Enzymatic Studies on the Conversion of Citric Acid Fermentation to Polyol Fermentation in Hydrocarbon-assimilating Yeast Candida zeylanoides

Production of polyols such as erythritol, d-mannitol and d-arabitol by citric acid-producing yeasts occurred only when the medium-pH was controlled at acidic pH, as described in the previous papers.

In order to elucidate the conversion mechanism of citric acid fermentation to polyol fermentation, the effect of pH on the activities of enzymes involved in polyol synthesis and tricarboxylic acid cycle was studied. Shifting down of the medium-pH from 5.5 to 3.5 led immediately to the change of intracellular pH, from 6.5~6.7 to 5.5~5.7. Such the change affected remarkably on the activities of intracellular enzymes. Citrate synthase was significantly depressed at pH 5.7, but isocitrate lyase and phosphoenolpyruvate carboxykinase were reversely stimulated at this pH.

In some yeast strains incapable of polyol production, the change of medium-pH reflected directly on intracellular pH, whereby almost all enzymes were inhibited.

From these results, the conversion of citric acid production to polyol production was explained by the change in the enzyme activities caused by lowering of intracellular pH.     

The pentose phosphate pathway of glucose metabolism. Measurement of the non-oxidative reactions of the cycle

Methods for the quantitative determination of ribose 5-phosphate isomerase, ribulose 5-phosphate 3-epimerase, transketolase and transaldolase in tissue extracts are described. The determinations depend on the measurement of glyceraldehyde 3-phosphate by using the coupled system triose phosphate isomerase, α-glycero-phosphate dehydrogenase and NADH. By using additional purified enzymes transketolase, ribose 5-phosphate isomerase and ribulose 5-phosphate epimerase conditions could be arranged so that each enzyme in turn was made rate-limiting in the overall system. Transaldolase was measured with fructose 6-phosphate and erythrose 4-phosphate as substrates, and again glyceraldehyde 3-phosphate was measured by using the same coupled system. Measurements of the activities of the non-oxidative reactions of the pentose phosphate pathway were made in a variety of tissues and the values compared with those of the two oxidative steps catalysed by glucose 6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase.     

Partial purification and kinetic characterization of transaldolase fromDictyostelium discoideum

Transaldolase was purified 42-fold fromDictyostelium discoideum and the resulting preparation exhibited stoichiometry. Kinetic analyses consisted of initial velocity and product inhibition studies in both the forward and the reverse directions. The enzyme exhibited ping-pong kinetics with sedoheptulose 7-phosphate adding first and erythrose 4-phosphate releasing first. TheK_m values for sedoheptulose 7-phosphate, glyceraldehyde 3-phosphate, erythrose 4-phosphate, and fructose 6-phosphate were 0.46, 0.072, 0.10, and 1.6 mM, respectively. TheK_i values for sedoheptulose 7-phosphate and erythrose 4-phosphate were 3.6 and 0.062 mM, respectively. Inorganic phosphate inhibited enzymatic activity and showed mixed-type inhibition when fructose 6-phosphate was varied. AK_i value of 35.2 mM was determined for inorganic phosphate.     

Antibacterial Activity of l-Lysine α-Oxidase against rec+ and rec− Strains of Bacillus subtilis

2-Deoxyribose 5-phosphate production through coupling of the alcoholic fermentation system of baker’s yeast and deoxyriboaldolase-expressing Escherichia coli was investigated. In this process, baker’s yeast generates fructose 1,6-diphosphate from glucose and inorganic phosphate, and then the E. coli convert the fructose 1,6-diphosphate into 2-deoxyribose 5-phosphate via D-glyceraldehyde 3-phosphate. Under the optimized conditions with toluene-treated yeast cells, 356 mM (121 g/l) fructose 1,6-diphosphate was produced from 1,111 mM glucose and 750 mM potassium phosphate buffer (pH 6.4) with a catalytic amount of AMP, and the reaction supernatant containing the fructose 1,6-diphosphate was used directly as substrate for 2-deoxyribose 5-phosphate production with the E. coli cells. With 178 mM enzymatically prepared fructose 1,6-diphosphate and 400 mM acetaldehyde as substrates, 246 mM (52.6 g/l) 2-deoxyribose 5-phosphate was produced. The molar yield of 2-deoxyribose 5-phosphate as to glucose through the total two step reaction was 22.1%. The 2-deoxyribose 5-phosphate produced was converted to 2-deoxyribose with a molar yield of 85% through endogenous or exogenous phosphatase activity.     

