D-Xylulose-1-phosphate: enzymatic assay and production in isolated rat hepatocytes

A specific enzymatic assay for D-xylulose-1-phosphate (D-threopentulose-1-phosphate) was developed, based on the measurement of glycolaldehyde, which is formed by aldolase cleavage of the D-xylulose-1-phosphate. This assay was used to confirm the identity of the product of fructokinase phosphorylation of D-xylulose and the production of glycolaldehyde and D-xylulose-1-phosphate in D-xylulose-treated isolated rat hepatocytes. An alternative pathway of xylitol metabolism in the liver, through D-xylulose-1-phosphate to glycolaldehyde, is proposed. Because glycolaldehyde is a known oxalate precursor, this pathway may explain the synthesis of oxalate from xylitol.     

The expression of ketohexokinase is diminished in human clear cell type of renal cell carcinoma

For identification and targeting of tumor-associated marker proteins, the proteome of clear cell type of renal cell carcinoma (RCC) and normal kidney tissues was analyzed by 2-DE. Ketohexokinase (also called fructokinase), which catalyzes the phosphorylation of fructose to fructose 1-phosphate, was identified by MALDI-TOF MS and found to be expressed at low rates in the renal tumor tissues. We found a decreased amount of ketohexokinase mRNA in RCC compared to that observed in the normal kidney tissues by Northern blot. The activity of ketohexokinase in 20 clear cell RCC specimens and the 20 corresponding normal kidneys was investigated, and its activity was shown to be approximately 1.4-fold lower in the RCC specimens than in the normal kidney. Ketohexokinase activity in tumor stage pT3 RCC was 1.5-fold lower than in pT1 RCC. The level of ketohexokinase activity in histological grade 3 RCC was 1.8-fold lower than that in grade 1 cancer. In addition, using in situ hybridization, it was revealed that ketohexokinase in the normal kidney tissue was confined to the proximal tubular epithelial cells, while the expression of ketohexokinase in RCC tissues was extremely low. Our research results show that the expression of human ketohexokinase was diminished in clear cell RCC.     

Novel kinetic and structural properties of the class-I D-fructose 1,6-bisphosphate aldolase from Escherichia coli (Crookes'' strain).

Investigation of aldolase 1, the class-I D-fructose 1,6-bisphosphate aldolase (EC4.1.2.13) from Escherichia coli (Crookes' strain), showed it to have unusual kinetic and structural properties. The enzyme appeared to be larger than was previously supposed and may be a decamer with a mol. wt. of approx. 340000. Its fructose 1,6-bisphosphate-cleavage activity was unaffected by these compounds. The enhancement exhibited a strong dependence on pH. These novel kinetic properties do not seem to be shared by any other fructose 1,6-bisphosphate aldolase, but recall the activation by polycarboxylic acids of the deoxyribose 3-phosphate aldolases from some other organisms. In view of its unusual properties, it is unlikely that aldolase 1 from E. coli is closely related to the class-1 aldolases that have been detected in several other prokaryotes, or to the typical class-1 enzymes from eukaryotes.     

Levels of enzymes of the pentose phosphate pathway in Pachysolen tannophilus Y-2460 and selected mutants

The compositions of intracellular pentose phosphate pathway enzymes have been examined in mutants of Pachysolen tannophilus NRRL Y-2460 which possessed enhanced D-xylose fermentation rates. The levels of oxidoreductive enzymes involved in converting D-xylose to D-xylulose via xylitol were 1.5–14.7-fold higher in mutants than in the parent. These enzymes were still under inductive control by D-xylose in the mutants. The D-xylose reductase activity (EC 1.1.1.21) which catalyses the conversion of D-xylose to xylitol was supported with either NADPH or NADH as coenzyme in all the mutant strains. Other enzyme specific activities that generally increased were: xylitol dehydrogenase (EC 1.1.1.9), 1.2–1.6-fold; glucose-6-phosphate dehydrogenase (EC 1.1.1.49), 1.9–2.6-fold; D-xylulose-5-phosphate phosphoketolase (EC 4.1.2.9), 1.2–2.6-fold; and alcohol dehydrogenase (EC 1.1.1.1), 1.5–2.7-fold. The increase of enzymatic activities, 5.3–10.3-fold, occurring in D-xylulokinase (EC 2.7.1.17), suggested a pivotal role for this enzyme in utilization of D-xylose by these mutants. The best ethanol-producing mutant showed the highest ratio of NADH- to NADPH-linked D-xylose reductase activity and high levels of all other pentose phosphate pathway enzymes assayed.     

