Erythrose 4-phosphate Research Articles

Characterization of Aspergillus niger phosphoglucose isomerase. Use for quantitative determination of erythrose 4-phosphate

Phosphoglucose isomerase (PGI) was purified from Aspergillus niger and the in vitro kinetic properties of the enzyme were related to its functioning in vivo. A new assay method was developed to study the forward reaction making use of mannitol 1-P dehydrogenase as the coupling enzyme. In this simple assay system mannitol 1-P dehydrogenase converts fructose 6-P and NADH to mannitol 1-P and NAD +, respectively. At pH 7.5 the K m for glucose 6-P was 0.48 mM, whereas the K m for fructose 6-P was 0.32 mM. The pentose phosphate pathway intermediates 6-phosphogluconate and erythrose 4-P (E4P) were competitive inhibitors of PGI with K i values of approximately 0.2 mM and 1 μM respectively. In citric acid producing A. niger mycelium inhibition by 6-phosphogluconate is of minor physiological significance (10% inhibition). Since E4P could not be detected by an existing procedure, a novel assay was developed based on the strong inhibition of PGI by E4P. Although the new assay is very sensitive (detection limit 25 pmol), E4P could still not be detected in metabolite extracts indicating that a very low level of E4P is present in the cells. Using in vitro kinetics and concentrations of intracellular metabolites the in vivo activity of PGI was calculated and closely matched the steady state glycolytic flux observed during citric acid production.

Cloning of the transketolase gene and the effect of its dosage on aromatic amino acid production in Corynebacterium glutamicum.

Transketolase is a key enzyme of the non-oxidative pentose phosphate pathway. The effect of its overexpression on aromatic amino acid production was investigated in Corynebacterium glutamicum, a typical amino-acid-producing organism. For this purpose, the transketolase gene of the organism was cloned on the basis of its ability to complement a C. glutamicum transketolase mutant with pleiotropically shikimic-acid-requiring, ribose- and gluconic-acid-negative phenotype. The gene was shown by deletion mapping and complementation analysis to be located in a 3.2-kb XhoI-SalI fragment of the genome. Amplification of the gene by use of low-, middle-, and high-copy-number vectors in a C. glutamicum strain resulted in overexpression of transketolase activities as well as a protein of approximately 83kDa in proportion to the copy numbers. Introduction of the plasmids into a tryptophan and lysine co-producer resulted in copy-dependent increases in tryptophan production along with concomitant decreases in lysine production. Furthermore, the presence of the gene in high copy numbers enabled tyrosine, phenylalanine and tryptophan producers to accumulate 5%-20% more aromatic amino acids. These results indicate that overexpressed transketolase activity operates to redirect the glycolytic intermediates toward the nonoxidative pentose phosphate pathway in vivo, thereby increasing the intracellular level of erythrose 4-phosphate, a precursor of aromatic biosynthesis, in the aromatic-amino-acid-producing C. glutamicum strains.

Identification of a Catalytic Aspartyl Residue ofd-Ribulose 5-Phosphate 3-Epimerase by Site-directed Mutagenesis

Guided by comparative sequence considerations, we have examined the possibility of a catalytic role of Asp186 of D-ribulose 5-phosphate epimerase by site-directed mutagenesis of the recombinant spinach enzyme. Accordingly, D186A, D186N, and D186E mutants of the epimerase were constructed, purified, and characterized; as judged by their electrophoretic mobilities the mutants are properly assembled into octamers like the wild-type enzyme. Based on the extent of internal quenching of Trp fluorescence, the conformational integrity of the wild-type enzyme is preserved in the mutants. The wild-type kcat of 7.1 x 10(3) s-1 is lowered to 3.3 x 10(-4) s-1 in D186A, 0.13 s-1 in D186N, and 1.1 s-1 in D186E; as gauged by D186A, altogether lacking a functional side chain at position 186, the beta-carboxyl of Asp186 facilitates catalysis by >10(7)-fold. Relative to the wild-type enzyme, the Km for D-ribulose 5-phosphate is essentially unaltered with D186N and D186E but increased 10-fold with D186A. Apart from their impairments in epimerase activity, the mutants are unable to catalyze exchange between solvent protons and the C3 proton of substrates. This deficiency and the differential alterations of kinetic parameters among the mutants are consistent with Asp186 serving as an electrophile to facilitate alpha-proton abstraction. This study is the first to identify a catalytic group of the epimerase.