Change in the Regulation of Enzyme Synthesis under Catabolite Repression in Bacillus subtilis Pleiotropic Mutant Lacking Transketolase

The regulation of enzyme synthesis has changed in Bacillus subtilis pleiotropic mutant lacking transketolase (tkt). The tkt mutant is hypersensitive to d-glucose repression of the synthesis of d-mannitol catabolic enzymes, such as d-mannitol-1-phosphate dehydrogenase and d-mannitol transport system. d-Gluconate, d-xylose and l-arabinose are also effectors for repression in the tkt mutant. In contrast, the synthesis of sorbitol catabolic enzymes, such as sorbitol permease and sorbitol dehydrogenase, are almost insensitive to d-glucose repression. These changes in the regulation of enzyme synthesis seem to be related to some defect in the cell surface structure of the tkt mutant by which other pleiotropic properties are also generated.     

D-Glucosaminate Aldolase Activity of D-Glucosaminate Dehydratase from Pseudomonas fluorescens and Its Requirement for Mn2+ Ion

When D-glucosaminate dehydratase (GADH) was incubated with D-glucosaminate (GlcNA) in veronal buffer (VB; 0.01 M, pH 8.0), GlcNA was converted stoichiometrically to glyceraldehyde, pyruvate, and ammonia (aldolase reaction A). This reaction occurred in addition to the dehydratase reaction (conversion of GlcNA to 2-keto-3-deoxy-o-gluconate and ammonia: α,β-elimination reaction, B). The ratio of the activities (A:B) was about 1:4. However, in potassium phosphate buffer (KPB; 0.04 M, pH 8.0), the aldolase reaction was inhibited to 3–4% of that in VB, and also inhibited by various derivatives of glycerol, in particular, glycerol-3-phosphate (glycerol-3-P) and glyceraldehyde-3-phosphate (glyceraldehyde-3-P) in VB. The native enzyme was inhibited by incubation with 0.1 M EDTA, and the activity was restored by incubation of the EDTA-treated enzyme with (Mn²⁺ + pyridoxal 5′-phosphate (PLP)). When the EDTA-treated enzyme was incubated with (Mn²⁺ + PLP + glycerol-3-P), the activity of reaction B increased to 131% but that of reaction A decreased to 21%. These results suggested that Mn²⁺, PLP, and the phosphate group of glycerol-3-P are involved in formation of the active enzyme. In the case of the aldolase reaction, Mn²⁺ ion, which might be essential for the reaction, is chelated by the phosphate group of glycerol-3-P with resultant inhibition of the aldolase reaction.     

Enzymatic Conversion of d-Glucose to d-Fructose

Glucose isomerizing enzyme was partially purified after investigation on the properties of crude enzyme extract. The crude extract was partly inactivated by the contact with air. The addition of manganese was effective to improve the stability. Magnesium was essential to the enzyme action and cobalt accelerated the reaction.

The maximal activity was observed at pH about 7.6 and 50°C was optimal for the incubation time of 30 minutes. The enzyme solution reacted with d-xylose as well as d-glucose. The activity of the enzyme was inhibited at high glucose concentrations.

An enzyme which catalyzes the conversion of d-glucose to d-fructose has been demonstrated in cell-free extracts of Streptomyces phaeochromo genus grown in the presence of D-xylose. The enzyme preparation reacts with d-glucose and d-xylose, but not with other sugars tested. It appears to require magnesium for the maximal activity and the addition of cobaltous ion remarkably intensifies the heat tolerance of the enzyme. The maximal activity occurs at about pH 9.3~9.5. Equilibrium is reached when about 52% fructose is present in the reaction mixture. The enzyme has half-maximal activity when the concentration of d-glucose is about 0.3 M at pH 9 and 60°C.     

