The Isolation and Characterization of d-Glucose 6-Phosphate Cycloaldolase (NAD-Dependent) from Acer pseudoplatanus L. Cell Cultures: Its Occurrence in Plants

A soluble enzyme system from suspension cultures of Acer pseudoplatanus L. converts d-glucose 6-phosphate to myoinositol. A Mg²⁺-dependent phosphatase, present in the crude extract, hydrolyzes the product of the cyclization, myoinositol monophosphate, to free myoinositol. Further purification of the enzyme system by precipitation with (NH₄)₂SO₄ followed by diethylaminoethyl cellulose chromatography eliminates the phosphatase and makes it necessary to add alkaline phosphatase to the reaction mixture in order to assay for free myoinositol. Gel filtration on Sephadex G-200 increases the specific activity of the cycloaldolase to 8.8 × 10⁻⁴ units per milligram protein (1 unit = 1 micromole of myoinositol formed per minute). The cycloaldolase has an absolute requirement for nicotinamide adenine dinucleotide and a maximum activity at pH 8 with 0.1 mm nicotinamide adenine dinucleotide. The reaction rate is linear for 2.5 hours when d-glucose 6-phosphate is below 4 mm and has a K_m of 1.77 mm. The diethylaminoethyl cellulose-purified enzyme is stable for 6 to 8 weeks in the frozen state.     

Partial Purification and Properties of l-Glutamine d-Fructose 6-Phosphate Amidotransferase from Phaseolus aureus

l-Glutamine d-fructose 6-phosphate amidotransferase (EC 2.6.1.16) was extracted and purified 600-fold by acetone fractionation and diethylaminoethyl cellulose column chromatography from mung bean seeds (Phaseolus aureus). The partially purified enzyme was highly specific for l-glutamine as an amide nitrogen donor, and l-asparagine could not replace it. The enzyme showed a pH optimum in the range of 6.2 to 6.7 in phosphate buffer. Km values of 3.8 mm and 0.5 mm were obtained for d-fructose 6-phosphate and l-glutamine, respectively. The enzyme was competitively inhibited with respect to d-fructose 6-phosphate by uridine diphosphate-N-acetyl-d-glucosamine which had a Ki value of 13 μm. Upon removal of l-glutamine and its replacement by d-fructose 6-phosphate and storage over liquid nitrogen, the enzyme was completely desensitized to inhibition by uridine diphosphate-N-acetyl-d-glucosamine. This indicates that the inhibitor site is distinct from the catalytic site and that uridine diphosphate-N-acetyl-d-glucosamine acts as a feedback inhibitor of the enzyme.     

myo-Inositol-1-Phosphatase from the Pollen of Lilium longiflorum Thunb

A Mg²⁺-dependent, alkaline phosphatase has been isolated from mature pollen of Lilium longiflorum Thunb., cv. Ace and partially purified. It hydrolyzes 1l- and 1d-myo-inositol 1-phosphate, myo-inositol 2-phosphate, and β-glycerophosphate at rates decreasing in the order named. The affinity of the enzyme for 1l- and 1d-myo-inositol 1-phosphate is approximately 10-fold greater than its affinity for myo-inositol 2-phosphate. Little or no activity is found with phytate, d-glucose 6-phosphate, d-glucose 1-phosphate, d-fructose 1-phosphate, d-fructose 6-phosphate, d-mannose 6-phosphate, or p-nitrophenyl phosphate. 3-Phosphosphoglycerate is a weak competitive inhibitor. myo-Inositol does not inhibit the reaction. Optimal activity is obtained at pH 8.5 and requires the presence of Mg²⁺. At 4 millimolar, Co²⁺, Fe²⁺ or Mn²⁺ are less effective. Substantial inhibition is obtained with 0.25 molar Li⁺. With β-glycerophosphate as substrate the K_m is 0.06 millimolar and the reaction remains linear at least 2 hours. In 0.1 molar Tris, β-glycerophosphate yields equivalent amounts of glycerol and inorganic phosphate, evidence that transphosphorylation does not occur.     