The EIIGlc protein is involved in glucose-mediated activation of Escherichia coli gapA and gapB-pgk transcription.

The Escherichia coli gapB gene codes for a protein that is very similar to bacterial glyceraldehyde-3-phosphate dehydrogenases (GAPDH). In most bacteria, the gene for GAPDH is located upstream of the pgk gene encoding 3-phosphoglycerate kinase (PGK). This is the case for gapB. However, this gene is poorly expressed and encodes a protein with an erythrose 4-phosphate dehydrogenase activity (E4PDH). The active GAPDH is encoded by the gapA gene. Since we found that the nucleotide region upstream of the gapB open reading frame is responsible for part of the PGK production, we analyzed gapB promoter activity in vivo by direct measurement of the mRNA levels by reverse transcription. We showed the presence of a unique transcription promoter, gapB P0, with a cyclic AMP (cAMP) receptor protein (CRP)-cAMP binding site centered 70.5 bp upstream of the start site. Interestingly, the gapB P0 promoter activity was strongly enhanced when glucose was used as the carbon source. In these conditions, deletion of the CRP-cAMP binding site had little effect on promoter gapB P0 activity. In contrast, abolition of CRP production or of cAMP biosynthesis (crp or cya mutant strains) strongly reduced promoter gapB P0 activity. This suggests that in the presence of glucose, the CRP-cAMP complex has an indirect effect on promoter gapB P0 activity. We also showed that glucose stimulation of gapB P0 promoter activity depends on the expression of enzyme IIGlc (EIIGlc), encoded by the ptsG gene, and that the gapA P1 promoter is also activated by glucose via the EIIGlc protein. A similar glucose-mediated activation, dependent on the EIIGlc protein, was described by others for the pts operon. Altogether, this shows that when glucose is present in the growth medium expression of the E. coli genes required for its uptake (pts) and its metabolism (gapA and gapB-pgk) are coordinately activated by a mechanism dependent upon the EIIGlc protein.

Involvement of the gapA- and epd (gapB)-encoded dehydrogenases in pyridoxal 5'-phosphate coenzyme biosynthesis in Escherichia coli K-12.

We show that epd (gapB) mutants lacking an erythrose 4-phosphate (E4P) dehydrogenase are impaired for growth on some media and contain less pyridoxal 5'-phosphate (PLP) and pyridoxamine 5'-phosphate (PMP) than their epd+ parent. In contrast to a previous report, we found that gapA epd double mutants lacking the glyceraldehyde 3-phosphate and E4P dehydrogenases are auxotrophic for pyridoxine. These results implicate the GapA and Epd dehydrogenases in de novo PLP and PMP coenzyme biosynthesis.

The two 3-deoxy-D-arabino-heptulosonate-7-phosphate synthase isoenzymes from Saccharomyces cerevisiae show different kinetic modes of inhibition.

Activity of the tyrosine-inhibitable 3-deoxy-d-arabino-heptulosonate-7-phosphate synthase (EC 4.1.2.15) from Saccharomyces cerevisiae that was encoded by the ARO4 gene cloned on a high-copy-number plasmid was enhanced 64-fold as compared to the wild-type. The enzyme was purified to apparent homogeneity from the strain that harbored this recombinant plasmid. The estimated molecular weight of 42,000 of the enzyme corresponded to the calculated molecular mass of 40 kDa deduced from the DNA sequence. The enzyme could be inactivated by EDTA in a reaction that was reversed by several bivalent metal ions; presumably a metal cofactor is required for enzymatic catalysis. The Michaelis constant of the enzyme was 125 microM for phosphoenolpyruvate and 500 microM for erythrose 4-phosphate. The rate constant was calculated as 6 s-1, and kinetic data indicated a sequential mechanism of the enzymatic reaction. Tyrosine was a competitive inhibitor with phosphoenolpyruvate as substrate of the enzyme (Ki of 0.9 microM) and a noncompetitive inhibitor with erythrose 4-phosphate as substrate. This is in contrast to the ARO3-encoded isoenzyme, where phenylalanine is a competitive inhibitor with erythrose 4-phosphate as a substrate of the enzyme and a noncompetitive inhibitor with phosphoenolpyruvate as substrate.

