 25 mM glucose as asubstrate as described previously (23). Growth was monitored by measuring theoptical density at 600 nm (OD600). Glucose consumption was determined enzy-matically with hexokinase and glucose-6-phosphate dehydrogenase.

13C-labeling experiments. To identify the glucose degradation pathways invivo, experiments with [D-13C]glucose were performed with growing cultures in100-ml Erlenmeyer flasks filled with 20 ml medium. In the case of P. torridus, themedium contained [1-13C]- or [3-13C]glucose (25 mM each) and yeast extract(0.1%). The pH was adjusted to pH 0.3. Cells were harvested in late log phase,when significant amounts of glucose had been consumed. In the case of S.acidocaldarius, the synthetic medium (pH 2.5) contained [1-13C]- or [3-13C]glu-cose (25 mM each). Cell aliquots were harvested in mid-exponential growthphase. For both P. torridus and S. acidocaldarius, 2-ml portions of culture brothwere centrifuged at 8,000 � g and 10°C for 10 min. The dry biomass pellet washydrolyzed in 1.5 ml of 6 M HCl for 24 h at 110°C in a sealed 2-ml Eppendorftube and desiccated overnight in a heating block at 85°C under a constant airstream. The hydrolysate was dissolved in 50 �l of 99.8% dimethyl formamide andtransferred into a new Eppendorf cup within a few seconds. For derivatization,30 �l of N-methyl-N-(tert-butyldimethylsilyl)-trifluoroacetamide was added,which readily silylates hydroxyl groups, thiols, primary amines, amides, andcarboxyl groups (5), and the mixture was incubated at 550 rpm and 85°C for 60min. One microliter of the derivatized sample was injected into a 6890N Networkgas chromatograph (GC) system, combined with a 5975 inert XL mass selectivedetector (Agilent Technologies) and analyzed as described earlier (6, 31). TheGC temperature profile was 160°C for 1 min, increase to 320°C at 20°C perminute, and hold at 320°C for 1 min. The injector temperature was set at 230°C,the split ratio was 1:10, the flow rate was 1.5 ml/min, and the carrier gas washelium in a HP-5MS column (30 m by 0.25 mm; 0.25 �m coated) (AgilentTechnologies). The mass spectra of the derivatized amino acid alanine werecorrected for the natural abundance of all stable isotopes and unlabeled biomassfrom inoculum. The labeling pattern of alanine is a direct and quantitativeevidence for metabolic pathways leading from glucose to pyruvate.

Determination of enzyme activities. Enzyme activities were assayed spectro-photometrically in 1 ml of assay mixture containing cell extracts of P. torridusgrown on glucose and yeast extract. Cells were harvested in the late exponentialgrowth phase at an OD600 of about 1.6 (see Fig. 1). To determine a possibleglucose-specific induction, the specific activities were also measured in cells aftergrowth in the absence of glucose on medium containing yeast extract (0.2%).Exponentially grown cells, harvested at an OD600 of 0.9, were used. One unit ofenzyme activity is defined as 1 �mol substrate consumed or product formed permin. KDG was prepared enzymatically from gluconate with purified gluconatedehydratase from P. torridus. KDG was quantified by the thiobarbituric acidassay (29). Glucose dehydrogenase was tested by the method of Reher andSchonheit (19). Gluconate dehydratase was determined at 60°C by measuring theconversion of gluconate to KDG, which was quantified as described previously(29). The assay mixture contained 50 mM sodium phosphate buffer (pH 6.2), 7.5mM gluconate, and 20 mM MgCl2. KDG aldolase was tested at 60°C both in thedirection of aldol (KDG) cleavage and aldol (KDG) synthesis: KDG cleavagewas analyzed by measuring pyruvate formation from KDG using lactate dehy-drogenase as described previously (12). The assay mixture contained 50 mMsodium phosphate buffer (pH 6.2), 2 to 5 mM KDG, 0.3 mM NADH, and 5.5 Ulactate dehydrogenase. The formation of KDG from pyruvate and D/L-glyceral-

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dehyde was monitored at 60°C over a period of 20 min and quantified in thethiobarbituric acid assay at 546 nm (29). The assay mixture contained 50 mMsodium phosphate buffer (pH 6.2), 20 mM pyruvate, and 20 mM glyceraldehyde.KDPG aldolase was determined as described above for KDG aldolase, exceptthat KDG was replaced by KDPG in the cleavage direction and glyceraldehydewas replaced by glyceraldehyde-3-phosphate in the direction of aldol synthesis.KDG aldolase and KDPG aldolase activities were also determined with cellextracts of glucose-grown T. acidophilum and S. solfataricus. Glyceraldehydedehydrogenase, enolase, and pyruvate kinase were determined as described inreference 19.

Purification of GAD from P. torridus. For gluconate dehydratase (GAD)purification, fermentor-grown cells (18 g [wet weight]) were harvested in the lateexponential growth phase by centrifugation, suspended in 50 mM Tris-HCl(pH 8.1) and 10 mM MgCl2, and disrupted by passage through a Frenchpressure cell at 1.3 � 108 Pa. Cell debris was removed by centrifugation for90 min at 50,000 � g. The supernatant was adjusted to 3 M (NH4)2SO4 andincubated at 4 to 8°C for 17 h. The precipitate was removed by centrifugationat 50,000 � g for 90 min. The supernatant was adjusted to a pH of 8 and to 2 M(NH4)2SO4 and applied to a phenyl-Sepharose 26/10 column equilibrated with100 mM Tris-HCl adjusted to a pH of 8.1 with 2 M (NH4)2SO4 and 10 mMMgCl2. Protein was desorbed by a linear gradient from 2 M to 0 M (NH4)2SO4.The fractions with the highest GAD activity eluting at 1.3 to 0.7 M (NH4)2SO4

were pooled, dialyzed against 100 mM Tris-HCl (pH 8) and 10 mM MgCl2, andapplied to a UnoQ1 column equilibrated with the same buffer. The protein waseluted with a increasing gradient from 0 to 2 M NaCl. The fractions containingthe highest GAD activity (0.2 to 0.4 M NaCl) were pooled and concentrated to1,000 �l by ultrafiltration (cutoff, 10 kDa). The concentrated protein solution wasapplied to a Superdex 200 HiLoad 16/60 column equilibrated with 50 mMTris-HCl (pH 7.1) and 150 mM NaCl. The protein was eluted at a flow rate of 1ml/min. At this stage, GAD was essentially pure as judged by SDS-PAGE.