Ketohexokinase (ATP:D-fructose 1-phosphotransferase) from a halophilic archaebacterium, Haloarcula vallismortis: purification and properties.

Ketohexokinase (ATP:D-fructose 1-phosphotransferase [EC 2.7.1.3]), detected for the first time in a prokaryote, i.e., the extreme halophile Haloarcula vallismortis, was isolated and characterized from the same archaebacterium. This enzyme was characterized with respect to its molecular mass, amino acid composition, salt dependency, immunological cross-reactivity, and kinetic properties. Gel filtration and sucrose density gradient centrifugation revealed a native molecular mass of 100 kDa for halobacterial ketohexokinase, which is larger than its mammalian counterpart. The enzyme could be labeled by UV irradiation in the presence of [ gamma-32P]ATP, suggesting the involvement of a phosphoenzyme intermediate. Other catalytic features of the enzyme were similar to those of its mammalian counterparts. No antigenic cross-reactivity could be detected between the H. vallismortis ketohexokinase and the ketohexokinases from different rat tissues.     

Interaction of D-fructose and fructose 1-phosphate with yeast phosphofructokinase and its influence on glycolytic oscillations.

Fermentation of D-fructose- and D-glucose induced glycolytic oscillations of different period lengths in Saccharomyces carlsbergensis. Recent studies suggested, that D-fructose or one of its metabolites interacted with phosphofructokinase (ATP:D-fructo-6-phosphate 1-phosphofructokinase, EC 2.7.1.11), the core of the glycolytic 'oscillator'. In order to explore the kinetics of interaction, the influence of D-fructose and fructose 1-phosphate on purified yeast phosphofructokinase was studied. D-fructose concentrations up to 0.3 mM stimulated the enzyme, while a further increase led to competitive inhibition. The Hill coefficient for fructose 6-phosphate decreased from 2.8 to 1.0. Fructose 1-phosphate acted in a similar way, up to 1 mM activation and inhibition competitive to fructose 6-phosphate at higher concentration (2.0--3.5 mM) with the same effect on the Hill coefficient. The inhibition patterns obtained with D-fructose or fructose 1-phosphate suggest a sequential random reaction mechanism of yeast phosphofructokinase with fructose 6-phosphate and MgATP2-. The mode of interaction of phosphofructokinase with D-fructose and fructose 1-phosphate is discussed. The influence of both effectors resulted in altered enzyme kinetics, which may cause the different period lengths of glycolytic oscillations.     

Characterization of the D-xylulose 5-phosphate/D-fructose 6-phosphate phosphoketolase gene (xfp) from Bifidobacterium lactis

A D-xylulose 5-phosphate/D-fructose 6-phosphate phosphoketolase (Xfp) from the probiotic Bifidobacterium lactis was purified to homogeneity. The specific activity of the purified enzyme with D-fructose 6-phosphate as a substrate is 4.28 Units per mg of enzyme. K(m) values for D-xylulose 5-phosphate and D-fructose 6-phosphate are 45 and 10 mM, respectively. The native enzyme has a molecular mass of 550,000 Da. The subunit size upon sodium dodecyl sulfate-polyacrylamide gel electrophoresis (90,000 Da) corresponds with the size (92,529 Da) calculated from the amino acid sequence of the isolated gene (named xfp) encoding 825 amino acids. The xfp gene was identified on the chromosome of B. lactis with the help of degenerated nucleotide probes deduced from the common N-terminal amino acid sequence of both the native and denatured enzyme. Comparison of the deduced amino acid sequence of the cloned gene with sequences in public databases revealed high homologies with hypothetical proteins (26 to 55% identity) in 20 microbial genomes. The amino acid sequence derived from the xfp gene contains typical thiamine diphosphate (ThDP) binding sites reported for other ThDP-dependent enzymes. Two truncated putative genes, pta and guaA, were localized adjacent to xfp on the B. lactis chromosome coding for a phosphotransacetylase and a guanosine monophosphate synthetase homologous to products of genes in Mycobacterium tuberculosis. However, xfp is transcribed in B. lactis as a monocistronic operon. It is the first reported and sequenced gene of a phosphoketolase.     