Effect of reversible reactions on isotope label redistribution--analysis of the pentose phosphate pathway.

The pentose phosphate pathway plays several key roles in metabolism including supply of biosynthetic carbon skeletons and reducing power. Previous research has focused on determining the fluxes through the reactions of this pathway using carbon-labeled substrates and models that make certain assumptions about the reversibility of the transketolase and transaldolase reactions in the nonoxidative pathway. These assumptions, however, have resulted in inconsistencies between the predicted carbon label distributions using these models and those determined experimentally. A general metabolic reaction network model developed in this paper and applied to the pentose phosphate pathway not only incorporates reaction reversibility but also accounts for the effect of individually varying extents of reaction reversibility on labeled carbon fractional enrichment values for intermediate metabolites. In addition, an algorithm is presented that can be used to calculate the three individual transaldolase and transketolase extents of reversibility. The results of this method show that varying extents of reaction reversibility have an observable effect on the metabolite carbon label distributions which can in turn affect flux calculation for other parts of the metabolic network such as the tricarboxylic acid cycle. In addition, the observability of reversibility extent and accuracy of flux calculations depend on the particular choice of metabolite carbon enrichments measured. In particular, [6-13C]hexose 6-phosphate and [4-13C]erythrose 4-phosphate carbon enrichment values resulting from [1-13C]glucose feeding contained more information as compared to those from ribose 5-phosphate. This analysis was applied to literature data of metabolite carbon labeling that resulted from supplying either 13C- or 14C-enriched substrates to several cell types growing under various conditions. The specific activities of metabolite carbon atoms taken from rat epididymal adipose tissue, goosefish islet cells, Corynebacterium glutamicum, and Escherichia coli supplied with either [2-14C]glucose or [1-13C]glucose demonstrate how reversibility is present in the pentose phosphate pathway and the extents of reversibility can be estimated from labeled carbon data sets.

Competitive inhibition of spinach leaf phosphoglucose isomerase isoenzymes by erythrose 4-phosphate

In plant cells, the reversible isomerization between fructose 6-phosphate (Fru6P) and glucose 6-phosphate (Glc6P) is catalyzed by a cytosolic and a chloroplastic isoenzyme of phosphoglucose isomerase (PGI, EC 5.3.1.9). The extractable activities of both PGI isoenzymes are in large excess compared with the flux required for product synthesis, but the measured Glu6P/Fru6P ratio in illuminated chloroplasts and in whole leaves is always displaced from equilibrium. Cytosolic (PGI 2) and stromal (PGI 1) isoenzymes were purified from spinach leaves and used to investigate the possibility of metabolic regulation at this step. Several metabolites were found to inhibit PGI, but within the physiological concentration range, only erythrose 4-phosphate (Ery4P) inhibited significantly. The inhibition was competitive, with K i values below 10 μM for PGI 2 and 1. The physiological significance of the inhibition of PGI by Ery4P was assessed in isolated intact spinach chloroplasts. We conclude that, in vivo, this inhibition is probably responsible for the observed displacement from equilibrium in the chloroplasts, but limits the carbon flow towards starch synthesis only when Fru6P is low. In contrast, the inhibition by Ery4P is unlikely to play any role in the cytosolic carbon metabolism because both Fru6P concentration and PGI activity, are much higher than in the chloroplast stroma.

