Novel listerial glycerol dehydrogenase- and phosphoenolpyruvate-dependent dihydroxyacetone kinase system connected to the pentose phosphate pathway

Several bacteria use glycerol dehydrogenase to transform glycerol into dihydroxyacetone (Dha). Dha is subsequently converted into Dha phosphate (Dha-P) by an ATP- or phosphoenolpyruvate (PEP)-dependent Dha kinase. Listeria innocua possesses two potential PEP-dependent Dha kinases. One is encoded by 3 of the 11 genes forming the glycerol (gol) operon. This operon also contains golD (lin0362), which codes for a new type of Dha-forming NAD(+)-dependent glycerol dehydrogenase. The subsequent metabolism of Dha requires its phosphorylation via the PEP:sugar phosphotransferase system components enzyme I, HPr, and EIIA(Dha)-2 (Lin0369). P～EIIA(Dha)-2 transfers its phosphoryl group to DhaL-2, which phosphorylates Dha bound to DhaK-2. The resulting Dha-P is probably metabolized mainly via the pentose phosphate pathway, because two genes of the gol operon encode proteins resembling transketolases and transaldolases. In addition, purified Lin0363 and Lin0364 exhibit ribose-5-P isomerase (RipB) and triosephosphate isomerase activities, respectively. The latter enzyme converts part of the Dha-P into glyceraldehyde-3-P, which, together with Dha-P, is metabolized via gluconeogenesis to form fructose-6-P. Together with another glyceraldehyde-3-P molecule, the transketolase transforms fructose-6-P into intermediates of the pentose phosphate pathway. The gol operon is preceded by golR, transcribed in the opposite orientation and encoding a DeoR-type repressor. Its inactivation causes the constitutive but glucose-repressible expression of the entire gol operon, including the last gene, encoding a pediocin immunity-like (PedB-like) protein. Its elevated level of synthesis in the golR mutant causes slightly increased immunity against pediocin PA-1 compared to the wild-type strain or a pedB-like deletion mutant.     

Formation of a pentose phosphate cycle metabolite, erythrose-4-phosphate, from initial compounds of glycolysis by transketolase from the rat liver

Using ion-exchange chromatography of sucrose phosphates on Dowex-1, it was demonstrated that the highly purified rat liver transketolase (specific activity 1.7 mumol/min.mg protein) is capable of catalyzing the synthesis of erythrose-4-phosphate, a metabolite of the pentose phosphate pathway non-oxidizing step, from the initial participants of glycolysis, i. e., glucose-6-phosphate and fructose-6-phosphate. As can be evidenced from the reaction course, the second product of this synthesis is octulose-8-phosphate. The reaction was assayed by accumulation of erythrose-4-phosphate. The soluble fraction from rat liver catalyzes under identical conditions the synthesis of heptulose-7-phosphate (but not erythrose-4-phosphate), which points to the utilization of the erythrose-4-phosphate formed in the course of the transketolase reaction by transaldolase which is also present in the soluble fraction. The role of the transketolase reaction reversal from the synthesis of pentose phosphate derivatives to glycolytic products is discussed. The transketolase reaction provides for the relationship between glycolysis and the anaerobic step of the pentose phosphate pathway which share common metabolites, i. e. glucose-6-phosphate and fructose-6-phosphate.     

High control coefficient of transketolase in the nonoxidative pentose phosphate pathway of human erythrocytes: NMR, antibody, and computer simulation studies.

The degree of control exerted by transketolase over metabolite flux in the nonoxidative pentose phosphate pathway in human erythrocytes was investigated using transketolase antiserum to modulate the activity of that enzyme. 31P NMR enabled the simultaneous measurement of the levels of pentose phosphate pathway metabolites following incubation of hemolysates with ribose 5-phosphate. The variations in metabolic flux which occurred as the transketolase activity of hemolysate samples was altered indicated that a high degree of control was exerted by transketolase. Investigations using transaldolase-depleted hemolysates showed that transaldolase exhibits a lesser degree of control over pathway flux. Experimental data were compared with simulations generated by a computer model encompassing the reactions of the classical nonoxidative pentose phosphate pathway. The sensitivity coefficients (also called "control strengths" or "flux-control coefficients") calculated from the computer simulations were 0.74 and 0.03 for transketolase and transaldolase, respectively.     