Carbohydrate Metabolism-Mutants of a Bacillus Species

During the course of studies on the effects of mutation in carbohydrate metabolism on the synthesis of purine derivatives, it was found that three mutants of a Bacillus species, which lacked transketolase or d-ribulose 5-phosphate 3-epimerase, accumulated a large amount of d-ribose in the culture medium. The amount of d-ribose was about 35 mg per ml of the broth incubated for 6 days. d-Ribose in the broth was purified in crystalline form and was identified from its chemical and physical properties.     

Mechanisms of Browning Degradation of d-Fructose in Special Comparison with d-Glucose-Glycine Reaction

Degradation mechanisms of d-fructose by the interaction with amino acids or organic acids in aqueous solution at initial pH 5.5 heated at 100°C were investigated and a substantial difference in mechanisms between fructose degradation and glucose-glycine reaction was presented. d-Fructose browned more intensely than did d-glucose in lower concentration of glycine and/or in earier stage of reaction period. By catalytic action of carboxylate anions without any condensation with amino groups, d-fructose was decomposed to 3-deoxy-d-erythrohexosulose, 5-(hydroxymelhyl)-2-furaldehyde, and a less amount of pyruval-dehyde through caramelization. It was considered that the main path of fructose degradation was 1,2-enolization but 2,3-enolization would also occur to a little extent.     

Pathway and regulation of erythritol formation in Leuconostoc oenos.

It was recently observed that Leuconostoc oenos GM, a wine lactic acid bacterium, produced erythritol anaerobically from glucose but not from fructose or ribose and that this production was almost absent in the presence of O2. In this study, the pathway of formation of erythritol from glucose in L. oenos was shown to involve the isomerization of glucose 6-phosphate to fructose 6-phosphate by a phosphoglucose isomerase, the cleavage of fructose 6-phosphate by a phosphoketolase, the reduction of erythrose 4-phosphate by an erythritol 4-phosphate dehydrogenase and, finally, the hydrolysis of erythritol 4-phosphate to erythritol by a phosphatase. Fructose 6-phosphate phosphoketolase was copurified with xylulose 5-phosphate phosphoketolase, and the activity of the latter was competitively inhibited by fructose 6-phosphate, with a Ki of 26 mM, corresponding to the Km of fructose 6-phosphate phosphoketolase (22 mM). These results suggest that the two phosphoketolase activities are borne by a single enzyme. Extracts of L. oenos were also found to contain NAD(P)H oxidase, which must be largely responsible for the reoxidation of NADPH and NADH in cells incubated in the presence of O2. In cells incubated with glucose, the concentrations of glucose 6-phosphate and of fructose 6-phosphate were higher in the absence of O2 than in its presence, explaining the stimulation by anaerobiosis of erythritol production. The increase in the hexose 6-phosphate concentration is presumably the result of a functional inhibition of glucose 6-phosphate dehydrogenase because of a reduction in the availability of NADP.     

Replacement of a phenylalanine by a tyrosine in the active site confers fructose-6-phosphate aldolase activity to the transaldolase of Escherichia coli and human origin

Based on a structure-assisted sequence alignment we designed 11 focused libraries at residues in the active site of transaldolase B from Escherichia coli and screened them for their ability to synthesize fructose 6-phosphate from dihydroxyacetone and glyceraldehyde 3-phosphate using a newly developed color assay. We found one positive variant exhibiting a replacement of Phe(178) to Tyr. This mutant variant is able not only to transfer a dihydroxyacetone moiety from a ketose donor, fructose 6-phosphate, onto an aldehyde acceptor, erythrose 4-phosphate (14 units/mg), but to use it as a substrate directly in an aldolase reaction (7 units/mg). With a single amino acid replacement the fructose-6-phosphate aldolase activity was increased considerably (>70-fold compared with wild-type). Structural studies of the wild-type and mutant protein suggest that this is due to a different H-bond pattern in the active site leading to a destabilization of the Schiff base intermediate. Furthermore, we show that a homologous replacement has a similar effect in the human transaldolase Taldo1 (aldolase activity, 14 units/mg). We also demonstrate that both enzymes TalB and Taldo1 are recognized by the same polyclonal antibody.     