Response of the central metabolism ofCorynebacterium glutamicum to different flux burdens

To evaluate the importance of reactions within the central metabolism under different flux burdens the fluxes within the pentose phosphate pathway (PPP), as well as the other reactions of the central metabolism, were intensively analyzed and quantitated. For this purpose, Corynebacterium glutamicum was grown with [1-(13)C]glucose to metabolic and isotopic steady state and the fractional enrichments in precursor metabolites (e.g., pentose 5-phosphate) were quantified. Matrix calculus was used to express these data together with metabolite mass data. The detailed analysis of the dependence of (13)C enrichments on exchange fluxes enabled the transketolase-catalyzed exchange rate (2 pentose 5-phosphate <--> sedoheptulose 7-phosphate + glyceraldehyde 3-phosphate) to be quantified as 74.3% (molar metabolite flux) at a net flux of 10.3% and the exchange rate (pentose 5-phosphate + erythrose 4-phosphate <--> fructose 6-phosphate + glyceraldehyde 3-phosphate) to be quantified as 5.6% at a net flux of 8.1%. The flux entering the tricarboxylic acid cycle was 93.3%. The same comprehensive flux analysis as performed for the nonexcreting condition was done with the identical strain that had been forced to excrete L-glutamate. Because we had already quantified the fluxes for L-lysine excretion with an isogenic strain, three directly comparable flux situations are thus available. Consequently, this comparison permits a direct cause-and-effect relationship to be specified. In response to the different flux burdens of the cell, the PPP flux decreased from a maximum of 67% to 26%, with the glycolytic flux increasing accordingly. The carbon flux through isocitrate dehydrogenase increased from 20% to 36%. The bidirectional carbon flux between pyruvate and oxaloacetate decreased from 36% to 9%. Since the cause of the three different flux states was the allelic exchange in the final L-lysine assembling pathway or the glutamate export activity, respectively, the flexible response is the effect. This shows conclusively the enormous flexibility within the central metabolism of C. glutamicum to supply precursors upon their withdrawal for the synthesis of amino acids. (c) 1997 John Wiley & Sons, Inc. Biotechnol Bioeng 56: 168-180, 1997.

Ribose biosynthesis and evidence for an alternative first step in the common aromatic amino acid pathway in Methanococcus maripaludis.

An acetate-requiring mutant of Methanococcus maripaludis allowed efficient labeling of riboses following growth in minimal medium supplemented with [2-(13)C]acetate. Nuclear magnetic resonance and mass spectroscopic analysis of purified cytidine and uridine demonstrated that the C-1' of the ribose was about 67% enriched for 13C. This value was inconsistent with the formation of erythrose 4-phosphate (E4P) exclusively by the carboxylation of a triose. Instead, these results suggest that either (i) E4P is formed by both the nonoxidative pentose phosphate and triose carboxylation pathways or (ii) E4P is formed exclusively by the nonoxidative pentose phosphate pathway and is not a precursor of aromatic amino acids.

Dihydroxyacetone synthase from a methanol-utilizing carboxydobacterium, Acinetobacter sp. strain JC1 DSM 3803.

Acinetobacter sp. strain JC1 DSM 3803, a carboxydobacterium, grown on methanol was found to show dihydroxyacetone synthase, dihydroxyacetone kinase, and ribulose 1,5-bisphosphate carboxylase, but no hydroxypyruvate reductase and very low hexulose 6-phosphate synthase, activities. The dihydroxyacetone synthase was found to be expressed earlier than the ribulose 1,5-bisphosphate carboxylase. The dihydroxyacetone synthase was purified 19-fold in eight steps to homogeneity, with a yield of 9%. The final specific activity of the purified enzyme was 1.12 micromol of NADH oxidized per min per mg of protein. The molecular weight of the native enzyme was determined to be 140,000. Sodium dodecyl sulfate-gel electrophoresis revealed a subunit of molecular weight 73,000. The optimum temperature and pH were 30 degrees C and 7.0, respectively. The enzyme was inactivated very rapidly at 70 degrees C. The enzyme required Mg2+ and thiamine pyrophosphate for maximal activity. Xylulose 5-phosphate was found to be the best substrate when formaldehyde was used as a glycoaldehyde acceptor. Erythrose 4-phosphate, glycolaldehyde, and formaldehyde were found to act as excellent substrates when xylulose 5-phosphate was used as a glycoaldehyde donor. The Kms for formaldehyde and xylulose 5-phosphate were 1.86 mM and 33.3 microM, respectively. The enzyme produced dihydroxyacetone from formaldehyde and xylulose 5-phosphate. The enzyme was found to be expressed only in cells grown on methanol and shared no immunological properties with the yeast dihydroxyacetone synthase.