Revisiting the 13C-label distribution of the non-oxidative branch of the pentose phosphate pathway based upon kinetic and genetic evidence

The currently applied reaction structure in stoichiometric flux balance models for the nonoxidative branch of the pentose phosphate pathway is not in accordance with the established ping-pong kinetic mechanism of the enzymes transketolase (EC 2.2.1.1) and transaldolase (EC 2.2.1.2). Based upon the ping-pong mechanism, the traditional reactions of the nonoxidative branch of the pentose phosphate pathway are replaced by metabolite specific, reversible, glycolaldehyde moiety (C(2)) and dihydroxyacetone moiety (C(3)) fragments producing and consuming half-reactions. It is shown that a stoichiometric model based upon these half-reactions is fundamentally different from the currently applied stoichiometric models with respect to the number of independent C(2) and C(3) fragment pools in the pentose phosphate pathway and can lead to different label distributions for (13)C-tracer experiments. To investigate the actual impact of the new reaction structure on the estimated flux patterns within a cell, mass isotopomer measurements from a previously published (13)C-based metabolic flux analysis of Saccharomyces cerevisiae were used. Different flux patterns were found. From a genetic point of view, it is well known that several micro-organisms, including Escherichia coli and S. cerevisiae, contain multiple genes encoding isoenzymes of transketolase and transaldolase. However, the extent to which these gene products are also actively expressed remains unknown. It is shown that the newly proposed stoichiometric model allows study of the effect of isoenzymes on the (13)C-label distribution in the nonoxidative branch of the pentose phosphate pathway by extending the half-reaction based stoichiometric model with two distinct transketolase enzymes instead of one. Results show that the inclusion of isoenzymes affects the ensuing flux estimates.     

Properties and functions of the thiamin diphosphate dependent enzyme transketolase

This review highlights recent research on the properties and functions of the enzyme transketolase, which requires thiamin diphosphate and a divalent metal ion for its activity. The transketolase-catalysed reaction is part of the pentose phosphate pathway, where transketolase appears to control the non-oxidative branch of this pathway, although the overall flux of labelled substrates remains controversial. Yeast transketolase is one of several thiamin diphosphate dependent enzymes whose three-dimensional structures have been determined. Together with mutational analysis these structural data have led to detailed understanding of thiamin diphosphate catalysed reactions. In the homodimer transketolase the two catalytic sites, where dihydroxyethyl groups are transferred from ketose donors to aldose acceptors, are formed at the interface between the two subunits, where the thiazole and pyrimidine rings of thiamin diphosphate are bound. Transketolase is ubiquitous and more than 30 full-length sequences are known. The encoded protein sequences contain two motifs of high homology; one common to all thiamin diphosphate-dependent enzymes and the other a unique transketolase motif. All characterised transketolases have similar kinetic and physical properties, but the mammalian enzymes are more selective in substrate utilisation than the nonmammalian representatives. Since products of the transketolase-catalysed reaction serve as precursors for a number of synthetic compounds this enzyme has been exploited for industrial applications. Putative mutant forms of transketolase, once believed to predispose to disease, have not stood up to scrutiny. However, a modification of transketolase is a marker for Alzheimer’s disease, and transketolase activity in erythrocytes is a measure of thiamin nutrition. The cornea contains a particularly high transketolase concentration, consistent with the proposal that pentose phosphate pathway activity has a role in the removal of light-generated radicals.     

Microsequecing and cDNA cloning of the Calvin cycle/OPPP enzyme ribose-5-phosphate isomerase (EC 5.3.1.6) from spinach chloroplasts