Identification of the gene encoding GAD and characterization of GAD. Thegene encoding GAD was identified by matrix-assisted laser desorption ioniza-tion–time of flight (MALDI-TOF) mass spectrometry (MS) of the purified pro-tein (44-kDa band by SDS-PAGE) as reported previously (17, 20). The pHdependence of gluconate dehydratase was measured at 60°C between pH 4.0 andpH 7.5 using either 0.1 M sodium acetate (pH 4.0 to 6.0) or 0.1 M sodiumphosphate (pH 6.2 to 7.5) as a buffer. The temperature dependence was deter-mined between 49°C and 83°C. The long-term thermostability was tested insealed vials containing 6 �g protein in 100 �l sodium phosphate buffer (pH 6.2)with 50 mM MgCl2, which were incubated at 60°C, 70°C, 80°C, and 90°C for upto 120 min. The vials were cooled for 10 min, and the remaining activity wastested. The substrate specificity was tested at 60°C in 50 mM sodium phosphatebuffer (pH 6.2). Kinetic constants were determined for gluconate and galacto-nate in the presence of 20 mM MgCl2, and kinetic constants for MgCl2 weredeternined in the presence of 10 mM gluconate.

Purification of 2-keto-3-deoxygluconate aldolase (KDGA) from P. torridus andidentification of the gene encoding KDGA. For KDGA purification, glucose-grown cells were harvested in the late exponential growth phase by centrifuga-tion. The cells (15 g [wet weight]) were suspended in 50 mM Tris-HCl (pH 8.1)and disrupted by passage through a French pressure cell. Cell debris was re-moved by centrifugation at 50,000 � g for 90 min. The supernatant was adjustedto 1 M (NH4)2SO4 and applied to a phenyl-Sepharose 26/10 column equilibratedwith 100 mM Tris-HCl (pH 8) and 1 M (NH4)2SO4. Protein was eluted by alinear gradient from 1 M to 0 M (NH4)2SO4. Fractions containing the highestKDGA activity [eluting at 0.3 to 0.1 M (NH4)2SO4] were diluted 30-fold in 50mM Tris-HCl (pH 7.6), and applied to a UnoQ5 column (5 ml), which wasequilibrated with the same buffer. Bound protein was eluted with a linear gra-dient up to 1 M NaCl. Fractions containing the KDGA activity (eluting at 0.05to 0.15 M NaCl) were concentrated by ultrafiltration (cutoff, 10 kDa) and appliedto a Superdex 200 HiLoad 16/60 column equilibrated with 50 mM Tris-HCl (pH7.1) containing 150 mM NaCl. The protein was eluted with the same buffer.Fractions containing the highest KDGA activity were diluted in 0.1 M Tris-HCl(pH 8.0) and applied to a UnoQ1 column equilibrated with 100 mM Tris-HCl(pH 8). KDGA was eluted with an increasing gradient up to 0.5 M NaCl. At thisstage, KDGA was essentially pure as judged by SDS-PAGE, yielding a singleprotein band at 32 kDa. After in-gel digestion of this band with trypsin, theeluted peptides were analyzed by MALDI-TOF mass spectrometry and used toidentify the KDGA-encoding gene as described previously (17, 20). In addition,N-terminal amino acid sequencing of the purified enzyme was performed by themethod of Meyer et al. (15).

Characterization of KDGA from P. torridus. The pH dependence and temper-ature dependence of KDGA were determined using the same assay conditions asdescribed for GAD. KDGA activity was analyzed in the direction of KDG

formation. The substrate specificity of KDG aldolase was tested at 60°C in 50mM sodium phosphate buffer (pH 6.2). Kinetic constants for KDG and KDPGwere determined using substrate concentrations up to 1 mM and 32 mM, re-spectively. In the direction of aldol formation, apparent Km and Vmax values weredetermined for glyceraldehyde and glyceraldehyde-3-phosphate with 25 mMpyruvate, and the values were determined for pyruvate at 10 mM glyceraldehyde.Other aldehyde substrates, i.e., glycolaldehyde, D-ribose, D-xylose, L-arabinose,D-arabinose, acetaldehyde, and crotonaldehyde, were each tested at 25 mM inthe presence of 25 mM pyruvate. The (aldol) condensation product formed byKDGA from D- or L-glyceraldehyde and pyruvate was identified by the methodof Lamble et al. (12), with the following modifications: 80 mM (each) D-glycer-aldehyde or L-glyceraldehyde was mixed with 160 mM sodium pyruvate in 250 �lof water containing 5 �g P. torridus KDGA. The reaction mixture was heated at50°C overnight in a shaking incubator. Samples were analyzed by high-perfor-mance liquid chromatography (HPLC) using an Aminex HPX-87H column (Bio-Rad) using 1 M formic acid as the eluent and linked to a refractive indexdetector.

