Gene expression and characterization of 2-keto-3-deoxygluconate kinase, a key enzyme in the modified Entner-Doudoroff pathway of the aerobic and acidophilic hyperthermophile Sulfolobus tokodaii

2-Keto-3-deoxygluconate kinase (KDGK) catalyzes the ATP-dependent phosphorylation of 2-keto-3-deoxygluconate, a key intermediate in the modified (semi-phosphorylative) Entner-Doudoroff (ED) glucose metabolic pathway. We identified the gene (ORF ID: ST2478) encoding KDGK in the hyperthermophilic archaeon Sulfolobus tokodaii based on the structure of a gene cluster in a genomic database and functionally expressed it in Escherichia coli. The expressed protein was purified from crude extract by heat treatment and two conventional column chromatography steps, and the partial amino acid sequence in the N-terminal region of the purified enzyme (MAKLIT) was identical to that obtained from the gene sequence. The purified enzyme was extremely thermostable and retained full activity after heating at 80 degrees C for 1 h. The enzyme utilized ATP or GTP, but not ADP or AMP, as a phosphoryl donor and 2-keto-3-deoxy-D-gluconate or 2-keto-D-gluconate as a phosphoryl acceptor. Divalent cations including Mg(2+), Co(2+), Ni(2+), Zn(2+) or Mn(2+) were required for activity, and the apparent Km values for KDG and ATP at 50 degrees C were 0.027 mM and 0.057 mM, respectively. The presence of KDGK means that the hyperthermophilic archaeon S. tokodaii metabolizes glucose via both modified (semi-phosphorylative) and non-phosphorylative ED pathways.     

Crystal structure and stereochemical studies of KD(P)G aldolase from Thermoproteus tenax

Carbon-carbon bond forming enzymes offer great potential for organic biosynthesis. Hence there is an ongoing effort to improve their biocatalytic properties, regarding availability, activity, stability, and substrate specificity and selectivity. Aldolases belong to the class of C-C bond forming enzymes and play important roles in numerous cellular processes. In several hyperthermophilic Archaea the 2-keto-3-deoxy-(6-phospho)-gluconate (KD(P)G) aldolase was identified as a key player in the metabolic pathway. The carbohydrate metabolism of the hyperthermophilic Crenarchaeote Thermoproteus tenax, for example, has been found to employ a combination of a variant of the Embden-Meyerhof-Parnas pathway and an unusual branched Entner-Doudoroff pathway that harbors a nonphosphorylative and a semiphosphorylative branch. The KD(P)G aldolase catalyzes the reversible cleavage of 2-keto-3-deoxy-6-phosphogluconate (KDPG) and 2-keto-3-deoxygluconate (KDG) forming pyruvate and glyceraldehyde 3-phosphate or glyceraldehyde, respectively. In T. tenax initial studies revealed that the pathway is specific for glucose, whereas in the thermoacidophilic Crenarchaeote Sulfolobus solfataricus the pathway was shown to be promiscuous for glucose and galactose degradation. The KD(P)G aldolase of S. solfataricus lacks stereo control and displays additional activity with 2-keto-3-deoxy-6-phosphogalactonate (KDPGal) and 2-keto-3-deoxygalactonate (KDGal), similar to the KD(P)G aldolase of Sulfolobus acidocaldarius. To address the stereo control of the T. tenax enzyme the formation of the two C4 epimers KDG and KDGal was analyzed via gas chromatography combined with mass spectroscopy. Furthermore, the crystal structure of the apoprotein was determined to a resolution of 2.0 A, and the crystal structure of the protein covalently linked to a pathway intermediate, namely pyruvate, was determined to 2.2 A. Interestingly, although the pathway seems to be specific for glucose in T. tenax the enzyme apparently also lacks stereo control, suggesting that the enzyme is a trade-off between required catabolic flexibility needed for the conversion of phosphorylated and nonphosphorylated substrates and required stereo control of cellular/physiological enzymatic reactions.     