Ribose-5-phosphate isomerase (RPI) catalyses the interconversion of ribose-5-phosphate and ribulose-5-phosphate in the reductive and oxidative pentose phosphate pathways in plants. RPI from spinach chloroplasts was purified and microsequenced. Via PCR with degenerate primers designed against microsequenced peptides, a hybridisation probe was obtained and used to isolate several cDNA clones which encode RPI. The nuclear-encoded 239 amino acid mature RPI subunit has a predicted size of 25.3 kDa and is translated as a cytosolic precursor possessing a 50 amino acid transit peptide. The processing site of the transit peptide was identified from protein sequence data. Spinach leaves possess only one type of homodimeric RPI enzyme which is localized in chloroplasts and is encoded by a single nuclear gene. Molecular characterization of RPI supports the view that a single amphibolic RPI enzyme functions in the oxidative and reductive pentose phosphate pathways of spinach plastids.Abbreviations RPI ribose-5-phosphate isomerase - OPPP oxidative pentose phosphate pathway - CNBr cyanogen bromide - R5P ribose-5-phosphate - Ru5P ribulose-5-phosphate     

The plastidic pentose phosphate translocator represents a link between the cytosolic and the plastidic pentose phosphate pathways in plants

Plastids are the site of the reductive and the oxidative pentose phosphate pathways, which both generate pentose phosphates as intermediates. A plastidic transporter from Arabidopsis has been identified that is able to transport, in exchange with inorganic phosphate or triose phosphates, xylulose 5-phosphate (Xul-5-P) and, to a lesser extent, also ribulose 5-phosphate, but does not accept ribose 5-phosphate or hexose phosphates as substrates. Under physiological conditions, Xul-5-P would be the preferred substrate. Therefore, the translocator was named Xul-5-P/phosphate translocator (XPT). The XPT shares only approximately 35% to 40% sequence identity with members of both the triose phosphate translocator and the phosphoenolpyruvate/phosphate translocator classes, but a higher identity of approximately 50% to glucose 6-phosphate/phosphate translocators. Therefore, it represents a fourth group of plastidic phosphate translocators. Database analysis revealed that plant cells contain, in addition to enzymes of the oxidative branch of the oxidative pentose phosphate pathway, ribose 5-phosphate isomerase and ribulose 5-phosphate epimerase in both the cytosol and the plastids, whereas the transketolase and transaldolase converting the produced pentose phosphates to triose phosphates and hexose phosphates are probably solely confined to plastids. It is assumed that the XPT function is to provide the plastidic pentose phosphate pathways with cytosolic carbon skeletons in the form of Xul-5-P, especially under conditions of a high demand for intermediates of the cycles.     

Biosynthesis of ribose-5-phosphate and erythrose-4-phosphate in archaea: a phylogenetic analysis of archaeal genomes

A phylogenetic analysis of the genes encoding enzymes in the pentose phosphate pathway (PPP), the ribulose monophosphate (RuMP) pathway, and the chorismate pathway of aromatic amino acid biosynthesis, employing data from 13 complete archaeal genomes, provides a potential explanation for the enigmatic phylogenetic patterns of the PPP genes in archaea. Genomic and biochemical evidence suggests that three archaeal species (Methanocaldococcus jannaschii, Thermoplasma acidophilum and Thermoplasma volcanium) produce ribose-5-phosphate via the nonoxidative PPP (NOPPP), whereas nine species apparently lack an NOPPP but may employ a reverse RuMP pathway for pentose synthesis. One species (Halobacterium sp. NRC-1) lacks both the NOPPP and the RuMP pathway but may possess a modified oxidative PPP (OPPP), the details of which are not yet known. The presence of transketolase in several archaeal species that are missing the other two NOPPP genes can be explained by the existence of differing requirements for erythrose-4-phosphate (E4P) among archaea: six species use transketolase to make E4P as a precursor to aromatic amino acids, six species apparently have an alternate biosynthetic pathway and may not require the ability to make E4P, and one species (Pyrococcus horikoshii) probably does not synthesize aromatic amino acids at all.     