Regulation of the synthesis of nucleoside diphosphate sugars in reticulo-endothelial tissues.

The kinetic and regulatory properties of enzymes involved in the biosynthesis of UDP-D-galactose, UDP-N-acetylglucosamine. GDP-alpha-D-mannose and GDP-beta-L-fucose from D-glucose 6-phosphate in various reticulo-endothelial tissues was studied. The tissues examined include bovine liver, thyroid, spleen, salivary gland, lung, intestine and mesenteric; pulmonary, portal and sub-maxillary lymphnodes. The maximum rates of specific enzymes in these pathways which were slow enough to be rate-limiting in the formation of glycoproteins in these tissues was determined. UDP-D-galactose 4-epimerase was consistently the rate-limiting reaction in the conversion of -d-glucose 6-phosphate to UDP-D-galactose in all of the tissues examined. The series of reactions leading to the formation of GDP-alpha-D-mannose and GDP-beta-L-fucose were limited by the activity of GDP-alpha-D-mannose pyrophosphorylase and GDP-alpha-D-mannose oxidoreductase, respectively. The formation of UDP-N-acetylglucosamine was limited by the rate of the amination reaction which converts -d-fructose 6-hosphate to D-glucosamine 6-phosphate in the presence of glutamine. Several of these rate-limiting enzymes were partially purified from mesenteric lymph node extracts, and their regulatory properties were examined. GDP-alpha-D-mannose was found to be a competitive inhibitor of GDP-alpha-D-mannose pyrophosphorylase. The apparent Km for GTP was 0.06 mM and the Ki for GDP-alpha-D-mannose was 0.03 mM. The concentrations of GTP and GDP-alpha-D-mannose in lymph node extracts were determined to be 0.095 and 0.012 mumol per g, respectively. UDP-N-acetylglucosamine and UDP-D-glucose inhibited D-fructose 6-phosphate amidotransferase in a manner competitive with D-fructose 6-phosphate. The Km for fructose 6-phosphate was 0.3 mM, while the Ki for UDP-D-glucose and UDP-N-acetyglucosamine were determined to be 0.4 mM and 0.045 mM, respectively. The concentrations of these metabolites in lymph node tissue were: UDP-D-glucose, 0.42; UDP-N-acetylglucosamine 0.095; and D-fructose 6-phosphate, 0.073 mumol per g wet weight of tissue. The results obtained in these studies show that specific rate-limiting enzymes in the pathways for the biosynthesis of nucleoside diphosphate sugars in reticulo-endothelial tissues may be subject to cumulative feedback inhibition by the nucleoside diphosphate sugars which are the final products of these systems and the initial precursors of the oligosaccharide units of glycoproteins in these tissues.     

The missing link in the fungal L-arabinose catabolic pathway,identification of the L-xylulose reductase gene

The fungal L-arabinose pathway consists of five enzymes, aldose reductase, L-arabinitol 4-dehydrogenase, L-xylulose reductase, xylitol dehydrogenase, and xylulokinase. All the genes encoding the enzymes of this pathway are known except for that of L-xylulose reductase (EC 1.1.1.10). We identified a gene encoding this enzyme from the filamentous fungus Trichoderma reesei (Hypocrea jecorina). The gene was named lxr1. It was overexpressed in the yeast Saccharomyces cerevisiae, and the enzyme activity was confirmed in a yeast cell extract. Overexpression of all enzymes of the L-arabinose pathway in S. cerevisiae led to growth of S. cerevisiae on L-arabinose; i.e., we could show that the pathway is active in a heterologous host. The lxr1 gene encoded a protein with 266 amino acids and a calculated molecular mass of 28 428 Da. The LXRI protein is an NADPH-specific reductase. It has activity with L-xylulose, D-xylulose, D-fructose, and L-sorbose. The highest affinity is toward L-xylulose (K(m) = 16 mM). In the reverse direction, we found activity with xylitol, D-arabinitol, D-mannitol, and D-sorbitol. It requires a bivalent cation for activity. It belongs to the protein family of short chain dehydrogenases. The enzyme is catalytically similar and homologous in sequence to a D-mannitol:NADP 2-dehydrogenase (EC 1.1.1.138).     