Partial Purification and Properties of d-Glucosamine 6-Phosphate N-Acetyltransferase from Phaseolus aureus

d-Glucosamine-6-P N-acetyltransferase (EC 2.3.1.4) from mung bean seeds (Phaseolus aureus) was purified 313-fold by protamine sulfate and isoelectric precipitation, ammonium sulfate and acetone fractionation, and CM Sephadex column chromatography. The partially purified enzyme was highly specific for d-glucosamine-6-P. Neither d-glucosamine nor d-galactosamine could replace this substrate. The partially purified enzyme preparation was inhibited up to 50% by 2 × 10⁻²m EDTA, indicating the requirement of a divalent cation. Among divalent metal ions tested, Mg²⁺ was required for maximum activity of the enzyme. Mn²⁺ and Zn²⁺ were inhibitory, while Co²⁺ had no effect on the enzyme activity. The pH optimum of the enzyme in sodium acetate and sodium citrate buffers was found to be 5.2. The effect of Mg²⁺ on the enzyme in sodium acetate and sodium citrate buffers was particularly noticeable in the range of optimum pH. Km values of 15.1 × 10⁻⁴m and 7.1 × 10⁻⁴m were obtained for d-glucosamine-6-P and acetyl CoA, respectively. The enzyme was completely inhibited by 1 × 10⁻⁴mp-hydroxymercuribenzoate, and this inhibition was partially reversed by l-cysteine; indicating the presence of sulfhydryl groups at or near the active site of the enzyme.     

Nonreversible d-Glyceraldehyde 3-Phosphate Dehydrogenase of Plant Tissues

Preparations of TPN-linked nonreversible d-glyceraldehyde 3-phosphate dehydrogenase (EC 1.2.1.9), free of TPN-linked reversible d-glyceraldehyde 3-phosphate dehydrogenase, have been obtained from green shoots, etiolated shoots, and cotyledons of pea (Pisum sativum), cotyledons of peanut (Arachis hypogea), and leaves of maize (Zea mays). The properties of the enzyme were similar from each of these sources: the Km values for d-glyceraldehyde 3-phosphate and TPN were about 20 μm and 3 μm, respectively. The enzyme activity was inhibited by l-glyceraldehyde 3-phosphate, d-erythrose 4-phosphate, and phosphohydroxypyruvate. Activity was found predominantly in photosynthetic and gluconeogenic tissues of higher plants. A light-induced, phytochrome-mediated increase of enzyme activity in a photosynthetic tissue (pea shoots) was demonstrated. Appearance of enzyme activity in a gluconeogenic tissue (endosperm of castor bean, Ricinus communis) coincided with the conversion of fat to carbohydrate during germination. In photosynthetic tissue, the enzyme is located outside the chloroplast, and at in vivo levels of triose-phosphates and pyridine nucleotides, the activity is probably greater than that of DPN-linked reversible d-glyceraldehyde 3-phosphate dehydrogenase. Several possible roles for the enzyme in plant carbohydrate metabolism are considered.     

The incorporation of labelled amino sugars by Bacillus subtilis

1. Glucosamine 6-phosphate deaminase [2-amino-2-deoxy-d-glucose 6-phosphate ketol-isomerase (deaminating), EC 5.3.1.10] of Bacillus subtilis has been partially purified. Its K_m is 3·0mm. 2. Extracts of B. subtilis contain N-acetylglucosamine 6-phosphate deacetylase (K_m 1·4mm), glucosamine 1-phosphate acetylase and amino sugar kinases (EC 2.7.1.8 and 2.7.1.9). 3. Glucosamine 6-phosphate synthetase (l-glutamine–d-fructose 6-phosphate aminotransferase, EC 2.6.1.16) is repressed by growth of B. subtilis in the presence of glucosamine, N-acetylglucosamine, N-propionylglucosamine or N-formylglucosamine. Glucosamine 6-phosphate deaminase and N-acetylglucosamine 6-phosphate deacetylase are induced by N-acetylglucosamine. Amino sugar kinases are induced by glucose, glucosamine and N-acetylglucosamine. The synthesis of glucosamine 1-phosphate acetylase is unaffected by amino sugars. 4. Glucose in the growth medium prevents the induction of glucosamine 6-phosphate deaminase and of N-acetylglucosamine 6-phosphate deacetylase caused by N-acetylglucosamine; glucose also alleviates the repression of glucosamine 6-phosphate synthetase caused by amino sugars. 5. Glucosamine 6-phosphate deaminase increases in bacteria incubated beyond the exponential phase of growth. This increase is prevented by glucose.     