Cloning and expression of KDGA from P. torridus in E. coli and purificationof the recombinant enzyme. On the basis of MALDI-TOF MS analysis and of theN-terminal amino acid sequence, a single open reading frame (ORF), Pto1279(see Results), was identified in the sequenced genome of P. torridus. The ORFwas characterized as the kdgA gene, encoding 2-keto-3-deoxygluconate aldolase,by its functional overexpression in E. coli as follows. The gene was amplified fromgenomic DNA of P. torridus by PCR and cloned into pET19b via two restrictionsites (NdeI and BamHI) created with the primers 5�-GAATTCATATGTACAAGGGTATAGTATG-3� and 5�-GAT TAGGATCCAAAATATTAATTTATATTTCAA-3� (restriction sites are underlined). The recombinant plasmidpET19b-kdgA was transferred into E. coli BL21 CodonPlus (DE3)-RIL cells.Transformed cells were grown in Luria-Bertani medium at 37°C, and kdgAexpression was induced by the addition of 1 mM isopropyl-1-thio-�-D-galacto-pyranoside (IPTG). After 4 h, cells were harvested by centrifugation, followed byresuspension in 50 mM Tris-HCl (pH 8.2) containing 300 mM NaCl and 5 mMimidazole. Cells were disrupted by passage through a French pressure cell. Aftercentrifugation, the supernatant was incubated at 58°C for 45 min and centrifugedat 100,000 � g for 1 h. The supernatant, exhibiting KDG aldolase activity, wasapplied to a Ni-nitrilotriacetic acid (Ni-NTA) column equilibrated with 50 mMTris-HCl (pH 8.2) containing 300 mM NaCl and 5 mM imidazole. Bound proteinwas specifically eluted with increasing imidazole concentrations, yielding pureKDGA as judged by a single protein band at 33 kDa on SDS-polyacrylamide gels.

RESULTS

Growth of P. torridus on glucose. Growth of P. torridus onglucose requires low concentrations of yeast extract. With 25mM glucose and 0.2% yeast extract and at pH 0.9, the cellsgrew with a doubling time of about 10 h up to a final celldensity with an OD600 of 1.6. In the absence of glucose, thecells grew on yeast extract with a similar doubling time to a celldensity with an OD600 of 1. As shown in Fig. 1, significantglucose consumption occurred only in the later growth phase.This indicates that initial growth was based almost exclusivelyon components of the yeast extract which apparently preventedglucose consumption.

In vivo operation of the ED pathway in P. torridus and S.acidocaldarius. To analyze the sugar-degrading pathway of P.torridus by in vivo 13C-labeling experiments (31), cells weregrown on 13C-labeled glucose at pH 0.3 at a reduced yeastextract concentration of 0.1%. Under these conditions, thecells grew on yeast extract up to an OD600 of 0.3 followed bythe phase of glucose consumption up to an OD600 of 0.8 (notshown). Cells were harvested in the late exponential phase atan OD600 of 0.8, i.e., when significant amounts of glucose havebeen consumed. L-Alanine obtained by protein hydrolysis aftergrowth on natural, [1-13C]- or [3-13C]glucose was analyzed byGC-MS. When [1-13C]- or [3-13C]glucose was used, 13C labelwas found exclusively in the C-1 or C-3 position of alanine,respectively, derived directly from pyruvate, while phos-

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phoenolpyruvate (PEP) was unlabeled (not shown). These la-beling patterns clearly show that glucose was exclusively catab-olized through an ED-like pathway in vivo. The activity of anEmbden-Meyerhof pathway was excluded due to the absenceof 13C label at the C-1 or C-3 position of alanine when [1-13C]-or [3-13C]glucose was used as the carbon source, respectively(Table 1). From the fact that about 30% of the alanine con-tained 13C label, it is concluded that about 40% of the alanineis taken up by the cell from the yeast extract in the medium and60% (two times 30% due to the unlabeled PEP) originatesfrom glucose with pyruvate as the immediate precursor (Table1).

For comparison, similar in vivo 13C-labeling experimentswere performed with S. acidocaldarius, for which a branchedED pathway was proposed. The labeling distribution in pro-tein-derived L-alanine was analyzed after growth of S. acido-caldarius in a minimal medium with [1-13C]- or [3-13C]glucose

as the sole carbon source. Similar to P. torridus, the labelingpattern indicates the exclusive operation of an ED pathway invivo with more than 50% alanine containing 13C label (Table1). Assuming that alanine was produced from pyruvate origi-nating exclusively from the ED pathway, a fractional label ofmaximally 50% can be expected with the other 50% synthe-sized from unlabeled PEP. The slightly higher observed 60%label might be due to additional label introduced by otherpathways, presumably tricarboxylic acid (TCA) cycle and glu-coneogenesis.

Enzyme activities of nonphosphorylative ED pathway in P.torridus. In accordance with previous data (19), extracts of P.torridus grown on glucose and yeast extract contained activitiesof enzymes of a nonphosphorylative ED pathway, includingglucose dehydrogenase, gluconate dehydratase, KDG aldolase,glyceraldehyde dehydrogenase, glycerate kinase, (2-phospho-glycerate-forming) enolase, and pyruvate kinase. To test thepossible induction of these enzymes by glucose, the activitieswere also measured in cells after growth with yeast extract inthe absence of glucose. The specific activities of all ED en-zymes were similar both in the presence and absence of glucose(Table 2), suggesting that the enzymes were not inducible byglucose but constitutively expressed. Accordingly, the activitiesof ED enzymes did not change in growing cultures after tran-sition from growth on yeast extract components to growth onglucose (data not shown).

FIG. 1. Growth of P. torridus on 25 mM glucose and 0.2% yeastextract. The cultures were incubated at pH 0.9 and 60°C in a 8,000-mlfermentor filled with 5,000 ml medium and stirred at 200 rpm. Growthon glucose and yeast extract (f), growth on yeast extract in the absenceof glucose (F), and glucose consumption (�) are shown.