Enzymatic Regeneration of NAD in Enantioselective Oxidation of Secondary Alcohols: Glutamate Dehydrogenase Versus NADH Dehydrogenase

To improve yield and productivity of ketose in NAD-dependent polyol oxidations, two enzymatic methods for regeneration of the oxidized coenzyme form have been compared and partly optimized for the batch conversion of xylitol into D-xylulose and D-sorbitol into D-fructose. Polyol oxidation was catalyzed by xylitol dehydrogenase from the yeast Galactocandida mastotermitis. Reduction of OM₂ (apparently to H₂O) by partially purified NADH dehydrogenase complex from Corynebacterium callunae could drive alcohol oxidations better than reductive amination of EaL-ketoglutarate by glutamate dehydrogenase. A fed-batch procedure was developed that overcame inhibition of glutamate dehydrogenase by α-ketoglutarate (K_is 25 mM), thus increasing the productivity of ketose almost 2-fold. For D-fructose production from D-sorbitol (0.1-0.3M) yields of < 90% and productivities up to 1.30g/(L.h) have been obtained. High conversion of up to 50g/L xylitol into D-xylulose for which xylitol dehydrogenase exhibits an about 80-fold higher specificity constant than for D-fructose required complexation of the ketose product with borate. In comparison with reductive amination by glutamate dehydrogenase, advantages of using NADH-dehydrogenase catalyzed regeneration of NAD for ketose production are (i) avoidance of byproduct formation, (ii) cheaper substrate (02 versus α-ketoglutarate), and (iii) easier process control (batch versus fed-batch).     

Mitochondrial aldehyde dehydrogenase from higher plants

Aldehyde dehydrogenase has been purified to homogeneity from mitochondria of potato tubers and pea epicotyls. Although the enzyme had a high affinity for glycolaldehyde it also had a high affinity for a number of other aliphatic and arylaldehydes. It is proposed that the codification glycolaldehyde dehydrogenase (EC 1.2.1.22) should be abandoned in favour of mitochondrial aldehyde dehydrogenase (EC 1.2.1.3). The purified enzyme showed esterase activity and had properties similar to those reported for the mammalian mitochondrial aldehyde dehydrogenase. Although the natural substrate(s) for the enzyme is not known, the kinetic properties of the enzyme are consistent with it playing a role in the oxidation of acetaldehyde, glycolaldehyde and indoleacetaldehyde.     

Purification and characterization of the class-II D-fructose 1,6-bisphosphate aldolase from Escherichia coli (Crookes'' strain).

A new form of the class-II D-fructose 1,6-bisphosphate aldolase (EC 4.1.2.13) of Escherichia coli (Crookes' strain) was isolated from an extract of glycerol-grown bacteria. It has a higher molecular weight (approx. 80000)than previous preparations of the enzyme and closely resembles the typical class-II aldolase from yeast in size and amino acid composition. On the other hand, its kinetic behaviour is not typical of a class-II aldolase. The enzyme has no requirement for thiol compounds either for stability or activity, added K+ ions have no effect, and the optimum pH for the cleavage activity is unusually high. The class-II enzymes from the prokaryote E. coli and the eukaryote yeast show no immunological identity. However, the similarity of their structures suggests that they have evolved from a common ancestor.     