Fructose-6-phosphate aldolase is a novel class I aldolase from Escherichia coli and is related to a novel group of bacterial transaldolases

We have cloned an open reading frame from the Escherichia coli K-12 chromosome that had been assumed earlier to be a transaldolase or a transaldolase-related protein, termed MipB. Here we show that instead a novel enzyme activity, fructose-6-phosphate aldolase, is encoded by this open reading frame, which is the first report of an enzyme that catalyzes an aldol cleavage of fructose 6-phosphate from any organism. We propose the name FSA (for fructose-six phosphate aldolase; gene name fsa). The recombinant protein was purified to apparent homogeneity by anion exchange and gel permeation chromatography with a yield of 40 mg of protein from 1 liter of culture. By using electrospray tandem mass spectroscopy, a molecular weight of 22,998 per subunit was determined. From gel filtration a size of 257,000 (+/- 20,000) was calculated. The enzyme most likely forms either a decamer or dodecamer of identical subunits. The purified enzyme displayed a V(max) of 7 units mg(-)1 of protein for fructose 6-phosphate cleavage (at 30 degrees C, pH 8.5 in 50 mm glycylglycine buffer). For the aldolization reaction a V(max) of 45 units mg(-)1 of protein was found; K(m) values for the substrates were 9 mm for fructose 6-phosphate, 35 mm for dihydroxyacetone, and 0.8 mm for glyceraldehyde 3-phosphate. FSA did not utilize fructose, fructose 1-phosphate, fructose 1,6-bisphosphate, or dihydroxyacetone phosphate. FSA is not inhibited by EDTA which points to a metal-independent mode of action. The lysine 85 residue is essential for its action as its exchange to arginine (K85R) resulted in complete loss of activity in line with the assumption that the reaction mechanism involves a Schiff base formation through this lysine residue (class I aldolase). Another fsa-related gene, talC of Escherichia coli, was shown to also encode fructose-6-phosphate aldolase activity and not a transaldolase as proposed earlier.     

Physiology of polyol formation by free and immobilized cells of the osmotolerant yeast Pichia farinosa

Summary Some physiological data of cells of Pichia farinosa immobilized on sintered glass Raschig rings were compared with data from free cells. Glucose consumption and productivity of total polyols (arabitol, glycerol and erythritol) showed a simultaneous inter-lag phase. Enzymes that catalyse steps of the pentosephosphate pathway (glucose-6-phosphate dehydrogenase, 6-phosphogluconate dehydrogenase, transaldolase and polyol dehydrogenase) showed a distinct increase after transfer of the cells into production medium. The activity of glycerol-3-phosphate dehydrogenase was generally low. Only alcohol dehydrogenase presented the inter-lag phase mentioned above.Offprint requests to: H.-J. Rehm     

Substrate-dependent inactivation of transaldolase activity with tetranitromethane

Transaldolase (Type III) from Candida utilis was found to be inactivated by tetranitromethane only in the presence of the substrates fructose 6-phosphate and sedoheptulose 7-phosphate. This reaction was prevented by the addition of erythrose 4-phosphate or glyceraldehyde 3-phosphate, which are known to accept dihydroxyacetone from the transaldolase-dihydroxyacetone complex, releasing free transaldolase. These results strongly suggest that tetranitromethane does not react with free transaldolase but only with the Schiff-base intermediate. After 1 min of incubation with the reagent at pH 6.0, 4 moles of nitroformate were produced per mole of inactivated enzyme. The modification, probably a nitration or an oxidation of certain amino acid residues of the complex by tetranitromethane, caused a dissociation of the dihydroxyacetone moiety from the complex without any recovery of the enzymatic activity. The fact that the reaction with tetranitromethane takes place only in the presence of substrates indicates that a substrate-mediated change of conformation occurs in transaldolase. Chemical and spectrophotometric evidence is presented showing that tetranitromethane did not modify tyrosine, cysteine, and tryptophan residues in the inactivated enzyme. From amino acid analyses it appears that histidine, serine, proline, methionine, tyrosine, and phenylalanine residues were not altered by this reagent. The possible mechanisms of modification of the transaldolasedihydroxyacetone complex and the chemical nature of the modification by tetranitromethane are discussed.     