Deoxycytidylate deaminase. Properties of the enzyme from cultured kidney cells of baby hamster

dCMP deaminase was partially purified from BHK-21/C13 cells grown in culture. The molecular weight of the enzyme was estimated by gel filtration and gradient centrifugation to be 130000 and 115000 respectively. The enzyme had a pH optimum of 8.4. Its activity versus substrate concentration curve was sigmoid, the substrate concentration at half-maximal velocity being 4.4mm. dCTP activated the deaminase maximally at 40μm, gave a hyperbolic curve for activity versus dCMP concentration and a K_m value for dCMP of 0.91mm. dCTP activation required the presence of Mg²⁺ or Mn²⁺ ions. dTTP inhibited the deaminase maximally at 15μm; the inhibition required the presence of Mg²⁺ or Mn²⁺ ions. The enzyme was very heat-labile but could be markedly stabilized by dCTP at 0.125mm and ethylene glycol at 20% (v/v).     

Further studies on the regulation of amino sugar metabolism in Bacillus subtilis

1. Glucosamine 6-phosphate deaminase [2-amino-2-deoxy-d-glucose 6-phosphate ketol-isomerase (deaminating), EC 5.3.1.10] of Bacillus subtilis has been partially purified. Its K_m is 3·0mm. 2. Extracts of B. subtilis contain N-acetylglucosamine 6-phosphate deacetylase (K_m 1·4mm), glucosamine 1-phosphate acetylase and amino sugar kinases (EC 2.7.1.8 and 2.7.1.9). 3. Glucosamine 6-phosphate synthetase (l-glutamine–d-fructose 6-phosphate aminotransferase, EC 2.6.1.16) is repressed by growth of B. subtilis in the presence of glucosamine, N-acetylglucosamine, N-propionylglucosamine or N-formylglucosamine. Glucosamine 6-phosphate deaminase and N-acetylglucosamine 6-phosphate deacetylase are induced by N-acetylglucosamine. Amino sugar kinases are induced by glucose, glucosamine and N-acetylglucosamine. The synthesis of glucosamine 1-phosphate acetylase is unaffected by amino sugars. 4. Glucose in the growth medium prevents the induction of glucosamine 6-phosphate deaminase and of N-acetylglucosamine 6-phosphate deacetylase caused by N-acetylglucosamine; glucose also alleviates the repression of glucosamine 6-phosphate synthetase caused by amino sugars. 5. Glucosamine 6-phosphate deaminase increases in bacteria incubated beyond the exponential phase of growth. This increase is prevented by glucose.     

Stable changes to calcium fluxes in mitochondria isolated from rat livers perfused with α-adrenergic agonists and with glucagon

1. Human uterine cervical stroma was found to contain a Ca²⁺-independent neutral proteinase against casein and N-benzoyl-dl-arginine p-nitroanilide (Bz-dl-Arg-Nan). This enzyme was tightly bound to an insoluble material (20000g pellet) and was solubilized by high concentrations of NaCl or KCl. High concentrations of them in the reaction system, however, inhibited reversibly the activity of this enzyme. 2. The neutral proteinase was partially purified by extraction with NaCl, gel filtration on Sephadex G-200 and affinity chromatography on casein–Sepharose. 3. The optimal pH of this partially purified enzyme was 7.4–8.0 against casein and Bz-dl-Arg-Nan. The molecular weight of the enzyme was found to be about 1.4×10⁵ by gel filtration on Sephadex G-200. 4. The enzyme was significantly inhibited by di-isopropyl phosphorofluoridate (0.1mm). High concentration of phenylmethanesulphonyl fluoride (5mm), 7-amino-1-chloro-3-l-tosylamidoheptan-2-one (0.5mm), antipain (10μm) or leupeptin (10μm) was also found to be inhibitory, but chymostatin (40μg/ml), soya-bean trypsin inhibitor (2.5mg/ml), human plasma (10%, v/v), p-chloromercuribenzoate (1mm), EDTA (10mm) and 1-chloro-4-phenyl-3-l-tosylamidobutan-2-one (1mm) had no effect on the enzyme. 5. The neutral proteinase hydrolysed casein, Bz-dl-Arg-Nan and heat-denatured collagen, but was inactive towards native collagen and several synthetic substrates, such as 4-phenylazobenzyloxycarbonyl-Pro-Leu-Gly-Pro-d-Arg, 3-carboxypropionyl-Ala-Ala-Ala p-nitroanilide and 2,4-dinitrophenyl-Pro-Gln-Gly-Ile-Ala-Gly-Gln-d-Arg, and also proteoglycan. The enzyme did not act as a plasminogen activator. 6. These properties suggested that a neutral proteinase in the human uterine cervix was different from enzymes previously reported.     