Statistical analysis of pentose phosphate pathway genes from eubacteria and eukarya reveals translational selection as a major force in shaping codon usage pattern

Comparative analysis of metabolic pathways among widely diverse species provides an excellent opportunity to extract information about the functional relation of organisms and pentose phosphate pathway exemplifies one such pathway. A comparative codon usage analysis of the pentose phosphate pathway genes of a diverse group of organisms representing different niches and the related factors affecting codon usage with special reference to the major forces influencing codon usage patterns was carried out. It was observed that organism specific codon usage bias percolates into vital metabolic pathway genes irrespective of their near universality. A clear distinction in the codon usage pattern of gram positive and gram negative bacteria, which is a major classification criterion for bacteria, in terms of pentose phosphate pathway was an important observation of this study. The codon utilization scheme in all the organisms indicates the presence of translation selection as a major force in shaping codon usage. Another key observation was the segregation of the H. sapiens genes as a separate cluster by correspondence analysis, which is primarily attributed to the different codon usage pattern in this genus along with its longer gene lengths. We have also analyzed the amino acid distribution comparison of transketolase protein primary structures among all the organisms and found that there is a certain degree of predictability in the composition profile except in A. fumigatus and H. sapiens, where few exceptions are prominent. In A. fumigatus, a human pathogen responsible for invasive aspergillosis, a significantly different codon usage pattern, which finally translated into its amino acid composition model portraying a unique profile in a key pentose phosphate pathway enzyme transketolase was observed.     

The pentose phosphate pathway of glucose metabolism. Hormonal and dietary control of the oxidative and non-oxidative reactions of the cycle in liver

1. Measurements were made of the non-oxidative reactions of the pentose phosphate cycle in liver (transketolase, transaldolase, ribulose 5-phosphate epimerase and ribose 5-phosphate isomerase activities) in a variety of hormonal and nutritional conditions. In addition, glucose 6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase activities were measured for comparison with the oxidative reactions of the cycle; hexokinase, glucokinase and phosphoglucose isomerase activities were also included. Starvation for 2 days caused significant lowering of activity of all the enzymes of the pentose phosphate cycle based on activity in the whole liver. Re-feeding with a high-carbohydrate diet restored all the enzyme activities to the range of the control values with the exception of that of glucose 6-phosphate dehydrogenase, which showed the well-known ;overshoot' effect. Re-feeding with a high-fat diet also restored the activities of all the enzymes of the pentose phosphate cycle and of hexokinase; glucokinase activity alone remained unchanged. Expressed as units/g. of liver or units/mg. of protein hexokinase, glucose 6-phosphate dehydrogenase, transketolase and pentose phosphate isomerase activities were unchanged by starvation; both 6-phosphogluconate dehydrogenase and ribulose 5-phosphate epimerase activities decreased faster than the liver weight or protein content. 2. Alloxan-diabetes resulted in a decrease of approx. 30-40% in the activities of 6-phosphogluconate dehydrogenase, ribose 5-phosphate isomerase, ribulose 5-phosphate epimerase and transketolase; in contrast with this glucose 6-phosphate dehydrogenase, transaldolase and phosphoglucose isomerase activities were unchanged. Treatment of alloxan-diabetic rats with protamine-zinc-insulin for 3 days caused a very marked increase to above normal levels of activity in all the enzymes of the pentose phosphate pathway except ribulose 5-phosphate epimerase, which was restored to the control value. Hexokinase activity was also raised by this treatment. After 7 days treatment of alloxan-diabetic rats with protamine-zinc-insulin the enzyme activities returned towards the control values. 3. In adrenalectomized rats the two most important changes were the rise in hexokinase activity and the fall in transketolase activity; in addition, ribulose 5-phosphate epimerase activity was also decreased. These effects were reversed by cortisone treatment. In addition, in cortisone-treated adrenalectomized rats glucokinase activity was significantly lower than the control value. 4. In thyroidectomized rats both ribose 5-phosphate isomerase and transketolase activities were decreased; in contrast with this transaldolase activity did not change significantly. Hypophysectomy caused a 50% fall in transketolase activity that was partially reversed by treatment with thyroxine and almost fully reversed by treatment with growth hormone for 8 days. 5. The results are discussed in relation to the hormonal control of the non-oxidative reactions of the pentose phosphate cycle, the marked changes in transketolase activity being particularly outstanding.     