TABLE 1. Mass isotopomer distribution in alanine in P. torridus and S. acidocaldariusa

Species Glucose label Fractional label m0b m1 m2 m3

P. torridus 100% �1-13C�glucose Alanine (M-57)�c 0.719 0.272 0.005 0.000Alanine (M-85)� 0.965 0.032 0.000

100% �3-13C�glucose Alanine (M-57)� 0.683 0.301 0.010 0.002Alanine (M-85)� 0.698 0.292 0.007

S. acidocaldarius 100% �1-13C�glucose Alanine (M-57)� 0.389 0.604 0.008 0.000Alanine (M-85)� 0.984 0.017 0.000

100% �3-13C�glucose Alanine (M-57)� 0.307 0.655 0.036 0.003Alanine (M-85)� 0.317 0.663 0.020

a The data are from 100% �1-13C�- and �3-13C�glucose growth experiments.b m0 is the fractional abundance of the fragments with the lowest mass, and m1 to m3 (mi 0) are the abundances of molecules with higher masses.c Cracking of the derivatized alanine leads to the following fragments: (M-57)� with loss of a tert-butyl group; (M-85)� with loss of the CO of alanine (C-1 position)

and a tert-butyl group. Thus, (M-57)� and (M-85)� correspond to the 1-3 fragment and the 2-3 fragment of alanine, respectively.

TABLE 2. Specific activities of enzymes of the nonphosphorylativeEntner-Doudoroff pathway in the presence and absence of glucosea

Enzyme activitySp act (U mg1)

� Glucose Glucose

Glucose dehydrogenase 0.49 0.42Gluconate dehydratase 0.03 0.04KDG aldolase 0.07 0.07Glyceraldehyde dehydrogenase 0.14 0.10Glycerate kinase 0.10 0.09Enolase 0.04 0.04Pyruvate kinase 0.03 0.03

a The enzymes were from cell extracts of P. torridus cells grown on 25 mMglucose and 0.2% yeast extract or 0.2% yeast extract in the absence of glucose.Enzyme assays were as described in Materials and Methods. KDG aldolase wasassayed in the direction of KDG cleavage.

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KDG and KDPG aldolase activities in cell extracts of P.torridus, T. acidophilum, and S. acidocaldarius. Cell extracts ofP. torridus exhibited aldolase activity catalyzing the cleavage ofKDG (0.07 U/mg; Km of 0.4 mM) at a 25-fold-higher ratecompared to the cleavage of KDPG (0.003 U/mg). This highpreference for KDG over KDPG suggests that in P. torridus, aKDG-specific aldolase (KDGA) is the relevant aldolase forglucose degradation in vivo as part of the nonphosphorylatedEDpathway.Similarresultswereobtainedwiththethermoacido-philic euryarchaeon Thermoplasma acidophilum. Extracts ofglucose-grown cells converted KDG (0.26 U/mg; Km 0.28 mM)at 130-fold-higher activities compared to KDPG (0.002 U/mg),indicating that as in P. torridus, a KDG-specific aldolase isoperative in vivo in T. acidophilum.

For comparison, aldolase activities were also measured inthe thermoacidophilic crenarchaeon Sulfolobus acidocaldarius,for which a branched ED pathway was proposed with a bifunc-tional KD(P)G aldolase as a key enzyme. Extracts of glucose-grown cells of S. acidocaldarius catalyzed the cleavage of KDG(0.18 U/mg; Km of 2.6 mM) and KDPG (0.2 U/mg; Km of 0.19mM) at similar rates, which is in accordance with an in vivofunction of a bifunctional KD(P)G aldolase.

Characterization of key enzymes of the nonphosphorylativeED pathway in P. torridus. The enzyme activities in cell extractsof P. torridus, in particular the unusual KDG-specific aldolase,suggest the operation of a nonphosphorylative ED pathway inthis euryarchaeon. To further characterize the key enzymes ofthis pathway, the first euryarchaeal gluconate dehydratase andthe KDG-specific aldolase activity were purified and charac-terized, and the genes encoding them were identified.

GAD. Gluconate dehydratase (GAD) was purified 190-foldto apparent homogeneity involving five purification steps (Ta-ble 3). SDS-PAGE of the purified enzyme revealed one sub-unit at 44 kDa (Fig. 2A). The molecular mass as estimated bygel filtration was 340 kDa, indicating a homooctameric struc-ture of the native enzyme. By peptide mass fingerprinting ofthe purified enzyme, a single ORF, Pto0485, was identifiedin the genome of P. torridus; the matched peptides cover 34%of the protein. Thus, Pto0485 represents the gad gene encodinggluconate dehydratase in P. torridus. GAD catalyzed the con-version of gluconate to KDG following Michaelis-Menten ki-netics with Vmax and Km values of 15 U/mg and 2.5 mM,respectively. Gluconate dehydratase also catalyzed dehydra-tion of the D-galactonate at about 1 U/mg with a Km for ga-lactonate of 2 mM. Xylonate (10 mM) was not utilized. Theenzyme required Mg2� with an apparent Km of 4.2 mM. ThepH optimum of GAD was at pH 6, and the enzyme showed50% of activity at pH 8.1 and at pH 4.5, which corresponds to

the measured internal pH of P. torridus of pH 4.6 (28). GADshowed moderately thermophilic properties with a tempera-ture optimum at 70°C and a substantial thermostability at 60°C,not losing activity upon incubation for 2 h. At 70°C, its half-lifewas about 15 min.

KDGA. 2-Keto-3-deoxygluconate aldolase (KDGA) activityfrom P. torridus was purified 340-fold to apparent homogeneityby four chromatographic steps (Table 4). SDS-PAGE of thepurified enzyme revealed one subunit at 32 kDa (Fig. 2B). Themolecular mass as estimated by gel filtration was 120 kDa,indicating a homotetrameric structure of the native enzyme.The pH optimum of KDGA was at pH 5.5, and 50% of activitywas found at pH 4.5 and 7.5. The enzyme has a temperatureoptimum at 65°C and showed high thermostability. At 70°C,the enzyme did not lose activity upon incubation for 2 h. Thehalf-lives of the enzyme at 80°C and 90°C were 20 min and 15min, respectively.