Further Studies on myo-Inositol-1-phosphatase from the Pollen of Lilium longiflorum Thunb

myo-Inositol-1-phosphatase has been purified to homogeneity from Lilium longiflorum pollen using an alternative procedure which includes pH change and phenyl Sepharose column chromatography. Sodium dodecyl sulfate-polyacrylamide gel electrophoretic analysis shows that the enzyme is a dimer (subunit molecular weight, 29,000 daltons). The enzyme is stable at low pH values and is inactivated only below pH 3.0. In addition to 1l-and 1d-myo-inositol-1-phosphate, it shows high specificity for 1l-chiro-inositol-3-phosphate. As observed earlier with other primary phosphate esters, d-glucitol-6-phosphate and d-mannitol-6-phosphate are hydrolyzed very slowly. No activity is observed with inorganic pyrophosphate or myo-inositol pentaphosphate as substrate. The enzyme is inhibited by fluoride, sulfate, molybdate, and thiol-directed reagents. Partial protection against N-ethylmaleimide inhibition by substrate and Mg²⁺ together suggests sulfhydryl involvement at the active site.     

Identification,Purification, and Characterization of a Novel Amino Acid Racemase,Isoleucine 2-Epimerase,from Lactobacillus Species

Accumulation of d-leucine, d-allo-isoleucine, and d-valine was observed in the growth medium of a lactic acid bacterium, Lactobacillus otakiensis JCM 15040, and the racemase responsible was purified from the cells and identified. The N-terminal amino acid sequence of the purified enzyme was GKLDKASKLI, which is consistent with that of a putative γ-aminobutyrate aminotransferase from Lactobacillus buchneri. The putative γ-aminobutyrate aminotransferase gene from L. buchneri JCM 1115 was expressed in recombinant Escherichia coli and then purified to homogeneity. The enzyme catalyzed the racemization of a broad spectrum of nonpolar amino acids. In particular, it catalyzed at high rates the epimerization of l-isoleucine to d-allo-isoleucine and d-allo-isoleucine to l-isoleucine. In contrast, the enzyme showed no γ-aminobutyrate aminotransferase activity. The relative molecular masses of the subunit and native enzyme were estimated to be about 49 kDa and 200 kDa, respectively, indicating that the enzyme was composed of four subunits of equal molecular masses. The K_m and V_max values of the enzyme for l-isoleucine were 5.00 mM and 153 μmol·min⁻¹·mg⁻¹, respectively, and those for d-allo-isoleucine were 13.2 mM and 286 μmol·min⁻¹·mg⁻¹, respectively. Hydroxylamine and other inhibitors of pyridoxal 5′-phosphate-dependent enzymes completely blocked the enzyme activity, indicating the enzyme requires pyridoxal 5′-phosphate as a coenzyme. This is the first evidence of an amino acid racemase that specifically catalyzes racemization of nonpolar amino acids at the C-2 position.     

Substrate specificity of the Bacillus licheniformis lyxose isomerase YdaE and its application in in vitro catalysis for bioproduction of lyxose and glucose by two-step isomerization

Enzymatic processes are useful for industrially important sugar production, and in vitro two-step isomerization has proven to be an efficient process in utilizing readily available sugar sources. A hypothetical uncharacterized protein encoded by ydaE of Bacillus licheniformis was found to have broad substrate specificities and has shown high catalytic efficiency on d-lyxose, suggesting that the enzyme is d-lyxose isomerase. Escherichia coli BL21 expressing the recombinant protein, of 19.5 kDa, showed higher activity at 40 to 45°C and pH 7.5 to 8.0 in the presence of 1.0 mM Mn²⁺. The apparent K_m values for d-lyxose and d-mannose were 30.4 ± 0.7 mM and 26 ± 0.8 mM, respectively. The catalytic efficiency (k_cat/K_m) for lyxose (3.2 ± 0.1 mM⁻¹ s⁻¹) was higher than that for d-mannose (1.6 mM⁻¹ s⁻¹). The purified protein was applied to the bioproduction of d-lyxose and d-glucose from d-xylose and d-mannose, respectively, along with the thermostable xylose isomerase of Thermus thermophilus HB08. From an initial concentration of 10 mM d-lyxose and d-mannose, 3.7 mM and 3.8 mM d-lyxose and d-glucose, respectively, were produced by two-step isomerization. This two-step isomerization is an easy method for in vitro catalysis and can be applied to industrial production.     