Effect of transketolase modifications on carbon flow to the purine-nucleotide pathway in Corynebacterium ammoniagenes

Transketolase, one of the enzymes in the nonoxidative branch of the pentose phosphate pathway, operates to shuttle ribose 5-phosphate and glycolytic intermediates together with transaldolase, and might be involved in the availability of ribose 5-phosphate, a precursor of nucleotide biosynthesis. The tkt and tal genes encoding transketolase and transaldolase, respectively, were cloned from the typical nucleotide- and nucleoside-producing organism Corynebacterium ammoniagenes by a PCR approach using oligonucleotide primers derived from conserved regions of each amino acid sequence from other organisms. Enzymatic and molecular analyses revealed that the two genes were clustered on the genome together with the glucose 6-phosphate dehydrogenase gene (zwf). The effect of transketolase modifications on the production of inosine and 5'-xanthylic acid was investigated in industrial strains of C. ammoniagenes. Multiple copies of plasmid-borne tkt caused about tenfold increases in transketolase activity and resulted in 10-20% decreased yields of products relative to the parents. In contrast, site-specific disruption of tkt enabled both producers to accumulate 10-30% more products concurrently with a complete loss of transketolase activity and the expected phenotype of shikimate auxotrophy. These results indicate that transketolase normally shunts ribose 5-phosphate back into glycolysis in these biosynthetic processes and interception of this shunt allows cells to redirect carbon flux through the oxidative pentose pathway from the intermediate towards the purine-nucleotide pathway.     

Cloning and sequencing of a putative Escherichia coli [NiFe] hydrogenase-1 operon containing six open reading frames.

DNA encompassing the structural genes of an Escherichia coli [NiFe] hydrogenase has been cloned and sequenced. The genes were identified as those encoding the large and small subunits of hydrogenase isozyme 1 based on NH2-terminal sequences of purified subunits (kindly provided by K. Francis and K. T. Shanmugam). The structural genes formed part of a putative operon that contained four additional open reading frames. We have designated the operon hya and the six open reading frames hyaA through F. hyaA and hyaB encode the small and large structural subunits, respectively. The nucleotide-derived amino acid sequence of hyaC has a calculated molecular mass of 27.6 kilodaltons, contains 20% aromatic residues, and has four potential membrane-spanning regions. Open reading frames hyaD through F could encode polypeptides of 21.5, 14.9, and 31.5 kilodaltons, respectively. These putative peptides have no homology to other reported protein sequences, and their functions are unknown.     

The form II fructose 1,6-bisphosphatase and phosphoribulokinase genes form part of a large operon in Rhodobacter sphaeroides: primary structure and insertional mutagenesis analysis

Fructose 1,6-bisphosphatase (FBPase) and phosphoribulokinase (PRK) are two key enzymes of the reductive pentose phosphate pathway or Calvin cycle of photosynthetic carbon dioxide assimilation. Early studies had indicated that the properties of enzymes isolated from photosynthetic bacteria were clearly distinct from those of enzymes obtained from the chloroplasts of higher plants [for a review, see Tabita (1988)]. The eucaryotic enzymes, which are light activated by the thioredoxin/ferredoxin system (Buchanan, 1980), were each shown to contain a putative regulatory amino acid sequence (Marcus et al., 1988; Porter et al., 1988). The enzymes from photosynthetic bacteria are not controlled by the thioredoxin/ferredoxin system but exhibit complex kinetic properties and, in the case of PRK, there is an absolute requirement of NADH for activity. In the photosynthetic bacterium Rhodobacter sphaeroides, the structural genes of the Calvin cycle, including the genes that encode FBPase (fbp) and PRK (prk), are found in two distinct clusters, and the fbp and prk genes are closely associated in each cluster. In the present investigation, we have determined the nucleotide sequence of the fbpB and prkB genes of the form II cluster and have compared the deduced amino acid sequences to previously determined sequences of light-activated enzymes from higher plants and from other eucaryotic and procaryotic sources. In the case of FBPase, there are several regions that are conserved in the R. sphaeroides enzymes, including a protease-sensitive area located in a region equivalent to residues 51-71 of mammalian FBPase.(ABSTRACT TRUNCATED AT 250 WORDS)     