Expression, purification, and characterization of natural mutants of human aldolase B. Role of quaternary structure in catalysis

Fructaldolases (EC 4.1.2.13) are ancient enzymes of glycolysis that catalyze the reversible cleavage of phosphofructose esters into cognate triose (phosphates). Three vertebrate isozymes of Class I aldolase have arisen by gene duplication and display distinct activity profiles with fructose 1,6-bisphosphate and with fructose 1-phosphate. We describe the biochemical and biophysical characterization of seven natural human aldolase B variants, identified in patients suffering from hereditary fructose intolerance and expressed as recombinant proteins in E. coli, from which they were purified to homogeneity. The mutant aldolases were all missense variants and could be classified into two principal groups: catalytic mutants, with retained tetrameric structure but altered kinetic properties (W147R, R303W, and A337V), and structural mutants, in which the homotetramers readily dissociate into subunits with greatly impaired enzymatic activity (A149P, A174D, L256P, and N334K). Investigation of these two classes of mutant enzyme suggests that the integrity of the quaternary structure of aldolase B is critical for maintaining its full catalytic function.     

Modulation of the interaction between aldolase and glycerol-phosphate dehydrogenase by fructose phosphates

Kinetics of fructose-1,6-disphosphate aldolase (EC 4.1.2.13) catalyzed conversion of fructose phosphates was analyzed by coupling the aldolase reactions to the metabolically sequential enzyme, glycerol-3-phosphate dehydrogenase (EC 1.1.1.8), which interacts with aldolase. At low enzyme concentration poly(ethylene glycol) was added to promote complex formation of aldolase and glycerol-phosphate dehydrogenase resulting in a 3-fold increase in KM of fructose-1,6-bisphosphate and no change in Vmax. Kinetic parameters for fructose-1-phosphate conversion changed inversely upon complex formation: Vmax increased while KM remained unchanged. Gel penetration and ion-exchange chromatographic experiments showed positive modulation of the interaction of aldolase and dehydrogenase by fructose-1,6-bisphosphate. The dissociation constant of the heterologous enzyme complex decreased 10-fold in the presence of this substrate. Fructose-1-phosphate or dihydroxyacetone phosphate had no effect on the dissociation constant of the aldolase-dehydrogenase complex. In addition, titration of fluorescein-labelled glycerol-phosphate dehydrogenase with aldolase indicated that both fructose-1,6-bisphosphate and fructose-2,6-biphosphate enhanced the affinity of aldolase to glycerol-phosphate dehydrogenase. The results of the kinetic and binding experiments suggest that binding of the C-6 phosphate group of fructose-1,6-bisphosphate to aldolase complexed with dehydrogenase is sterically impeded while saturation of the C-6 phosphate group site increases the affinity of aldolase for dehydrogenase. The possible molecular mechanism of the fructose-1,6-bisphosphate modulated interaction is discussed.     

Production of xylitol by metabolically engineered strains of Bacillus subtilis

Xylitol-phosphate dehydrogenase (XPDH) genes from several Gram-positive bacteria were isolated and expressed in Bacillus subtilis. The substrate specificities of the recombinant XPDH enzymes were compared and it was found that the XPDH enzymes of Lactobacillus rhamnosus and Clostridium difficile had the highest selectivity towards D-xylulose 5-phosphate. Expression of these two XPDH enzymes in D-ribulose and D-xylulose producing B. subtilis strain resulted in strains of B. subtilis capable of converting D-glucose into xylitol at around 23% yield.     

Cooperative effect of fructose bisphosphate and glyceraldehyde-3-phosphate dehydrogenase on aldolase action

The combination of binding and kinetic approaches is suggested to study (i) the mechanism of substrate-modulated dynamic enzyme associations; (ii) the specificity of enzyme interactions. The effect of complex formation between aldolase and glyceraldehyde-3-phosphate dehydrogenase (D-glyceraldehyde-3-phosphate:NAD+ oxidoreductase (phosphorylating), EC 1.2.1.12) on aldolase catalysis was investigated under pseudo-first-order conditions. No change in kcat but a significant increase in KM of fructose 1,6-bisphosphate for aldolase was found when both enzymes were obtained from muscle. In contrast, kcat rather than KM changed if dehydrogenase was isolated from yeast. Next, the conversion of fructose 1-phosphate was not affected by interactions between enzyme couples isolated from muscle. The influence of fructose phosphates on the enzyme-complex formation was studied by means of covalently attached fluorescent probe. We found that the interaction ws not perturbed by the presence of fructose 1-phosphate; however, fructose 1,6-bisphosphate altered the dissociation constant of the enzyme complex. A molecular model for fructose 1,6-bisphosphate-modulated enzyme interaction has been evaluated which suggests that high levels of fructose bisphosphate would drive the formation of the 'channelling' complex between aldolase and glyceraldehyde-3-phosphate dehydrogenase.     