Serine Transhydroxymethylase of Cauliflower (Brassica oleracea var. botrytis L.): Partial Purification and Properties

Serine transhydroxymethylase (EC 2.1.2.1) has been purified 46-fold from cauliflower (Brassica oleracea var. botrytis L.). The enzyme was completely dependent on the presence of tetrahydrofolic acid for the conversion of serine to glycine. The addition of pyridoxal phosphate gave a large increase in the reaction rate. A double pH optimum was observed with maxima at 7.5 and 9.5. The enzyme is specific for l-serine. The d-isomer is neither a substrate nor an inhibitor. The Michaelis constants for l-serine, tetrahydrofolic acid, and pyridoxal phosphate were 300 μm, 760 μm, and 24 μm, respectively. The addition of K⁺ also stimulated the reaction rate considerably. The effect was quite specific since all other metal ions tested either had very little: influence or were extremely inhibitory.     

VTC4 Is a Bifunctional Enzyme That Affects Myoinositol and Ascorbate Biosynthesis in Plants

Myoinositol synthesis and catabolism are crucial in many multiceullar eukaryotes for the production of phosphatidylinositol signaling molecules, glycerophosphoinositide membrane anchors, cell wall pectic noncellulosic polysaccharides, and several other molecules including ascorbate. Myoinositol monophosphatase (IMP) is a major enzyme required for the synthesis of myoinositol and the breakdown of myoinositol (1,4,5)trisphosphate, a potent second messenger involved in many biological activities. It has been shown that the VTC4 enzyme from kiwifruit (Actinidia deliciosa) has similarity to IMP and can hydrolyze l-galactose 1-phosphate (l-Gal 1-P), suggesting that this enzyme may be bifunctional and linked with two potential pathways of plant ascorbate synthesis. We describe here the kinetic comparison of the Arabidopsis (Arabidopsis thaliana) recombinant VTC4 with d-myoinositol 3-phosphate (d-Ins 3-P) and l-Gal 1-P. Purified VTC4 has only a small difference in the V_max/K_m for l-Gal 1-P as compared with d-Ins 3-P and can utilize other related substrates. Inhibition by either Ca²⁺ or Li⁺, known to disrupt cell signaling, was the same with both l-Gal 1-P and d-Ins 3-P. To determine whether the VTC4 gene impacts myoinositol synthesis in Arabidopsis, we isolated T-DNA knockout lines of VTC4 that exhibit small perturbations in abscisic acid, salt, and cold responses. Analysis of metabolite levels in vtc4 mutants showed that less myoinositol and ascorbate accumulate in these mutants. Therefore, VTC4 is a bifunctional enzyme that impacts both myoinositol and ascorbate synthesis pathways.Myoinositol is a six-member carbon ring polyol that is synthesized by both eukaryotes and prokaryotes (for review, see Michell, 2007). In multiceullar eukaryotes, myoinositol becomes incorporated into many crucial cellular compounds, including those involved in signal transduction such as phosphatidylinositol phosphates and myoinositol phosphates (InsPs; for review, see Boss et al., 2006), gene expression (InsPs; for review, see Alcazar-Roman and Wente, 2007), auxin perception and phosphorus storage (myoinositol hexakisphosphate [InsP₆]; for review, see Raboy and Bowen, 2006; Tan et al., 2007), membrane tethering (glycerophosphoinositide anchors; for review, see Fujita and Jigami, 2007), stress tolerance (ononitol, pinitol; for review, see Taji et al., 2006), and oligosaccharide synthesis (galactinol; for review, see Karner et al., 2004; Fig. 1). Its primary breakdown product, d-GlcUA, is utilized for the synthesis of cell wall pectic noncellulosic compounds (for review, see Loewus, 2006) and, in some organisms, ascorbate (for review, see Linster and Van Schaftingen, 2007). Thus, myoinositol synthesis and catabolism affect metabolites involved in many different and critical biochemical pathways.Open in a separate windowFigure 1.Myoinositol synthesis and metabolism pathway. De novo synthesis of myoinositol (i.e. the Loewus pathway) is catalyzed by myoinositol phosphate synthase (MIPS) and IMP, where its immediate precursor is d-Ins 3-P = l-Ins 1-P. IMP also regenerates myoinositol from the second messenger d-Ins(1,4,5)P₃. Oxidation of inositol by myoinositol oxygenase (MIOX) produces d-GlcUA (d-GlcA), which is a possible entry point