Metabolic pathway promiscuity in the archaeon Sulfolobus solfataricus revealed by studies on glucose dehydrogenase and 2-keto-3-deoxygluconate aldolase

The hyperthermophilic Archaeon Sulfolobus solfataricus metabolizes glucose by a non-phosphorylative variant of the Entner-Doudoroff pathway. In this pathway glucose dehydrogenase and gluconate dehydratase catalyze the oxidation of glucose to gluconate and the subsequent dehydration of gluconate to 2-keto-3-deoxygluconate. 2-Keto-3-deoxygluconate (KDG) aldolase then catalyzes the cleavage of 2-keto-3-deoxygluconate to glyceraldehyde and pyruvate. The gene encoding glucose dehydrogenase has been cloned and expressed in Escherichia coli to give a fully active enzyme, with properties indistinguishable from the enzyme purified from S. solfataricus cells. Kinetic analysis revealed the enzyme to have a high catalytic efficiency for both glucose and galactose. KDG aldolase from S. solfataricus has previously been cloned and expressed in E. coli. In the current work its stereoselectivity was investigated by aldol condensation reactions between D-glyceraldehyde and pyruvate; this revealed the enzyme to have an unexpected lack of facial selectivity, yielding approximately equal quantities of 2-keto-3-deoxygluconate and 2-keto-3-deoxygalactonate. The KDG aldolase-catalyzed cleavage reaction was also investigated, and a comparable catalytic efficiency was observed with both compounds. Our evidence suggests that the same enzymes are responsible for the catabolism of both glucose and galactose in this Archaeon. The physiological and evolutionary implications of this observation are discussed in terms of catalytic and metabolic promiscuity.     

Promiscuity in the part-phosphorylative Entner-Doudoroff pathway of the archaeon Sulfolobus solfataricus

The hyperthermophilic archaeon Sulfolobus solfataricus metabolises glucose and galactose by a 'promiscuous' non-phosphorylative variant of the Entner-Doudoroff pathway, in which a series of enzymes have sufficient substrate promiscuity to permit the metabolism of both sugars. Recently, it has been proposed that the part-phosphorylative Entner-Doudoroff pathway occurs in parallel in S. solfataricus as an alternative route for glucose metabolism. In this report we demonstrate, by in vitro kinetic studies of D-2-keto-3-deoxygluconate (KDG) kinase and KDG aldolase, that the part-phosphorylative pathway in S. solfataricus is also promiscuous for the metabolism of both glucose and galactose.     

Structure of Thermus thermophilus 2-Keto-3-deoxygluconate kinase: evidence for recognition of an open chain substrate

2-Keto-3-deoxygluconate kinase (KDGK) catalyzes the phosphorylation of 2-keto-3-deoxygluconate (KDG) to 2-keto-3-deoxy-6-phosphogluconate (KDGP). The genome sequence of Thermus thermophilus HB8 contains an open reading frame that has a 30% identity to Escherichia coli KDGK. The KDGK activity of T.thermophilus protein (TtKDGK) has been confirmed, and its crystal structure has been determined by the molecular replacement method and refined with two crystal forms to 2.3 angstroms and 3.2 angstroms, respectively. The enzyme is a hexamer organized as a trimer of dimers. Each subunit is composed of two domains, a larger alpha/beta domain and a smaller beta-sheet domain, similar to that of ribokinase and adenosine kinase, members of the PfkB family of carbohydrate kinases. Furthermore, the TtKDGK structure with its KDG and ATP analogue was determined and refined at 2.1 angstroms. The bound KDG was observed predominantly as an open chain structure. The positioning of ligands and the conservation of important catalytic residues suggest that the reaction mechanism is likely to be similar to that of other members of the PfkB family, including ribokinase. In particular, the Asp251 is postulated to have a role in transferring the gamma-phosphate of ATP to the 5'-hydroxyl group of KDG.     