Kinetic properties. KDGA catalyzed the cleavage of KDGto pyruvate and glyceraldehyde with apparent Vmax and Km

values of 50 U/mg and 0.3 mM, respectively. The enzyme alsocatalyzed the conversion of KDPG to pyruvate and glyceral-dehyde-3-phosphate with apparent Vmax and Km of 0.63 U/mgand 8 mM, respectively. Thus, the catalytic efficiency (kcat/Km)for KDG was almost 2,000-fold higher than that for KDPG,indicating that KDGA from P. torridus is highly specific forKDG, making the use of KDPG as a physiological substratehighly unlikely (Table 5).

KDGA catalyzed the aldol condensation reaction, i.e., KDGformation from D- or L-glyceraldehyde and pyruvate with ap-

TABLE 3. Purification of gluconate dehydratase from P. torridus

FractionTotal

activity(U)

Totalprotein

(mg)

Sp act(U/mg)

Purificationfactor(fold)

Yield(%)

Cell extract 67.6 2,464 0.027 1 100(NH4)2SO4

precipitation90.3 816 0.11 4.1 130

Phenyl Sepharose 47.5 24.5 1.9 72.3 70UnoQ1 1.7 0.46 3.6 133 2.5Gel filtration 0.93 0.182 5.1 189 1.4

FIG. 2. SDS-PAGE of purified gluconate dehydratase (A) andKDG aldolase (B). Lane 1, molecular mass markers, lane 2, purifiedenzyme. The positions of molecular mass markers (in kilodaltons) areshown to the left of each gel.

TABLE 4. Purification of KDG aldolase from P. torridus

FractionTotal

activity(U)

Totalprotein

(mg)

Sp act(U/mg)

Purificationfactor(fold)

Yield(%)

Cell extract 164 1,179 0.14 1 100Phenyl Sepharose 47 42 1.1 7.8 29UnoQ5 37 9.1 4.1 29 23Gel filtration 6.2 0.2 31 221 3.8UnoQ1 6.0 0.125 48 343 3.6

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parent Vmax values of 67 U/mg (D form) and 59 U/mg (L form)and apparent Km values of 4.6 mM (glyceraldehyde) and 2.7mM (pyruvate). HPLC analysis of the aldol formed from D-glyceraldehyde and pyruvate revealed the formation of bothKDG and its C-4 epimer 2-keto-3-deoxygalactonate (KDGal)at a ratio of 60% and 40%, respectively. Almost identical datawere obtained with L-glyceraldehyde and pyruvate. The dataindicate that KDGA from P. torridus lacks facial stereoselec-tivity of aldol formation as has been reported previously for thebifunctional KD(P)G aldolase from Sulfolobus solfataricus(12). Besides glyceraldehyde (100%), glycolaldehyde (13%),D-ribose (4%), and D-xylose (4%) were accepted as aldehydesubstrates with pyruvate. KDGA enzyme also catalyzed at lowactivity the condensation of glyceraldehyde-3-phosphate andpyruvate. The reaction showed a pronounced substrate inhibi-tion above 4 mM GAP. No activity was found at 15 mM GAP.At 4 mM GAP and 20 mM pyruvate, the specific activity was 7U/mg.

Identification of the KDGA-encoding gene. The gene encod-ing KDGA, kdgA, was identified in the genome of P. torridus bypeptide mass fingerprinting of the purified enzyme. A singleORF, Pto1279, was detected with matching peptides covering51% of the encoded protein. This ORF, previously annotatedas dihydrodipicolinate synthase, codes for a protein of 266amino acids with a calculated molecular mass of 30.2 kDa. Thedetermination of N-terminal amino acid sequence (MYKGIVCPMITPLDAHGNIDYNATN) of KDGA purified from P.torridus revealed that the translation start codon was an ATG24 bp upstream of the annotated ATG start codon of Pto1279.Thus, the corrected ORF Pto1279 encodes a larger protein(274 amino acids, 31.3 kDa) containing eight additional aminoacids at the N terminus.

Recombinant KDGA. The function encoded by Pto1279(kdgA gene) was determined by its functional overexpression inE. coli. The ORF was cloned into a pET vector and expressedin E. coli as a His-tagged fusion protein, which was purified bya heat step and Ni-NTA affinity chromatography. The purifiedrecombinant KDGA was characterized as a 120-kDa homotet-ramer showing catalytic properties similar to those of the en-zyme purified from P. torridus (Table 5).

DISCUSSION

In the present communication, the glucose degradationpathway in P. torridus was analyzed by in vivo labeling experi-ments and by detailed enzyme studies. The data indicate thatP. torridus utilizes a strictly nonphosphorylative Entner-Dou-

doroff pathway with a novel KDG-specific aldolase as a keyenzyme. The nonphosphorylative ED pathway of P. torridusand its key enzymes, gluconate dehydratase (GAD) andKDGA, will be discussed in comparison with the branched EDpathway of Sulfolobus.

The nonphosphorylative ED pathway in P. torridus. The in vivo13C-labeling pattern of protein-derived L-alanine after growth ofP. torridus with specifically 13C-labeled glucose clearly indicatesthe exclusive operation of an ED-like pathway. Cell extracts con-tained all enzyme activities of a nonphosphorylative ED pathway.The two key enzymes, gluconate dehydratase (GAD) and aKDG-specific aldolase (KDGA), were characterized, and theKDGA-encoding genes were identified (see below).