Characterization of multiple acid phosphatases in bovine liver cytosol and lysosome. Inactivation of cytosolic enzymes by disulfides and its redox regulation by thioltransferase

Cytosolic and lysosomal acid phosphatases have the ability to hydrolyze orthophosphoric monoesters below pH 5–6. However, it is thought they may have different intracellular roles. To clarify their properties, substrate specificity, inhibitor sensitivity and the modulation of enzyme by redox conditions were determined using bovine liver enzymes. DEAE-cellulose chromatography following (NH 4) 2SO 4 fractionation revealed three forms of cytosolic acid phosphatases as in the KCl gradient (0–500 mM). After Sephadex G-75 gel filtration, the enzymes appeared as single bands on SDS-polyacrylamide gel electrophoresis (SDS-PAGE). Their activities for d-erythrose 4-phosphate co-purified with p-nitrophenylphosphatase activities in all steps. In contrast the lysosomal enzyme was purified by Octyl-Sepharose column chromatography after n-butanol treatment, (NH 4) 2SO 4 fractionation, Bio gel P-200 gel filtration and DE-52 chromatography. The relative molecular masses ( M r) determined by SDS-PAGE indicated that M r of the cytosolic enzymes (16 000) was less than that of lysosomal enzyme (160 000). The cytosolic enzymes were active against sugar phosphates and were inhibited by 1 mM Cu 2+. In addition, the cytosolic enzymes were inactivated by 5 mM oxidized glutathione and protected by 10 mM reduced glutathione (in the presence or absence of thioltransferase), suggesting that sensitive cysteinyl residue(s) existed. The lysosomal enzyme was active against various substrates and was strongly inhibited by 1 mM Cu 2+ and 2 mM fluoride. The results presented here suggest that cytosolic enzymes have different properties from those of lysosomal enzyme with respect to substrates, inhibitors and regulation of activity.

Comparative Enzymatic Properties of GapB-encoded Erythrose-4-Phosphate Dehydrogenase of Escherichia coliand Phosphorylating Glyceraldehyde-3-phosphate Dehydrogenase

GapB-encoded protein of Escherichia coli and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) share more than 40% amino acid identity. Most of the amino acids involved in the binding of cofactor and substrates to GAPDH are conserved in GapB-encoded protein. This enzyme shows an efficient non-phosphorylating erythrose-4-phosphate dehydrogenase activity (Zhao, G., Pease, A. J., Bharani, N., and Winkler, M. E. (1995) J. Bacteriol. 177, 2804-2812) but a low phosphorylating glyceraldehyde-3-phosphate dehydrogenase activity, whereas GAPDH shows a high efficient phosphorylating glyceraldehyde-3-phosphate dehydrogenase activity and a low phosphorylating erythrose-4-phosphate dehydrogenase activity. To identify the structural factors responsible for these differences, comparative kinetic and binding studies have been carried out on both GapB-encoded protein of Escherichia coli and GAPDH of Bacillus stearothermophilus. The KD constant of GapB-encoded protein for NAD is 800-fold higher than that of GAPDH. The chemical mechanism of erythrose 4-phosphate oxidation by GapB-encoded protein is shown to proceed through a two-step mechanism involving covalent intermediates with Cys-149, with rates associated to the acylation and deacylation processes of 280 s-1 and 20 s-1, respectively. No isotopic solvent effect is observed suggesting that the rate-limiting step is not hydrolysis. The rate of oxidation of glyceraldehyde 3-phosphate is 0.12 s-1 and is hydride transfer limiting, at least 2000-fold less efficient compared with that of erythrose 4-phosphate. Thus, it can be concluded that it is only the structure of the substrates that prevails in forming a ternary complex enzyme-NAD-thiohemiacetal productive (or not) for hydride transfer in the acylation step. This conclusion is reinforced by the fact that the rate of oxidation for erythrose 4-phosphate by GAPDH is 0.1 s-1 and is limited by the acylation step, whereas glyceraldehyde 3-phosphate acylation is efficient and is not rate-determining (>/=800 s-1). Substituting Asn for His-176 on GapB-encoded protein, a residue postulated to facilitate hydride transfer as a base catalyst, decreases 40-fold the kcat of glyceraldehyde 3-phosphate oxidation. This suggests that the non-efficient positioning of the C-1 atom of glyceraldehyde 3-phosphate relative to the pyridinium of the cofactor within the ternary complex is responsible for the low catalytic efficiency. No phosphorylating activity on erythrose 4-phosphate with GapB-encoded protein is observed although the Pi site is operative as proven by the oxidative phosphorylation of glyceraldehyde 3-phosphate. Thus the binding of inorganic phosphate to the Pi site likely is not productive for attacking efficiently the thioacyl intermediate formed with erythrose 4-phosphate, whereas a water molecule is an efficient nucleophile for the hydrolysis of the thioacyl intermediate. Compared with glyceraldehyde-3-phosphate dehydrogenase activity, this corresponds to an activation of the deacylation step by >/=4.5 kcal.mol-1. Altogether these results suggest subtle structural differences between the active sites of GAPDH and GapB-encoded protein that could be revealed and/or modulated by the structure of the substrate bound. This also indicates that a protein engineering approach could be used to convert a phosphorylating aldehyde dehydrogenase into an efficient non-phosphorylating one and vice versa.