into ascorbate synthesis. The major route to ascorbate in plants is the Smirnoff-Wheeler pathway and utilizes GDP-d-Man. VTC4 has homology to the animal IMPs and has been shown to catalyze the conversion of l-Gal 1-P to l-Gal in the Smirnoff-Wheeler pathway. Inositol is also the precursor for the synthesis of several compounds indicated in gray. The asterisk indicates the inositol signaling pathway.Although organisms incorporate myoinositol into various compounds, there is only one biosynthetic route to produce myoinositol in what has been referred to as the Loewus pathway (Eisenberg et al., 1964; Chen and Charalampous, 1966; Sherman et al., 1969; Loewus and Loewus, 1980; Loewus et al., 1980). The conversion of Glc-6-P to InsP is catalyzed by the myoinositol phosphate synthase (EC 5.5.1.4; for review, see GhoshDastidar et al., 2006). The product of this reaction can be referred to as either l-myoinositol 1-P or d-myoinositol 3-P (d-Ins 3-P), which are equivalent compounds. The conversion of d-Ins 3-P to free myoinositol is catalyzed by the myoinositol monophosphatase (IMP; EC 3.1.3.25; for review, see Torabinejad and Gillaspy, 2006). We have been interested in the function of IMP in both de novo myoinositol synthesis and during myoinositol second messenger recycling from myoinositol phosphate signaling molecules, such as d-myoinositol 1-P (d-Ins 1-P; Fig. 1). IMP is encoded by multiple genes in plants (e.g. three IMP genes have been examined in tomato [Solanum lycopersicum]; Gillaspy et al., 1995). The three different tomato IMPs are highly conserved enzymes that act specifically on monophosphorylated substrates and are inhibited by LiCl (Gillaspy et al., 1995; Berdy et al., 2001). IMP gene expression is developmentally regulated, as is the accumulation of IMP proteins, with maximal levels being present in plant tissues undergoing rapid cell divisions, such as seedlings and developing anthers (Gillaspy et al., 1995; Suzuki et al., 2007).In contrast, Arabidopsis (Arabidopsis thaliana) contains one potential IMP gene (At3g02870), which was previously identified as functioning in ascorbate synthesis and named VTC4 (Laing et al., 2004; Conklin et al., 2006). Two other genes (At1g31190 and At4g39120) encode proteins that we have classified as IMP-like (IMPL), because of their greater homology to the prokaryotic IMPs, such as the SuhB (Matsuhisa et al., 1995; Chen and Roberts, 2000) and CysQ (Neuwald et al., 1992; Peng and Verma, 1995) proteins. Prokaryotic IMPLs are known to dephosphorylate d-Ins 1-P and other substrates in vitro; however, the function of these IMPL proteins is currently unknown (for review, see Roberts, 2006).Intriguing data suggest that animal IMP is a bifunctional enzyme. The animal IMP hydrolyzes d-Gal 1-P, which is involved in Gal metabolism (Parthasarathy et al., 1997). Furthermore, expression of human IMP can suppress Gal toxicity in yeast (Mehta et al., 1999). Efforts to isolate an l-Gal 1-P phosphatase required for ascorbate synthesis in plants revealed that the kiwi (Actinidia deliciosa) and Arabidopsis VTC4 can hydrolyze l-Gal 1-P (Laing et al., 2004). This fact prompted the proposal that VTC4 functions mainly to hydrolyze l-Gal 1-P during ascorbate synthesis and that other, unidentified enzymes might be responsible for de novo myoinositol synthesis in plants. This idea is supported by the fact that a vtc4 loss-of-function mutant contains lower ascorbate levels (Conklin et al., 2006).Since VTC4 and the IMPLs are the best candidates for enzymes with IMP activity, it is crucial to understand whether these enzymes impact myoinositol synthesis in plants in vivo. To determine whether VTC4 is bifunctional and functions during InsP hydrolysis as well as l-Gal 1-P hydrolysis, we expressed recombinant Arabidopsis VTC4 protein and compared the kinetic constants for both d-Ins 3-P and l-Gal 1-P. In contrast to previously reported results, we report here that VTC4 hydrolyzes both substrates well and thus should be considered a bifunctional enzyme. We investigated loss-of-function vtc4 mutant plants and confirm that these plants contain lower ascorbate levels. We also find reduced myoinositol levels in vtc4 mutants, supporting a direct role for VTC4 in InsP hydrolysis in plants.     