The enzymes of the nonphosphorylative ED pathway in P.torridus were found not to be regulated by glucose. This con-stitutive expression might be explained since the organism usesdifferent pathways for glucose degradation and gluconeo-genesis, i.e., modified ED pathway and reversed EM path-way, respectively. Theses pathways do not share commonintermediates and reactions which might cause a futile cycle,and thus, they can exist in parallel without being regulated.In accordance, we measured several enzymes of the reversedEM pathway in P. torridus, i.e., phosphoglycerate mutase(0.07), phosphoglycerate kinase (0.03), glyceraldehyde-3-phosphate dehydrogenase (0.03), triosephosphate isomerase(0.64), and phosphoglucose isomerase (0.03) and found thatthe specific activities (given in U/mg at 55°C) were also con-stitutive and not regulated by glucose. Despite the constitutiveformation of ED enzymes in P. torridus, glucose was not sig-nificantly metabolized in the first growth phase (Fig. 1). Thisindicates that components of the yeast extract are used ascarbon sources in this phase and that the presence of thesecomponents apparently prevents glucose utilization, e.g., byinhibition of glucose transport into the cell.

Gluconate dehydratase. GAD was characterized as a 340-kDa homooctameric protein encoded by Pto0485. The enzymeshowed high sequence identity (44%) to GAD (SSO3198)from S. solfataricus. Both GADs share a similar subunit sizeand an octameric oligomeric structure (11). However, a mo-nomeric structure of the S. solfataricus GAD has also beenreported (13). GADs from both organisms showed similar cat-alytic properties, including Mg2� dependence of activity andthe utilization of galactonate in addition to gluconate, indicat-ing substrate promiscuity. The ratio of catalytic efficiency forgluconate and galactonate utilization was about 10 to 1, whichis on the order of that reported for the promiscuous GAD fromS. solfataricus (ratio of 6:1) (13).

Orthologs of P. torridus GAD with high sequence identitywere also found in Thermoplasma species and in the crenar-chaeon Thermoproteus tenax (1). GAD from P. torridus andother archaeal GADs belong to the mandelate racemase (MR)subfamily of the enolase superfamily; they contain the charac-teristic signature patterns of this family, including conservedglutamate residues as ligands for Mg2� and conserved arginineand aspartate residues involved in general acid base cataly-sis in the dehydration mechanism (8, 14). The archaealGADs do not show similarities to the bacterial GAD fromAchromobacter xylosoxidans (10) and to 6-phosphogluconatedehydratases of the classical ED pathway in bacteria; these

TABLE 5. Kinetic properties of KDGA from P. torridus

Parameter Substrate P. torridusa S. solfataricusb

kcat (s1) KDG 26.7 (24.2) 28.3KDPG 0.34 (0.36) 64.3

Km (mM) KDG 0.3 (0.4) 25.7KDPG 8 (2.5) 0.1

kcat/Km ratio KDG 89 (52) 1.1KDPG 0.04 (0.14) 643

a Values in parentheses were determined with recombinant KDGA.b For comparison, kinetic data from bifunctional KD(P)GA from S. solfatari-

cus (14) were included.

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bacterial enzymes belong to the dihydroxyacid dehydratase/6-phosphogluconate dehydratase (ILVD/EDD) superfamily.

KDG aldolase. Cell extracts of P. torridus contained an al-dolase activity which shows a high preference for KDG overKDPG, giving the first indication of a KDG-specific aldolase tobe operative in glucose degradation in vivo. The purified KDGaldolase (KDGA) from P. torridus was characterized as a novelKDG-specific aldolase. KDGA is a homotetrameric proteincomposed of 32-kDa subunits. This molecular composition wasalso reported for bifunctional KD(P)GA from Sulfolobus spe-cies and Thermoproteus tenax. However, KDGA differs fromKD(P)GA with respect to substrate specificity for KDG andKDPG and to phylogenetic affiliation.

Substrate specificity. KDGA from P. torridus cleaved KDGat a 1,000- to 2,000-fold-higher catalytic efficiency compared tothat of the phosphorylated aldol KDPG. Thus, KDGA repre-sents an aldolase with a novel substrate specificity being highlyspecific for (nonphosphorylated) KDG. Also, in the directionof aldol synthesis, KDGA catalyzed the formation of KDGfrom pyruvate and glyceraldehyde with high preference overKDPG synthesis from pyruvate and glyceraldehyde-3-phos-phate.

In contrast, KD(P)GA from S. solfataricus cleaves bothKDPG and KDG; however, it has a high preference for KDPG(14) (Table 5). In the direction of aldol formation, KD(P)GA

from S. acidocaldarius and from Thermoproteus tenax showed apreferred formation of KDPG over KDG (1, 30).

KDGA from P. torridus showed substrate promiscuity, asmeasured in the direction of aldol synthesis. The enzyme cat-alyzed the formation of both KDG and its C-4 epimer 2-keto-3-deoxygalactonate (KDGal) from D- or L-glyceraldehyde andpyruvate at a similar ratio, indicating lack of stereospecificcontrol in formation and cleavage of KDG and KDGal. In thisrespect, KDGA from P. torridus is similar to KD(P)GA fromSulfolobus solfataricus.

Phylogenetic affiliation. By mass spectrometry analysis ofpurified P. torridus KDGA, a single ORF, Pto1279, originallyannotated as dihydrodipicolinate synthase, was identified asthe gene (kdgA) encoding KDGA. The start codon of Pto1279was incorrectly annotated as identified by N-terminal aminoacid sequencing of the enzyme purified from P. torridus. Thecorrect Pto1279 gene encodes a protein that is 8 amino acidslonger. Heterologous overexpression of Pto1279 yielded a re-combinant KDGA with kinetic properties similar to those ofthe KDGA purified from P. torridus.