Evidence for a pentose phosphate pathway in Helicobacter pylori

Abstract Evidence for the presence of enzymes of the pentose phosphate pathway in Helicobacter pylori was obtained using ³¹P nuclear magnetic resonance spectroscopy. Activities of enzymes which are part of the oxidative and non-oxidative phases of the pathway were observed directly in incubations of bacterial lysates with pathway intermediates. Generation of NADPH and 6-phosphogluconate from NADP⁺ and glucose 6-phosphate indicated the presence of glucose 6-phosphate dehydrogenase and 6-phosphogluconolactonase. Reduction of NADP⁺ with production of ribulose 5-phosphate from 6-phosphogluconate revealed 6-phosphogluconate dehydrogenase activity. Phosphopentose isomerase and transketolase activities were observed in incubations containing ribulose 5-phosphate and xylulose 5-phosphate, respectively. The formation of erythrose 4-phosphate from xylulose 5-phosphate and ribose 5-phosphate suggested the presence of transaldolase. The activities of this enzyme and triosephosphate isomerase were observed directly in incubations of bacterial lysates with dihydroxyacetone phosphate and sedoheptulose 7-phosphate. Glucose-6-phosphate isomerase activity was measured in incubations with fructos 6-phosphate. The presence of these enzymes in H. pylori suggested the existence of a pentose phosphate pathway in the bacterium, possibly as a mechanism to provide NADPH for reductive biosynthesis and ribose 5-phosphate for synthesis of nucleic acids.     

The pentose phosphate pathway of glucose metabolism. Hormonal and dietary control of the oxidative and non-oxidative reactions and related enzymes of the cycle in adipose tissue

1. Measurements were made of the activities of the enzymes of the pentose phosphate pathway concerned in both the oxidative (glucose 6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase) and the non-oxidative (ribose 5-phosphate isomerase, ribulose 5-phosphate epimerase, transketolase and transaldolase) reactions of this pathway, together with hexokinase and phosphoglucose isomerase, in adipose tissue in a variety of nutritional and hormonal conditions. 2. Starvation for 2 days caused a significant decrease in the activities of all the enzymes of the pentose phosphate pathway, with the exception of glucose 6-phosphate dehydrogenase, when expressed as activity/2 fat-pads; only the activities of ribose 5-phosphate isomerase and ribulose 5-phosphate epimerase were significantly decreased on the basis of activity/mg. of protein. Re-feeding with a high-carbohydrate or high-fat diet for 3 days restored the activity of all the enzymes of the pentose phosphate pathway to the range of the control values, with the exception of transketolase, which showed a marked ;overshoot' in rats re-fed with carbohydrate. Starvation for 3 days caused a marked decrease in the activities of glucose 6-phosphate dehydrogenase and transketolase. 3. On the basis of activity/two fat-pads, alloxan-diabetes caused a marked decrease, to about half the control value, in the activities of all the enzymes concerned in the pentose phosphate pathway, transketolase showing the smallest decrease; hexokinase and phosphoglucose isomerase activities were also decreased. Treatment with insulin for 3 and 7 days raised the activities to normal or supranormal values, transketolase showing the most marked ;overshoot' effect. On the basis of activity/mg. of protein the activity of none of the enzymes was significantly decreased in alloxan-diabetes; transketolase and transaldolase activities were raised above the control values. With insulin treatment for 3 or 7 days the activities of all the enzymes were significantly increased, except that of ribulose 5-phosphate epimerase at the shorter time-interval. Glucagon treatment did not alter any of the enzyme activities expressed on either basis. 4. Thyroidectomy caused a decrease of 30-40% in the activities of enzymes of the pentose phosphate pathway, except for transketolase activity, which fell to 50% of the control value. Little change occurred in adipose-tissue weight or protein content. 5. Adrenalectomy caused a decrease of 40% in the activity of glucose 6-phosphate dehydrogenase and of 20-30% in the activities of the remaining enzymes of the pentose phosphate pathway; hexokinase activity was also decreased. Treatment with cortisone for 3 days did not significantly raise the activity from that found in adrenalectomized rats. Treatment of normal rats with high doses of cortisone had no significant effect on the activities of the enzymes of the pentose phosphate pathway in adipose tissue. 6. The changes in enzyme activities are discussed in relation to: (a) the concept of constant-proportion groups of enzymes; (b) the known changes in the flux of glucose through alternative metabolic pathways; (c) the pattern of change found in liver with similar hormonal and dietary conditions.