Studies on Glucose-Isomerizing Enzyme of Bacteria

d-Glucose-isomerizing enzyme from Escherichia intermedia HN-500, which converts d-glucose to d-fructose in the presence of arsenate, was purified by treating with manganous sulfate, rivanol, and DEAE-Sephadex column chromatography. About 180-fold purified enzyme preparation was obtained by the above procedures. The purified preparation was free from the activities of d-glucose-, d-galactose-, glucose-6-phosphate-, mannitol-, and sorbitol-dehydrogenases and was homogeneous on polyacrylamide gel in zone electrophoresis. Optima of pH and temperature for the enzyme were found to be pH 7.0 and 50°C, respectively. The enzyme was completely inactivated by heating at 60°C for ten minutes and stable in the pH range of 7.0~9.0 at 30°C. Activation energy for the isomerizing enzyme was calculated to be 15,300 calories per mole degree from Arrhenius' equation. Either in the absence or presecne of arsenate, d-mannose, d-xylose, d-mannitol and d-sorbitol could not be isomerized by the purified enzyme at all, but the present enzyme isomerized exclusively glucose-6-phosphate and fructose-6-phosphate in the absence of arsenate.     

Studies on Chrysanthemic Acid

Ribose-5-phosphate ketol-isomerase, an enzyme isomerizing ribose-5-phosphate to ribulose-5-phosphate, is isolated from Candida utilis which is grown in a medium containing xylose. The enzyme is also purified by means of fractionation with ammonium sulfate, acetone, and by DEAE-cellulose column chromatography.

The enzyme has its optimum pH at 7.5 and optimum temperature at 50°C.

Michaelis-Menten constant for d-ribose-5-phosphate is 7.38 × 10^?4 M and activation energy of the enzyme reaction is 10,525 calories.

The enzyme activity is inhibited by p-CMB, EDTA and sodium pyrophosphate, and activated by the addition of magnesium ion.

Extract of Candida utilis contains polyol: NAD oxidoreductase which catalyzes the conversion of polyols to the corresponding ketoses.

By fractionation with ammonium sulfate and on DEAE-cellulose column chromatography, the purity of enzyme has been increased about 14-fold.

The relatively high activity with both xylitol and sorbitol suggests that they may be the natural substances for the enzyme.

Evidence suggests that this enzyme relates to the metabolism of d-xylose in Candida utilis.     

Evaluation of acceptor selectivity of Lactococcus lactis ssp. lactis trehalose 6-phosphate phosphorylase in the reverse phosphorolysis and synthesis of a new sugar phosphate

Trehalose 6-phosphate phosphorylase (TrePP), a member of glycoside hydrolase family 65, catalyzes the reversible phosphorolysis of trehalose 6-phosphate (Tre6P) with inversion of the anomeric configuration to produce β-d-glucose 1-phosphate (β-Glc1P) and d-glucose 6-phosphate (Glc6P). TrePP in Lactococcus lactis ssp. lactis (LlTrePP) is, alongside the phosphotransferase system, involved in the metabolism of trehalose. In this study, recombinant LlTrePP was produced and characterized. It showed its highest reverse phosphorolytic activity at pH 4.8 and 40°C, and was stable in the pH range 5.0–8.0 and at up to 30°C. Kinetic analyses indicated that reverse phosphorolysis of Tre6P proceeded through a sequential bi bi mechanism involving the formation of a ternary complex of the enzyme, β-Glc1P, and Glc6P. Suitable acceptor substrates were Glc6P, and, at a low level, d-mannose 6-phosphate (Man6P). From β-Glc1P and Man6P, a novel sugar phosphate, α-d-Glcp-(1?1)-α-d-Manp6P, was synthesized with 51% yield.     

d-Sedoheptulose-7-phosphate: d-Glyceraldehyde-3-phosphate Glycolaldehydetransferase and d-Ribulose-5-phosphate 3-Epimerase Mutants of a Bacillus Species

Determination of enzyme activities on the non-oxidative section of the pentose phosphate pathway in d-ribose-forming mutants of a Bacillus species revealed that two strains, which were isolated as shikimic acid-requiring mutants, lacked d-sedoheptulose-7-phosphate: d-glyceraldehyde glycolaldehydetransferase (EC 2.2.1.1) and one strain, which was isolated as d-gluconate-non-utilizing mutant, lacked d-ribulose-5-phosphate 3-epimerase (EC 5.1.3.1). These three strains were also found to have a kind of pleiotropic property, hardly growing on d-glucose.     