The structural basis for substrate promiscuity in 2-keto-3-deoxygluconate aldolase from the Entner-Doudoroff pathway in Sulfolobus solfataricus

The hyperthermophilic Archaea Sulfolobus solfataricus grows optimally above 80 degrees C and metabolizes glucose by a non-phosphorylative variant of the Entner-Doudoroff pathway. In this pathway glucose dehydrogenase and gluconate dehydratase catalyze the oxidation of glucose to gluconate and the subsequent dehydration of gluconate to D-2-keto-3-deoxygluconate (KDG). KDG aldolase (KDGA) then catalyzes the cleavage of KDG to D-glyceraldehyde and pyruvate. It has recently been shown that all the enzymes of this pathway exhibit a catalytic promiscuity that also enables them to be used for the metabolism of galactose. This phenomenon, known as metabolic pathway promiscuity, depends crucially on the ability of KDGA to cleave KDG and D-2-keto-3-deoxygalactonate (KDGal), in both cases producing pyruvate and D-glyceraldehyde. In turn, the aldolase exhibits a remarkable lack of stereoselectivity in the condensation reaction of pyruvate and D-glyceraldehyde, forming a mixture of KDG and KDGal. We now report the structure of KDGA, determined by multiwavelength anomalous diffraction phasing, and confirm that it is a member of the tetrameric N-acetylneuraminate lyase superfamily of Schiff base-forming aldolases. Furthermore, by soaking crystals of the aldolase at more than 80 degrees C below its temperature activity optimum, we have been able to trap Schiff base complexes of the natural substrates pyruvate, KDG, KDGal, and pyruvate plus D-glyceraldehyde, which have allowed rationalization of the structural basis of promiscuous substrate recognition and catalysis. It is proposed that the active site of the enzyme is rigid to keep its thermostability but incorporates extra functionality to be promiscuous.     

The Nonphosphorylative Entner-Doudoroff Pathway in the Thermoacidophilic Euryarchaeon Picrophilus torridus Involves a Novel 2-Keto-3-Deoxygluconate- Specific Aldolase