Localization of the Human Transaldolase Gene (TALDO) to Chromosome 1p33–p34.1 by Fluorescencein SituHybridization and PCR Analysis of Somatic Cell Hybrids

Transaldolase catalyzes the transfer of a C3 fragment corresponding to dihydroxyacetone from sedoheptulose 7-phosphate to glyceraldehyde 3-phosphate, forming erythrose 4-phosphate and fructose 6-phosphate in the pentose phosphate pathway. The pathway provides mainly D-ribose 5-phosphate for nucleic acid synthesis and NADPH for lipid biosynthesis. 10 refs., 1 fig.

Metabolic engineering and control analysis for production of aromatics: Role of transaldolase

Aromatic metabolites in Escherichia coli and other microorganisms are derived from two common precursors: phosphoenolpyruvate (PEP) and erythrose 4-phosphate (E4P). During growth on glucose, the levels of both E4P and PEP are insufficient for high throughput of aromatics because of the low carbon flux through the pentose pathway and the use of PEP in the phosphotransferase system. It has been shown that transketolase and PEP synthase are effective in relieving this limitation and promoting high throughput of aromatics. To determine whether transaldolase, another E4P-producing enzyme, is also a limiting factor in directing carbon flux to the aromatic pathway, E. coli transaldolase gene (tal) was cloned and overexpressed in an aroB strain which excretes 3-deoxy-D-arabinoheptulosonate-7-phosphate (DAHP), the first intermediate in the aromatic pathway. We found that overexpression of transaldolase did significantly increase the production of DAHP from glucose. This result further supports the contention that the supply of E4P is limiting when glucose is the carbon source. However, overexpression of transaldolase in strains which already overexpress transketolase did not show a further increase in production of aromatics. This result was attributed to the saturation of E4P supply when TktA was overexpressed. The flux control of DAHP production was discussed on the basis of Metabolic Control Analysis. (c) 1997 John Wiley & Sons, Inc.