Inhibition of Nicotinamide Phosphoribosyltransferase (NAMPT), an Enzyme Essential for NAD+ Biosynthesis,Leads to Altered Carbohydrate Metabolism in Cancer Cells

Nicotinamide phosphoribosyltransferase (NAMPT) has been extensively studied due to its essential role in NAD⁺ biosynthesis in cancer cells and the prospect of developing novel therapeutics. To understand how NAMPT regulates cellular metabolism, we have shown that the treatment with FK866, a specific NAMPT inhibitor, leads to attenuation of glycolysis by blocking the glyceraldehyde 3-phosphate dehydrogenase step (Tan, B., Young, D. A., Lu, Z. H., Wang, T., Meier, T. I., Shepard, R. L., Roth, K., Zhai, Y., Huss, K., Kuo, M. S., Gillig, J., Parthasarathy, S., Burkholder, T. P., Smith, M. C., Geeganage, S., and Zhao, G. (2013) Pharmacological inhibition of nicotinamide phosphoribosyltransferase (NAMPT), an enzyme essential for NAD⁺ biosynthesis, in human cancer cells: metabolic basis and potential clinical implications. J. Biol. Chem. 288, 3500–3511). Due to technical limitations, we failed to separate isotopomers of phosphorylated sugars. In this study, we developed an enabling LC-MS methodology. Using this, we confirmed the previous findings and also showed that NAMPT inhibition led to accumulation of fructose 1-phosphate and sedoheptulose 1-phosphate but not glucose 6-phosphate, fructose 6-phosphate, and sedoheptulose 7-phosphate as previously thought. To investigate the metabolic basis of the metabolite formation, we carried out biochemical and cellular studies and established the following. First, glucose-labeling studies indicated that fructose 1-phosphate was derived from dihydroxyacetone phosphate and glyceraldehyde, and sedoheptulose 1-phosphate was derived from dihydroxyacetone phosphate and erythrose via an aldolase reaction. Second, biochemical studies showed that aldolase indeed catalyzed these reactions. Third, glyceraldehyde- and erythrose-labeling studies showed increased incorporation of corresponding labels into fructose 1-phosphate and sedoheptulose 1-phosphate in FK866-treated cells. Fourth, NAMPT inhibition led to increased glyceraldehyde and erythrose levels in the cell. Finally, glucose-labeling studies showed accumulated fructose 1,6-bisphosphate in FK866-treated cells mainly derived from dihydroxyacetone phosphate and glyceraldehyde 3-phosphate. Taken together, this study shows that NAMPT inhibition leads to attenuation of glycolysis, resulting in further perturbation of carbohydrate metabolism in cancer cells. The potential clinical implications of these findings are also discussed.     

Simple Procedures for the Preparation of d-Ribose-5-phosphate Ketol-Isomerase,d-Ribulose-5-phosphate 3-Epimerase and d-Sedoheptulose-7-phosphate: d-Glyceraldehyde-3-phosphate Glycolaldehydetransferase

d-Ribose-5-phophate ketol-isomerase (EC 5.3.1,6), d-ribuIose-5-phosphate 3-epimerase (EC 5.1.3.1) and d-sedoheptulose-7-phosphate: d-gIyceraldehyde-3-phosphate glycolaldehyde-transferase (EC 2.2.1,1) have been partially purified. d-Ribose-5-phosphate ketol-isomerase was purified from spinach by column chromatography with DEAE-cellulose and DEAE-Sephadex A-50; d-ribulose-5-phosphate 3-epimerase was purified from baker’s yeast by column chromatography with DEAE-cellulose; and d-sedoheptulose-7-phosphate: d-glyceraldehyde-3-phosphate glycolaldehydetransferase was purified from a Bacillus species No. 102 mutant G3–46–22–6 by column chromatography with DEAE-cellulose. The preparations were used for the determination of the activities of these enzymes in the parent and d-ribose-forming mutants of a Bacillus species.