The pathway of glucose degradation in the thermoacidophilic euryarchaeon Picrophilus torridus has been studied 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 the proteinogenic amino acid alanine, indicating the exclusive operation of an Entner-Doudoroff (ED)-type pathway 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-kDa subunits, encoded by Pto0485. KDG aldolase, KDGA, is a homotetramer of 32-kDa subunits. This enzyme was highly specific for KDG with up to 2,000-fold-higher catalytic efficiency compared to 2-keto-3-deoxy-6-phosphogluconate (KDPG) and thus differs from the bifunctional KDG/KDPG aldolase, KD(P)GA of crenarchaea catalyzing 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 spectrometry (MS) as Pto1279, and the correct translation start codon, an ATG 24 bp upstream of the annotated start codon of Pto1279, was determined by N-terminal amino acid analysis. The kdgA gene was functionally overexpressed in Escherichia coli. Phylogenetic analysis revealed that KDGA is only distantly related to KD(P)GA, both enzymes forming separate families within the dihydrodipicolinate synthase superfamily. From the data we conclude that P. torridus degrades glucose via a strictly nonphosphorylative ED pathway with a novel KDG-specific aldolase, thus excluding the operation of the branched ED pathway involving a bifunctional KD(P)GA as a key enzyme.Comparative analyses of sugar-degrading pathways in members of the domain Archaea revealed that all species analyzed so far degrade glucose and glucose polymers to pyruvate via modification of the classical Embden-Meyerhof (EM) and Entner-Doudoroff (ED) pathways found in bacteria and eukarya. Modified EM pathways were reported for hyperthermophilic archaea, including, e.g., the strictly fermentative Thermococcales and Desulfurococcales, the sulfur-reducing Thermoproteus tenax, and the microaerophilic Pyrobaculum aerophilum. These pathways differ from the classical EM pathway by the presence of several novel enzymes and enzyme families, catalyzing, e.g., the phosphorylation of glucose 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 aerobic archaea, including halophiles, and thermoacidophilic crenarchaea, such as Sulfolobus species, and the euryarchaea Thermoplasma acidophilum and Picrophilus torridus. The anaerobic Thermoproteus tenax, which degrades glucose predominantly via a modified EM pathway, also utilizes—to a minor extent (<20%)—a modified ED pathway for glucose degradation. The following ED pathway modifications have been reported in archaea (25). A semiphosphorylative ED pathway was reported in halophilic archaea. Accordingly, glucose is converted to 2-keto-3-deoxy-6-gluconate (KDG) via glucose dehydrogenase and gluconate dehydratase. KDG is then phosphorylated by KDG kinase to 2-keto-3-deoxy-6-phosphogluconate (KDPG), which is split by KDPG aldolase to pyruvate and glyceraldehyde-3-phosphate (GAP). GAP is further converted to form another pyruvate via common reactions of the EM pathway, i.e., phosphorylative GAP dehydrogenase, phosphoglycerate kinase, phosphoglycerate mutase, enolase, and pyruvate kinase. The net ATP yield of this pathway is 1 ATP/mol glucose.From initial enzyme studies of the thermoacidophilic archaea Sulfolobus solfataricus, Thermoplasma acidophilum, and Thermoproteus tenax, a nonphosphorylative ED pathway was proposed (25). In this modification of the ED pathway, glucose is converted to KDG via glucose dehydrogenase and gluconate dehydratase, as in the semiphosphorylative pathway, but then the steps differ as follows: KDG is cleaved into pyruvate and glyceraldehyde via 2-keto-3-deoxygluconate-specific aldolase (KDGA). The subsequent oxidation of glyceraldehyde to glycerate involves either NAD(P)⁺-dependent dehydrogenases or oxidoreductases. Glycerate is then phosphorylated by a specific kinase to 2-phosphoglycerate, which is finally converted to pyruvate via enolase and pyruvate kinase. This modification of the ED pathway was called “nonphosphorylative” since it is not coupled with net ATP synthesis.However, recent comparative genomic studies and refined enzyme analyses suggest that the crenarchaea Sulfolobus and Thermoproteus utilize a so-called branched ED pathway, in which a semiphosphorylated route is simultaneously operative in addition to the nonphosphorylative route (25, 32). Accordingly, the semiphosphorylated route involves—via KDG kinase—the phosphorylation of KDG to KDPG, which is then cleaved to pyruvate and GAP by means of a bifunctional KDG/KDPG aldolase, KD(P)GA. GAP is then converted to another pyruvate via nonphosphorylative GAP dehydrogenase (GAPN), phosphoglycerate mutase, enolase, and pyruvate kinase. The net ATP yield of the branched ED pathway is zero. In support of this pathway, the genes encoding gluconate dehydratase, bifunctional KD(P)GA, KDG kinase, and GAPN were found to be clustered in Sulfolobus solfataricus (see Discussion) and Thermoproteus tenax. The key enzyme of the proposed branched ED pathway is the bifunctional KD(P)GA, which catalyzes the cleavage of KDG to pyruvate and glyceraldehyde and cleavage of KDPG to pyruvate and glyceraldehyde-3-phosphate. This bifunctional aldolase, which has been characterized from S. solfataricus, was found to be identical to a previously described KDG aldolase of the same organism; however, its catalytic property to also utilize KDPG as a substrate has been recognized only recently. In fact, the bifunctional KD(P)GA showed a higher catalytic efficiency for KDPG than for KDG (1, 14). Crystal structures of bifunctional KD(P)GAs of 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 reported to be promiscuous and therefore represents an equivalent degradation route for both glucose and its C-4 epimer, galactose. Accordingly, glucose dehydrogenase, gluconate dehydratase, KDG kinase, and bifunctional KD(P)GA were found to catalyze the conversion of both glucose and galactose and the corresponding subsequent intermediates, i.e., gluconate/galactonate, KDG/KDGal (KDGal stands for 2-keto-3-deoxygalactonate), and KDPG/KDPGal (KDPGal stands for 2-keto-3-deoxy-6-phosphogalactonate) (4, 12-14).In contrast to crenarchaea, the modified ED pathway in the thermoacidophilic euryarchaea Thermoplasma acidophilum and Picrophilus torridus has not been studied in detail. Enzyme measurements in cell extracts and the characterization of few enzymes suggest the operation of a nonphosphorylative ED pathway in these organisms (2, 3, 17, 19, 25). However, in vivo evidence for the operation of an ED-type pathway, e.g., by ¹³C-labeling experiments with growing cultures, has not been provided yet. Furthermore, the KDG aldolase activity measured in cell extracts of P. torridus and T. acidophilum has not been purified and characterized, in particular with respect to substrate specificity, and the genes encoding these enzymes have not been identified. The biochemical analysis of this aldolase is crucial to define the enzyme as a KDG-specific aldolase, indicative of a nonphosphorylative ED pathway, or as bifunctional KD(P)GA, indicative of the branched ED pathway as proposed for the crenarchaea Sulfolobus and Thermoproteus.In this communication we studied the sugar-degrading pathway in P. torridus by in vivo labeling experiments with [¹³C]glucose, by enzyme measurements, and by characterization of two key enzymes, gluconate dehydratase and KDG aldolase. The data indicate that P. torridus utilizes a strict nonphosphorylative ED pathway, involving a novel KDG-specific aldolase as a key enzyme, and thus exclude the operation of a branched ED pathway, as in crenarchaea involving a bifunctional KD(P)GA as a key enzyme.     