Nucleoside Diphosphate-sugar 4-Epimerases I. Uridine Diphosphate Glucose 4-Epimerase of Wheat Germ

Uridine diphosphate (UDP)-glucose 4-epimerase (EC 5.1.3.2) has been purified over 1000-fold from extracts of wheat germ by MnCl₂ treatment, (NH₄)₂SO₄ fractionation, Sephadex column chromatography, and adsorption onto and elution from calcium phosphate gel. The enzyme has a pH optimum of 9.0. Km values are 0.1 mm for UDP-d-galactose and 0.2 mm for UDP-d-glucose. NAD is required for activity; K_a = 0.04 mm. NADH is an inhibitor strictly competitive with NAD; K_i = 2 μm. Wheat germ also contains UDP-l-arabinose 4-epimerase (EC 5.1.3.5) and thymidine diphosphate (TDP)-glucose 4-epimerase which are distinct from UDP-glucose 4-epimerase.     

Importance of the pentose phosphate pathway for d-glucose catabolism in the obligatory aerobic yeast Rhodotorula gracilis

d-Glucose catabolism of a phosphofructokinase-deficient yeast Rhodotorula gracilis has been studied. By using d-glucose specifically ¹⁴C-labelled at different positions and measuring the distribution of the label in various fractions of cell metabolism, the following results were found. 1. The pentose phosphate pathway, being the main pathway of d-glucose catabolism, simultaneously converts glucose molecules into pentose phosphates oxidatively by using two NADP-linked dehydrogenases and via the non-oxidative transketolase–transaldolase pathway. 2. From the correlation of the ¹⁴CO₂ liberation and the d-glucose consumption and from the fact that the pentose phosphate moiety in nucleic acids is almost equally labelled from d-[1-¹⁴C]- and d-[6-¹⁴C]-glucose, it is concluded that of the glucose utilized about 80% undergoes transformation via the non-oxidative pentose phosphate pathway. Only about 20% of glucose is directly decarboxylated to pentose phosphate. 3. For further degradation it is postulated that the pentose phosphates are split into C₂ fragments and glyceraldehyde 3-phosphates. 4. All three loci of oxidative decarboxylation appear to be effective in Rh. gracilis, the oxidative part of the pentose phosphate pathway, the decarboxylation of pyruvate in the later part of the glycolytic pathway as well as the oxidation in the tricarboxylic acid cycle. 5. d-Glucose molecules taken up are only partially oxidized to CO₂: about four-fifths of each glucose molecule metabolized is incorporated into cell constituents. 6. The quantitative interrelations of the fluxes of d-glucose subunits along the catabolic pathways have been estimated and are discussed.     

Enzymes of glucose metabolism in normal mouse pancreatic islets

1. Glucose-phosphorylating and glucose 6-phosphatase activities, glucose 6-phosphate dehydrogenase, 6-phosphogluconate dehydrogenase, NADP⁺-linked isocitrate dehydrogenase, `malic' enzyme and pyruvate carboxylase were assayed in homogenates of normal mouse islets. 2. Two glucose-phosphorylating activities were detected; the major activity had K<sub>m</sub> 0.075mm for glucose and was inhibited by glucose 6-phosphate (non-competitive with glucose) and mannoheptulose (competitive with glucose). The other (minor) activity had a high K<sub>m</sub> for glucose (mean value 16mm) and was apparently not inhibited by glucose 6-phosphate. 3. Glucose 6-phosphatase activity was present in amounts comparable with the total glucose-phosphorylating activity, with K<sub>m</sub> 1mm for glucose 6-phosphate. Glucose was an inhibitor and the inhibition showed mixed kinetics. No inhibition of glucose 6-phosphate hydrolysis was observed with mannose, citrate or tolbutamide. The inhibition by glucose was not reversed by mannoheptulose. 4. 6-Phosphogluconate dehydrogenase had K<sub>m</sub> values of 2.5 and 21μm for NADP<sup>+</sup> and 6-phosphogluconate respectively. 5. Glucose 6-phosphate dehydrogenase had K<sub>m</sub> values of 4 and 22μm for NADP<sup>+</sup> and glucose 6-phosphate. The K<sub>m</sub> for glucose 6-phosphate was considerably below the intra-islet concentration of glucose 6-phosphate at physiological extracellular glucose concentrations. The enzyme had no apparent requirement for cations. Of a number of possible modifiers of glucose 6-phosphate dehydrogenase, only NADPH was inhibitory. The inhibition by NADPH was competitive with NADP<sup>+</sup> and apparently mixed with respect to glucose 6-phosphate. 6. NADP<sup>+</sup>–isocitrate dehydrogenase was present but the islet homogenate contained little, if any, `malic' enzyme. The presence of pyruvate carboxylase was also demonstrated. 7. The results obtained are discussed with reference to glucose phosphorylation and glucose 6-phosphate oxidation in the intact mouse islet, and the possible nature of the β-cell glucoreceptor mechanism.     