Inhibitory Effect of Shikimic Acid on PEP Carboxylase Activity

The shikimic acid pathway leads to the biosynthesis of aromatic amino acids in plants, bacteria and fungi. In plants, this route is located to some extent, (if not entirely) in the stroma of chloroplasts, although some research groups report the existence of a cytoplasmic counterpart (Hrazdina and Jensen 1992). The first reaction of this pathway is the condensation of one molecule of erythrose 4phosphate with one molecule of phosphoenolpyruvate catalyzed by deoxyarabino heptulosonate phosphate (DAHP) synthase, and shikimic acid is one of the intermediates. Most of the aromatic compounds synthesized by plants, including flavonoids, are derived from phenylalanine and tyrosine. Also, it has been concluded that the bulk of cytosolic phenylalanine is used as precursor for the synthesis of secondary metabolites rather than for the biosynthesis of proteins (Schmid and Amrhein 1995). Flavonoids are polyphenolic structures present in different plant tissues which display various roles, ranging from plant-microorganism interaction (Kent Peters et al. 1986, Kosslak et al. 1987) to ultraviolet radiation protection (Dakora 1995, Kootstra 1994, Stafford 1991). We have previously reported the inhibitory effects of different flavonoids on the activity of PEPC from leaves of the C4 plant Amaranthus viridis (Pairoba et al. 1996). In C4 plants this enzyme is present in the cytosol of mesophyll cells and catalyzes the carboxylation reaction of PEP to render oxaloacetate, the first reaction of the Hatch-Slack cycle. Since PEP is a common substrate for the shikimic

Pathway engineering for the production of aromatic compounds in Escherichia coli.

Glucose is the preferred substrate for certain fermentation processes. During its internalization and concomitant formation of glucose-6-phosphate through the glucose phosphotransferase system (PTS), one molecule of phosphoenolpyruvate (PEP) is consumed. Together with erythrose 4-phosphate (E4P), PEP is condensed to form 3-deoxy-D-arabino-heptulosonate 7-phosphate (DAHP), the first intermediate of the common segment of the aromatic pathway. From this metabolic route, several commercially important aromatic compounds can be obtained. We have selected Escherichia coli mutants that can transport glucose efficiently by a non-PTS uptake system. In theory, this process should increase the availability of PEP for other biosynthetic reactions. Using these mutants, in a background where the DAHP synthase (the enzyme that catalyzes the condensation of PEP and E4P into DAHP) was amplified, we were able to show that at least some of the PEP saved during glucose transport, can be redirected into the aromatic pathway. This increased carbon commitment to the aromatic pathway was enhanced still further upon amplification of the E. coli tktA gene that encodes for a transketolase involved in the biosynthesis of E4P.

Determination of the fluxes in the central metabolism ofCorynebacterium glutamicum by nuclear magnetic resonance spectroscopy combined with metabolite balancing

To determine the in vivo fluxes of the central metabolism we have developed a comprehensive approach exclusively based on the fundamental enzyme reactions known to be present, the fate of the carbon atoms of individual reactions, and the metabolite balance of the culture. No information on the energy balance is required, nor information on enzyme activities, or the directionalities of reactions. Our approach combines the power of (1)H-detected (13)C nuclear magnetic resonance spectroscopy to follow individual carbons with the simplicity of establishing carbon balances of bacterial cultures. We grew a lysine-producing strain of Corynebacterium glutamicum to the metabolic and isotopic steady state with [1-(13)C]glucose and determined the fractional enrichments in 27 carbon atoms of 11 amino acids isolated from the cell. Since precursor metabolites of the central metabolism are incorporated in an exactly defined manner in the carbon skeleton of amino acids, the fractional enrichments in carbons of precursor metabolites (oxaloacetate, glyceraldehyde 3-phosphate, erythrose 4-phosphate, etc.) became directly accessible. A concise and generally applicable mathematical model was established using matrix calculus to express all metabolite mass and carbon labeling balances. An appropriate all-purpose software for the iterative solution of the equations is supplied. Applying this comprehensive methodology to C. glutamicum, all major fluxes within the central metabolism were determined. The result is that the flux through the pentose phosphate pathway is 66.4% (relative to the glucose input flux of 1.49 mmol/g dry weight h), that of entry into the tricarboxylic acid cycle 62.2%, and the contribution of the succinylase pathway of lysine synthesis 13.7%. Due to the large amount and high quality of measured data in vivo exchange reactions could also be quantitated with particularly high exchange rates within the pentose phosphate pathway for the ribose 5-phosphate transketolase reaction. Moreover, the total net flux of the anaplerotic reactions was quantitated as 38.0%. Most importantly, we found that in vivo one component within these anaplerotic reactions is a back flux from the carbon 4 units of the tricarboxylic acid cycle to the carbon 3 units of glycolysis of 30.6%. (c) 1996 John Wiley & Sons, Inc.