Soluble expression of archaeal proteins in Escherichia coli by using fusion-partners

Expression of archaeal proteins in soluble form is of importance because archaeal proteins are usually produced as insoluble inclusion bodies in Escherichia coli. In this study, we investigated the use of soluble fusion tags to enhance the solubility of two archaeal proteins, d-gluconate dehydratase (GNAD) and 2-keto-3-deoxy-D-gluconate kinase (KDGK), key enzymes in the glycolytic pathway of the thermoacidophilic archaeon Sulfolobus solfataricus. These two proteins were produced as inclusion bodies in E. coli when polyhistidine was used as a fusion tag. To reduce inclusion body formation in E. coli, GNAD and KDGK were fused with three partners, thioredoxin (Trx), glutathione-S-transferase (GST), and N-utilization substance A (NusA). With the use of fusion-partners, the solubility of the archaeal proteins was remarkably enhanced, and the soluble fraction of the recombinant proteins was increased in this order: Trx>GST>NusA. Furthermore, In the case of recombinant KDGKs, the enzyme activity of the Trx-fused proteins was 200-fold higher than that of the polyhistidine-fusion protein. The strategy presented in this work may contribute to the production of other valuable proteins from hyperthermophilic archaea in E. coli.     

Identification and characterization ofThermoplasma acidophilum 2-keto-3-deoxy-D-gluconate kinase: A new class of sugar kinases

The thermoacidophilic archaeonThermoplasma acidophilum has long been known to utilized-glucosevia the non-phosphorylated Entner-Doudoroff (nED) pathway. We now report the identification of a gene encoding 2-keto-3-deoxy-d-gluconate (KDG) kinase. The discovery of this gene implies the presence of a glycolysis pathway, other than the nED pathway. It was found that Ta0122 in theT. acidophilum genome corresponded to KDG kinase. This enzyme shares no similarity with known KDG kinases, and belongs to a novel class of sugar kinases. Of the five sugars tested only KDG was utilized as a substrate.     

Eukaryotic and bacterial gene clusters related to an alternative pathway of nonphosphorylated L-rhamnose metabolism

The Entner-Doudoroff (ED) pathway is a classic central pathway of d-glucose metabolism in all three phylogenetic domains. On the other hand, Archaea and/or bacteria possess several modified versions of the ED pathway, in which nonphosphorylated intermediates are involved. Several fungi, including Pichia stipitis and Debaryomyces hansenii, possess an alternative pathway of L-rhamnose metabolism, which is different from the known bacterial pathway. Gene cluster related to this hypothetical pathway was identified by bioinformatic analysis using the metabolic enzymes involved in analogous sugar pathways to the ED pathway. Furthermore, the homologous gene cluster was found not only in many other fungi but also several bacteria, including Azotobacter vinelandii. Four putative metabolic genes, LRA1-4, were cloned, overexpressed in Escherichia coli, and purified. Substrate specificity and kinetic analysis revealed that nonphosphorylated intermediates related to L-rhamnose are significant active substrates for the purified LRA1-4 proteins. Furthermore, L-2-keto-3-deoxyrhamnonate was structurally identified as both reaction products of dehydration by LRA3 and aldol condensation by LRA4. These results suggested that the LRA1-4 genes encode L-rhamnose 1-dehydrogenase, L-rhamnono-gamma-lactonase, L-rhamnonate dehydratase, and L-KDR aldolase, respectively, by which L-rhamnose is converted into pyruvate and L-lactaldehyde through analogous reaction steps to the ED pathway. There was no evolutionary relationship between L-KDR aldolases from fungi and bacteria.     