13C and deuterium isotope effects suggest an aldol cleavage mechanism for L-ribulose-5-phosphate 4-epimerase

On the basis of (13)C and deuterium isotope effects, L-ribulose-5-phosphate 4-epimerase catalyzes the epimerization of L-ribulose 5-phosphate to D-xylulose 5-phosphate by an aldol cleavage to the enediolate of dihydroxyacetone and glycolaldehyde phosphate, followed by rotation of the aldehyde group and condensation to the epimer at C-4. With the wild-type enzyme, (13)C isotope effects were 1.85% at C-3 and 1.5% at C-4 at pH 7, with the values increasing to 2.53 and 2.05% at pH 5.5, respectively. H97N and Y229F mutants at pH 7 gave values of 3.25 and 2.53% at C-3 and 2. 69 and 1.99% at C-4, respectively. Secondary deuterium isotope effects at C-3 were 2.5% at pH 7 and 3.1% at pH 5.5 with the wild-type enzyme, and 4.1% at pH 7 with H97N. At C-4, the corresponding values were 9.6, 14, and 19%. These data suggest that H97N shows no commitments, while the wild-type enzyme has an external commitment of approximately 1.4 at pH 7 and an internal commitment independent of pH of approximately 0.6. The Y229 mutant shows only the internal commitment of 0.6. The sequence of the epimerase is similar to those of L-fuculose-1-phosphate and L-rhamnulose-1-phosphate aldolases for residues in the active site of L-fuculose-1-phosphate aldolase, suggesting that Asp76, His95, His97, and His171 of the epimerase may be metal ion ligands, and Ser44, Gly45, Ser74, and Ser75 may form a phosphate binding pocket. The pH profile of V/K for L-ribulose 5-phosphate is bell-shaped with pK values of 5.94 and 8.24. The CD spectra of L-ribulose 5-phosphate and D-xylulose 5-phosphate differ sufficiently that the epimerization reaction can be followed at 300 nm.     

Kinetic evidence for interaction between aldolase and D-glyceraldehyde-3-phosphate dehydrogenase.

The possibility of interaction between purified rabbit muscle aldolase and D-glyceraldehyde-3-phosphate dehydrogenase was studied by rapid kinetic methods, by analyzing the kinetics of the consecutive reaction catalyzed by the coupled enzyme system. The Km of the intermediary product, glyceraldehyde 3-phosphate, produced by aldolase was determined in the coupled reaction for glyceraldehyde-3-phosphate dehydrogenase. Its value corresponds to that of the aldehyde (active) form of glyceraldehyde 3-phosphate, although in the given conditions the aldehyde leads to diol interconversion is faster than the enzymic reaction catalyzed by glyceraldehyde-3-phosphate dehydrogenase. We suggest that above a certain concentration of the enzymes the glyceraldehyde 3-phosphate produced by aldolase gets direct access to glyceraldehyde-3-phosphate dehydrogenase without participating in the aldehyde leads to diol interconversion which otherwise would occur if the substrate were to mix with the bulk medium.     

A comparison of wild-type and mutant ribitol dehydrogenases from Klebsiella aerogenes

A ribitol dehydrogenase (ribitol-NAD(+) oxidoreductase, EC. 1.1.1.56) having increased specificity and catalytic efficiency toward xylitol was isolated from mutant strains of Klebsiella aerogenes, which were selected for increased growth rate on xylitol over the ribitol dehydrogenase constitutive wild-type organism. 2. The mutant enzyme was purified to homogeneity and its general characteristics were compared with those of the previously purified wild-type enzyme. 3. Initial-velocity steady-state kinetic parameters were determined for both wild-type and mutant enzymes and the results compared. 4. The results are interpreted in terms of a model in which the mutant enzyme results from a small change of amino acid sequence, which affects both the stability and conformational equilibria of the molecule.