KDGA from P. torridus (Pto1279) showed about 50% se-quence identity to putative homologs from Thermoplasma spe-cies, T. acidophilum (Ta1157), and T. volcanium (TVN1228),suggesting the presence of functional KDG-specific aldolasesin these euryarchaea. This is supported by enzymatic measure-

FIG. 3. Multiple amino acid sequence alignment of KDGA from P. torridus and its homologs in T. acidophilum and T. volcanii witharchaeal bifunctional KD(P)GA, NAL of E. coli, and DHDPS of E. coli. The alignment was generated with ClustalX using the gonnet matrix.Consensus patterns (DHDPS_1 and DHDPS_2) are boxed. The dihydrodipicolinate synthetase family signature 1 consensus pattern is[GSA]-[LIVM]-[LIVMFY]-x(2)-G-[ST]-[TG]-G-E-[GASNF]-x(6)-[EQ] (PS00665), and the dihydrodipicolinate synthetase family signature 2consensus pattern is Y-[DNSAH]-[LIVMFAN]-P-x(2)-[STAV]-x(2,3)-[LIVMFT]-x(13,14)-[LIVMCF]-x-[SGA]-[LIVMFNS]-K-[DEQAFYH]-[STACI](PS00666). Signature 2 consensus pattern includes a lysine (marked by an arrow) which is involved in Schiff base formation. Catalytic residues (�) andresidues forming a putative phosphate-binding motif (#) for KDP according to references 16, 27, and 30 are indicated. Pto-KDGA, KDGA of P. torridus;Tac-KDGA and Tvo-KDGA, homologs of Pto-KDGA in T. acidophilum and T. volcanii Ta1157 and TVN1228; Sso-KD(P)GA, S. solfataricus gi:2879782;Tte-KD(P)GA, T. tenax gi:41033593; Eco-NAL, gi:128526; Eco-DHDPS, gi:145708.

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ments in T. acidophilum (this paper), showing aldolase activityin vivo with high preference for KDG over KDPG, suggestingthe operation of a KDG-specific aldolase encoded by Ta1157.KDGA from P. torridus showed only low sequence identities(20 to 25%) to characterized bifunctional KD(P)GA from Sul-folobus species and T. tenax. Both types of aldolases, KDGAand KD(P)GA, are class I aldolases, which belong to the di-hydropicolinate synthase (DHPDS)-like superfamily, whichalso include DHPDSs and N-acetylneuraminate lyases (NAL).A multiple-sequence alignment of KDGA from P. torridus andputative homologs from Thermoplasma species, from charac-terized bifunctional KD(P)GAs from Sulfolobus species and T.tenax, and from selected members of DHPDS and NAL fam-ilies is given in Fig. 3. Both KDGA and KD(P)GA containseveral conserved amino acids typical of enzymes in theDHPDS-like family, including conserved lysine residues indica-tive of class I aldolases forming a Schiff base intermediate in

the catalytic cycle. On the basis of crystal structures of Sulfolo-bus and Thermoproteus KD(P)GAs, amino acids for substratebinding were identified, including two conserved arginines anda conserved tyrosine (Fig. 3), which were proposed to form aputative phosphate binding pocket (16, 30; Gary Taylor [UnitedKingdom], unpublished results). These conserved amino acidsare absent in KDGA from P. torridus, which might reflect itspreference for nonphosphorylated substrate KDG.

The phylogenetic relationships of KDGA from P. torridus,characterized bifunctional KDG/KDPG aldolases, KD(P)GA,and other families (dihydrodipicolinate synthase [DHDPS] andN-acetylneuraminate lyase [NAL]) of the DHDPS superfamilyare shown in Fig. 4. In accordance with its unique substratespecificity being highly specific for KDG, KDGA from P. torridus(Pto1279) and putative homologs from Thermoplasma volcanium(TVN1228), and T. acidophilum (Ta1157), form a distinct familywithin the DHDPS superfamily. The KDGA family is largely

FIG. 4. Phylogenetic relationship of KDGA with selected members of the DHDPS-like protein family from bacteria and archaea. Characterizedenzymes are underlined. The numbers at the nodes are bootstrapping values according to neighbor joining (generated by using the neighbor-joiningalgorithm of ClustalX). Accession numbers for the proteins and enzymes from the different species are shown in parentheses as follows: for KDGA,Picrophilus torridus Pto1279, Thermoplasma volcanium GSS1 (gi:13542059) TVN1228, and Thermoplasma acidophilum DSM 1728 (gi:16082170)Ta1157; for NAL, Homo sapiens (gi:13540533), Haemophilus influenzae (Swiss Prot P44539), and Escherichia coli (Swiss Prot P06995); forKD(P)GA, Sulfolobus solfataricus (gi:2879782), Sulfolobus tokodaii strain 7 (gi:15922811), Sulfolobus acidocaldarius DSM 639 (gi:70606067),Thermoproteus tenax (gi:41033593), Pyrobaculum arsenaticum DSM 13514 (gi:145591599), and Metallosphaera sedula DSM 5348 (gi:146304062); forDHDPS, Methanocaldococcus jannaschii DSM 2661 (gi:15668419), Methanobrevibacter smithii ATCC 35061 (gi:148642891), Haloarcula marismor-tui ATCC 43049 (gi:55377124), Natronomonas pharaonis DSM 2160 (gi:76801395), Halorubrum lacusprofundi ATCC 49239 (gi:153895122),Haloquadratum walsbyi DSM 16790 (gi:110667468), Thermotoga maritima (gi:7531088), Escherichia coli (gi:145708), Nicotiana sylvestris (gi:14575543), and Mycobacterium tuberculosis AF2122/97 (gi:31793927); for proteins/enzymes not shown in groups, Halobacterium sp. NRC-1(gi:15789685), Picrophilus torridus DSM 9790 (gi:48478098) Pto1026, Thermoplasma acidophilum DSM 1728 (gi:16081713) Ta0619, Thermoplasmavolcanium GSS1 (gi:14324884), TVG0663048, and Ferroplasma acidarmanus Fer1 (gi:69268899). The scale bar corresponds to 0.1 substitution persite.