New Pathway for Nonphosphorylated Degradation of Gluconate by Aspergillus niger

A new nonphosphorylative pathway for gluconate degradation was found in extracts of a strain of Aspergillus niger. The findings indicate that gluconate is dehydrated into 2-keto-3-deoxy-gluconate (KDG), which then is cleaved into glyceraldehyde and pyruvate. 6-Phosphogluconate was not degraded under the same conditions. In addition, KDG was formed from glyceraldehyde and pyruvate. Very weak activity was obtained when glyceraldehyde 3-phosphate replaced glyceraldehyde in this reaction.     

Different glycolytic pathways for glucose and fructose in the halophilic archaeon Halococcus saccharolyticus

The glucose and fructose degradation pathways were analyzed in the halophilic archaeon Halococcus saccharolyticus by ¹³C-NMR labeling studies in growing cultures, comparative enzyme measurements and cell suspension experiments. H. saccharolyticus grown on complex media containing glucose or fructose specifically ¹³C-labeled at C1 and C3, formed acetate and small amounts of lactate. The ¹³C-labeling patterns, analyzed by ¹H- and ¹³C-NMR, indicated that glucose was degraded via an Entner-Doudoroff (ED) type pathway (100%), whereas fructose was degraded almost completely via an Embden-Meyerhof (EM) type pathway (96%) and only to a small extent (4%) via an ED pathway. Glucose-grown and fructose-grown cells contained all the enzyme activities of the modified versions of the ED and EM pathways recently proposed for halophilic archaea. Glucose-grown cells showed increased activities of the ED enzymes gluconate dehydratase and 2-keto-3-deoxy-gluconate kinase, whereas fructose-grown cells contained higher activities of the key enzymes of a modified EM pathway, ketohexokinase and fructose-1-phosphate kinase. During growth of H. saccharolyticus on media containing both glucose and fructose, diauxic growth kinetics were observed. After complete consumption of glucose, fructose was degraded after a lag phase, in which fructose-1-phosphate kinase activity increased. Suspensions of glucose-grown cells consumed initially only glucose rather than fructose, those of fructose-grown cells degraded fructose rather than glucose. Upon longer incubation times, glucose- and fructose-grown cells also metabolized the alternate hexoses. The data indicate that, in the archaeon H. saccharolyticus, the isomeric hexoses glucose and fructose are degraded via inducible, functionally separated glycolytic pathways: glucose via a modified ED pathway, and fructose via a modified EM pathway.Abbreviations. KDG 2-Keto-3-deoxygluconate - KDPG 2-Keto-3-deoxy-6-phosphogluconate - FBP Fructose-1,6-bisphosphate - TIM Triosephosphate isomerase - GAP Glyceraldehyde-3-phosphate - PEP Phosphoenolpyruvate - PTS Phosphotransferase - 1-PFK Fructose 1-phosphate kinase An erratum to this article can be found at     

Identification and characterization of Thermoplasma acidophilum glyceraldehyde dehydrogenase: a new class of NADP+-specific aldehyde dehydrogenase

Thermoacidophilic archaea such as Thermoplasma acidophilum and Sulfolobus solfataricus are known to metabolize D-glucose via the nED (non-phosphorylated Entner-Doudoroff) pathway. In the present study, we identified and characterized a glyceraldehyde dehydrogenase involved in the downstream portion of the nED pathway. This glyceraldehyde dehydrogenase was purified from T. acidophilum cell extracts by sequential chromatography on DEAE-Sepharose, Q-Sepharose, Phenyl-Sepharose and Affi-Gel Blue columns. SDS/PAGE of the purified enzyme showed a molecular mass of approx. 53 kDa, whereas the molecular mass of the native protein was 215 kDa, indicating that glyceraldehyde dehydrogenase is a tetrameric protein. By MALDI-TOF-MS (matrix-assisted laser-desorption ionization-time-of-flight MS) peptide fingerprinting of the purified protein, it was found that the gene product of Ta0809 in the T. acidophilum genome database corresponds to the purified glyceraldehyde dehydrogenase. The native enzyme showed the highest activity towards glyceraldehyde, but no activity towards aliphatic or aromatic aldehydes, and no activity when NAD+ was substituted for NADP+. Analysis of the amino acid sequence and enzyme inhibition studies indicated that this glyceraldehyde dehydrogenase belongs to the ALDH (aldehyde dehydrogenase) superfamily. BLAST searches showed that homologues of the Ta0809 protein are not present in the Sulfolobus genome. Possible differences between T. acidophilum (Euryarchaeota) and S. solfataricus (Crenarchaeaota) in terms of the glycolytic pathway are thus expected.     