Accumulation of d-Glucose from Pentoses by Metabolically Engineered Escherichia coli

Escherichia coli that is unable to metabolize d-glucose (with knockouts in ptsG, manZ, and glk) accumulates a small amount of d-glucose (yield of about 0.01 g/g) during growth on the pentoses d-xylose or l-arabinose as a sole carbon source. Additional knockouts in the zwf and pfkA genes, encoding, respectively, d-glucose-6-phosphate 1-dehydrogenase and 6-phosphofructokinase I (E. coli MEC143), increased accumulation to greater than 1 g/liter d-glucose and 100 mg/liter d-mannose from 5 g/liter d-xylose or l-arabinose. Knockouts of other genes associated with interconversions of d-glucose-phosphates demonstrate that d-glucose is formed primarily by the dephosphorylation of d-glucose-6-phosphate. Under controlled batch conditions with 20 g/liter d-xylose, MEC143 generated 4.4 g/liter d-glucose and 0.6 g/liter d-mannose. The results establish a direct link between pentoses and hexoses and provide a novel strategy to increase carbon backbone length from five to six carbons by directing flux through the pentose phosphate pathway.     

Effectors of rat-heart hexokinases and the control of rates of glucose phosphorylation in the perfused rat heart

1. The kinetic properties of the soluble and particulate hexokinases from rat heart have been investigated. 2. For both forms of the enzyme, the K_m for glucose was 45μm and the K_m for ATP 0·5mm. Glucose 6-phosphate was a non-competitive inhibitor with respect to glucose (K_i 0·16mm for the soluble and 0·33mm for the particulate enzyme) and a mixed inhibitor with respect to ATP (K_i 80μm for the soluble and 40μm for the particulate enzyme). ADP and AMP were competitive inhibitors with respect to ATP (K_i for ADP was 0·68mm for the soluble and 0·60mm for the particulate enzyme; K_i for AMP was 0·37mm for the soluble and 0·16mm for the particulate enzyme). P_i reversed glucose 6-phosphate inhibition with both forms at 10mm but not at 2mm, with glucose 6-phosphate concentrations of 0·3mm or less for the soluble and 1mm or less for the particulate enzyme. 3. The total activity of hexokinase in normal hearts and in hearts from alloxan-diabetic rats was 21·5μmoles of glucose phosphorylated/min./g. dry wt. of ventricle at 25°. The temperature coefficient Q₁₀ between 22° and 38·5° was 1·93; the ratio of the soluble to the particulate enzyme was 3:7. 4. The kinetic data have been used to predict rates of glucose phosphorylation in the perfused heart at saturating concentrations of glucose from measured concentrations of ATP, glucose 6-phosphate, ADP and AMP. These have been compared with the rates of glucose phosphorylation measured with precision in a small-volume recirculation perfusion apparatus, which is described. The correlation between predicted and measured rates was highly significant and their ratio was 1·07. 5. These findings are consistent with the control of glucose phosphorylation in the perfused heart by glucose 6-phosphate concentration, subject to certain assumptions that are discussed in detail.     

Identification of an l-Arabinose Reductase Gene in Aspergillus niger and Its Role in l-Arabinose Catabolism

The first enzyme in the pathway for l-arabinose catabolism in eukaryotic microorganisms is a reductase, reducing l-arabinose to l-arabitol. The enzymes catalyzing this reduction are in general nonspecific and would also reduce d-xylose to xylitol, the first step in eukaryotic d-xylose catabolism. It is not clear whether microorganisms use different enzymes depending on the carbon source. Here we show that Aspergillus niger makes use of two different enzymes. We identified, cloned, and characterized an l-arabinose reductase, larA, that is different from the d-xylose reductase, xyrA. The larA is up-regulated on l-arabinose, while the xyrA is up-regulated on d-xylose. There is however an initial up-regulation of larA also on d-xylose but that fades away after about 4 h. The deletion of the larA gene in A. niger results in a slow growth phenotype on l-arabinose, whereas the growth on d-xylose is unaffected. The l-arabinose reductase can convert l-arabinose and d-xylose to their corresponding sugar alcohols but has a higher affinity for l-arabinose. The K_m for l-arabinose is 54 ± 6 mm and for d-xylose 155 ± 15 mm.