Biosynthesis of 3-Amino-5-hydroxybenzoic Acid, the Precursor of mC7N Units in Ansamycin Antibiotics

The biosynthetic pathway of 3-amino-5-hydroxybenzoic acid (AHBA) formation was studied with cell-free extracts from the rifamycin B producer, Amycolatopsis mediterranei S699, and the ansatrienin A producer, Streptomyces collinus Tu1892. Phosphoenolpyruvate (PEP) plus erythrose 4-phosphate (E4P) gave AHBA in low but nevertheless significant (6%) yield. 3,4-Dideoxy-4-amino-d-arabino-heptulosonic acid 7-phosphate (aminoDAHP) was converted efficiently into AHBA (45%), as were 5-deoxy-5-amino-3-dehydroquinic acid (aminoDHQ, 41%) and 5-deoxy-5-amino-3-dehydroshikimic acid (aminoDHS, 95%). On the other hand, the normal shikimate pathway intermediate, 3-deoxy-d-arabino-heptulosonic acid 7-phosphate (DAHP) did not give rise to AHBA under these conditions. AminoDAHP (9%) was produced by incubation of [14C]PEP and E4P, but not of [14C]DAHP, with the cell-free extracts. The results demonstrate the operation of a new variant of the shikimate pathway in the formation of the mC7N units of ansamycin, and presumably also ...

Transketolase A of Escherichia coli K12. Purification and properties of the enzyme from recombinant strains.

Transketolase A was purified to apparent homogeneity from recombinant Escherichia coli K12 cells carrying the homologous cloned tktA gene on a pUC19-derived plasmid. These recombinant cells exhibited a transketolase activity in crude extracts of up to 9.7 U/mg compared to < or = 0.1 U/mg in wild-type cells. Transketolase A was purified from crude extracts of a recombinant strain by successive ammonium sulfate precipitations and two anion-exchange chromatography steps (Q-Sepharose FF, Fractogel EMD-DEAE column) and afforded an apparently homogeneous protein band on SDS/PAGE. The enzyme, both in its active and apoform, had a molecular mass of 145,000 Da (+/- 10,000 Da), judged by gel-filtration chromatography. Subunits of 73,000 Da (+/- 2000 Da) were determined on SDS/PAGE, thus, transketolase A most likely forms a homodimer. N-terminal amino acid sequencing of the protein verified the identity with the cloned gene tktA. The specific activity of the purified enzyme, determined at 30 degrees C with the substrates xylulose 5-phosphate (donor of C2 compound) and ribose 5-phosphate (acceptor) at an optimal pH (50 mM glycylglycine, pH 8.5), was 50.4 U/mg. Km values for the substrates xylulose 5-phosphate and ribose 5-phosphate were 160 microM and 1.4 mM, respectively. Km values for the other physiological substrates of transketolase A were 90 microM for erythrose 4-phosphate (best acceptor substrate), 2.1 mM for D,L-glyceraldehyde 3-phosphate, 1.1 mM for fructose 6-phosphate, and 4 mM for sedoheptulose 7-phosphate. Hydroxypyruvate served as alternative donor (Km = 18 mM). Unphosphorylated acceptor compounds were formaldehyde (Km = 31 mM), glycolaldehyde (14 mM), D,L-glyceraldehyde (10 mM) and D-erythrose (150 mM). The enzyme was competitively inhibited by D-arabinose 5-phosphate (K = 6 mM at a concentration of 2.5 mM D-arabinose 5-phosphate) or by the chelating agent EDTA. The inactive apoform of transketolase A was yielded by dialysis against buffer containing 10 mM EDTA, thus removing the cofactors thiamine diphosphate and divalent cations. The reconstitution of the apoenzyme proceded faster in the presence of manganese ions (Kd = 7 microM at 10 microM thiamine diphosphate) than with other divalent cations.