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separated by strong bootstrap support from bifunctionalKD(P)GA, which also form a distinct cluster. Besides character-ized KD(P)GAs (underlined), the cluster contained putativehomologs in Pyrobaculum and Methanosphaera. Distanthomologs with less sequence identity were found in Ferroplasmaacidarmanus, P. torridus (Pto1026), T. acidophilum (Ta0619),and T. volcanii (TVN0669). The function of the encoded pro-teins is not known. A functional involvement in glucose deg-radation appears unlikely as concluded from a proteomic studyof soluble proteins in glucose-grown Thermoplasma acidophi-lum (26). In this study, no gene product of ORF Ta0619 wasdetected, whereas the Ta1157-encoded gene product, which isa close homolog to P. torridus KDGA, was present. The datasuggest that in T. acidophilum, a KDG-specific aldolase en-

coded by Ta1157 is operative in glucose degradation, which isin accordance with the KDG-specific aldolase activity, mea-sured in cell extracts (see above).

Nonphosphorylative ED pathway versus branched ED path-way in archaea. In summary, the data reported here and inprevious work for P. torridus (2, 17, 19) present the first compre-hensive description of a strictly nonphosphorylative ED pathwayin archaea. A similar pathway is probably operative in closelyrelated Thermoplasma species based on enzyme and genomicstudies. A comparison of the nonphosphorylative ED pathway inP. torridus and the branched ED pathway of S. solfataricus is givenin Fig. 5. For a recent article on the branched ED pathway inThermoproteus tenax, see reference 32.

In both the nonphosphorylative ED pathway and branched

FIG. 5. Proposed nonphosphorylative Entner-Doudoroff pathway in Picrophilus torridus in comparison to the proposed branched Entner-Doudoroff pathway in Sulfolobus solfataricus. Enzymes and the genes encoding these enzymes and the promiscuous activities of GDH, GAD,KDGA, KDGK, and KD(P)GA are indicated. Abbreviations: GDH, glucose dehydrogenase; GAD, gluconate dehydratase; KDG, 2-keto-3-deoxygluconate; KDGal, 2-keto-3-deoxygalactonate; KDGA, KDG-specific aldolase; GADH, glyceraldehyde dehydrogenase, GLK, glyceratekinase (2-phosphoglycerate forming); ENO, enolase; PYK, pyruvate kinase; KDGK, KDG kinase; KDPG, 2-keto-3-deoxy-6-phosphogluconate;KDPGal, 2-keto-3-deoxy-6-phosphogalactonate; KD(P)GA, bifunctional KDGKDPG aldolase; GAPN, nonphosphorylative glyceraldehyde dehy-drogenase; GAOR, glyceraldehyde oxidoreductase; PGM, phosphoglycerate mutase.

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ED pathway in Picrophilus and Sulfolobus, the formation ofKDG is catalyzed by homologous glucose dehydrogenases andgluconate dehydratases. However, the subsequent routes ofgluconate degradation to 2-phosphoglycerate differ as follows.In the nonphosphorylative ED pathway of Picrophilus, KDG iscleaved by the novel KDG-specific aldolase to pyruvate andglyceraldehyde, which in turn is oxidized to glycerate via NADP-specific glyceraldehyde dehydrogenase, a novel enzyme of thealdehyde dehydrogenase superfamily (19). Glycerate is thenphosphorylated by a specific 2-phosphoglycerate-forming ki-nase (17).

In the branched ED pathway, two routes of KDG conversionto 2-phosphoglycerate have been proposed (25). In the non-phosphorylative route, KDG is split to glyceraldehyde andpyruvate via bifunctional KDGA. The oxidation of glyceralde-hyde is catalyzed by an oxidoreductase (9), rather than by theNADP�-dependent glyceraldehyde dehydrogenase as in P. tor-ridus. Glycerate phosphorylation to 2-phosphoglycerate is cat-alyzed by a specific kinase homologous to the P. torridus en-zyme. The semiphosphorylated route involves phosphorylationof KDG—via KDG kinase—to KDPG, which is cleaved topyruvate and glyceraldehyde-3-phosphate (GAP) by bifunc-tional KDPGA. GAP is then oxidized by nonphosphorylativeGAPN, forming 3-phosphoglycerate, which is converted to2-phosphoglycerate by means of a phosphoglycerate mutase.Finally, the conversion of 2-phosphoglycerate to pyruvate inboth the nonphosphorylative and branched ED pathways iscatalyzed by conventional enolase and pyruvate kinase.

Pathway promiscuity. The nonphosphorylative ED pathway inP. torridus contained promiscuous glucose/galactose dehydroge-nase (2), gluconate/galactonate dehydratase, and KDG/KDGalaldolase (this work). These data suggest that the nonphosphory-lative ED pathway in P. torridus provides an equivalent route forthe degradation of both glucose and galactose, as first proposedfor the branched ED pathway (Fig. 5) in S. solfataricus (4).

Gene organization. The genes encoding all enzymes of thenonphosphorylative ED pathway in Picrophilus were found tobe scattered along the chromosome, whereas several genesencoding enzymes of the branched ED pathway in Sulfolobus(and also in Thermoproteus), i.e., GAD, KD(P)GA, KDG ki-nase, and GAPN, are clustered (see the ORF numbers in Fig.5). The latter finding is in accordance with the in vivo operationof the branched ED pathway in the crenarchaea. Furthermore,in the P. torridus genome, homologous genes encoding KDGkinase and GAPN were absent, which is in accordance with theproposed strict nonphosphorylative ED pathway in P. torridus.

ACKNOWLEDGMENTS

We thank U. Sauer for financial support of T. Fuhrer.We thank U. Sauer for use of GC-MS. Further, we thank R.

Schmid (Osnabruck, Germany) for N-terminal amino acid analyses,S. Anemuller (Lubeck, Germany) for providing cell mass of T. aci-dophilum and H. Preidl and A. Brandenburger for expert technicalassistance. KDPG was a gift from E. Toone (Durham, NC). Theanalysis of KDG and KDGal formation by KDGA from P. torridus wasperformed by D. Hough and M. Danson (Bath, United Kingdom).Finally, we thank U. Johnsen for measuring the kinetic constants ofGAD and for help preparing the manuscript.

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