Genetic Control of the 2-Keto-3-Deoxy-d-Gluconate Metabolism in Escherichia coli K-12: kdg Regulon

2-Keto-3-deoxy-gluconate (KDG), an intermediate of the hexuronate pathway in Escherichia coli K-12, is utilized as the sole carbon source only in strains derepressed for the specific KDG-uptake system. KDG is metabolized to pyruvate and glyceraldehyde-3-phosphate via the inducible enzymes KDG-kinase and 2-keto-3-deoxy-6-phosphate-gluconate (KDPG) aldolase. However, another inducible pathway, where the KDG is the branch point, has been demonstrated. Genetic studies of the KDG degradative pathway reported in this paper led to the location of KDG kinase-negative and pleiotropic constitutive mutations. The kdgK locus, presumably the structural gene of the kinase, occurs at min 69 and is co-transducible with xyl. The mutants, simultaneously constitutive for the uptake, kinase, and aldolase, define a kdgR locus at min 36 between the co-transducible markers kdgA and oldD. As to the nature of the control exerted by the kdgR product, we have shown the following. (i) Thermosensitive mutants of the kdgR locus are inducible at low temperature but derepressed at 42 C for the three operons—kdgT (transport system), kdgK, and kdgA (KDPG aldolase). (ii) The kdgR⁺ allele is dominant to the kdgR constitutive allele. (iii) A deletion in kdgA extending into the regulatory gene, kdgR, leads to a constitutive expression of the nondeleted operons—kdgT and kdgK. These properties demonstrate that the kdg regulon is negatively controlled by the kdgR product. It is presumed that differences in operator and in promotor structures could explain the strong decoordination, respectively, in the induction and catabolic repression, of these three enzymes activities.     

Gluconate dehydratase from the promiscuous Entner-Doudoroff pathway in Sulfolobus solfataricus

An investigation has been carried out into gluconate dehydratase from the hyperthermophilic Archaeon Sulfolobus solfataricus. The enzyme has been purified from cell extracts of the organism and found to be responsible for both gluconate and galactonate dehydratase activities. It was shown to be a 45 kDa monomer with a half-life of 41 min at 95 degrees C and it exhibited similar catalytic efficiency with both substrates. Taken alongside the recent work on glucose dehydrogenase and 2-keto-3-deoxygluconate aldolase, this report clearly demonstrates that the entire non-phosphorylative Entner-Doudoroff pathway of S. solfataricus is promiscuous for the metabolism of both glucose and galactose.     

Improvement of L-lysine production by Methylophilus methylotrophus from methanol via the Entner-Doudoroff pathway, originating in Escherichia coli

To improve the amino acid production by metabolic engineering, eliminating the pathway bottleneck is known to be very effective. The metabolic response of Methylophilus methylotrophus upon the addition of glucose and of pyruvate was investigated in batch cultivation. We found that the supply of pyruvate is a bottleneck in L-lysine production in M. methylotrophus from methanol as carbon source. M. methylotrophus has a ribulose monophosphate (RuMP) pathway for methanol assimilation, and consequently synthesized fructose-6-phosphate is metabolized to pyruvate via the Entner-Doudoroff (ED) pathway, and the ED pathway is thought to be the main pathway for pyruvate supply. An L-lysine producer of M. methylotrophus with an enhanced ED pathway was constructed by the introduction of the E. coli edd-eda operon encoding the enzyme involving the ED pathway. In this strain, the overall enzymatic activity of ED pathway, which is estimated by measuring the activities of 6-phosphogluconate dehydrogenase plus 2-keto-3-deoxy-6-phosphogluconate aldolase, was about 20 times higher than in the parent. This strain produced 1.2 times more L-lysine than the parent producer. Perhaps, then, the supply of pyruvate was a bottleneck in L-lysine production in the L-lysine producer of M. methylotrophus.