Purification and characterization of 3-deoxy-D-manno-octulosonate 8-phosphate synthetase from Escherichia coli

3-Deoxy-D-manno-octulosonate (KDO)-8-phosphate synthetase has been purified 450-fold from frozen Escherichia coli B cells. The purified enzyme catalyzed the stoichiometric formation of KDO-8-phosphate and Pi from phosphoenolpyruvate (PEP) and D-arabinose-5-phosphate. The enzyme showed no metal requirement for activity and was inhibited by 1 mM Cd2+, Cu2+, Zn2+, and Hg2+. The inhibition by Hg2+ could be reversed by dithiothreitol. The optimum temperature for enzyme activity was determined to be 45 degrees C, and the energy of activation calculated by the Arrhenius equation was 15,000 calories (ca. 3,585 J) per mol. The enzyme activity was shown to be pH and buffer dependent, showing two pH optima, one at pH 4.0 to 6.0 in succinate buffer and one at pH 9.0 in glycine buffer. The isoelectric point of the enzyme was 5.1. KDO-8-phosphate synthetase had a molecular weight of 90,000 +/- 6,000 as determined by molecular sieving through G-200 Sephadex and by Ferguson analysis using polyacrylamide gels. Based on sodium dodecyl sulfate-polyacrylamide gel electrophoresis, the 90,000-molecular-weight native enzyme was composed of three identical subunits, each with an apparent molecular weight of 32,000 +/- 4,000. The enzyme had an apparent Km for D-arabinose-5-phosphate of 2 X 10(-5) M and an apparent Km for PEP of 6 X 10(-6) M. No other sugar or sugar-phosphate could substitute for D-arabinose-5-phosphate. D-Ribose-5-phosphate was a competitive inhibitor of D-arabinose-5-phosphate, with an apparent Ki of 1 X 10(-3) M. The purified enzyme has been utilized to synthesize millimole quantities of pure KDO-8-phosphate.     

Biosynthesis of riboflavin. An aliphatic intermediate in the formation of 6,7-dimethyl-8-ribityllumazine from pentose phosphate

6,7-Dimethyl-8-ribityllumazine synthase deficient mutants of Candida guilliermondii were divided into two groups on the basis of in vitro complementation. Mutants of complementation group I produce an intermediate X from ribose 5-phosphate in a reaction requiring Mg++ ions. Compound X was partially purified and was shown to be a phosphoric acid ester. 6,7-Dimethyl-8-ribityllumazine can be formed from Compound X by cell extracts from mutants of complementation group II. The reaction requires 5-amino-6-ribitylamino-2,4(1H,3H)-pyrimidinedione or its 5'-phosphate as second substrate. No divalent cations are required.     

Characterization of an enzyme which catalyzes isomerization and epimerization of D-erythrose 4-phosphate

The enzyme which catalyzes the conversion of D-erythrose 4-phosphate to D-erythrulose 4-phosphate and D-threose 4-phosphate has been purified to homogeneity from a crude extract of beef liver. Analysis of the purified enzyme by Sephadex G-100 gel filtration and sodium dodecyl sulfate/polyacrylamide gel electrophoresis revealed it to be a dimer of relative molecular mass 43 000. From the gas chromatography/mas spectrometry analyses of the enzymatic reaction products, it appeared that about 90% of the total amount of tetrose 4-phosphate was present as D-erythrulose 4-phosphate after equilibration. The purified enzyme, which is tentatively called 'erythrose-4-phosphate isomerase' had no significant isomerase activities on D-glyceraldehyde 3-phosphate, D-ribose 5-phosphate, D-glucose 6-phosphate and D-fructose 6-phosphate, but a strong D-ribulose-5-phosphate 3-epimerase activity was co-purified with the erythrose-4-phosphate isomerase activity through every step in the isolation. Both the erythrose-4-phosphate isomerase and D-ribulose-5-phosphate 3-epimerase activities were inactivated at the same rate at the elevated temperature, and also inhibited to the same extent by various inhibitors. It is likely, that both activities are catalyzed by the single enzyme protein.     

Purification and characterization of glutamine:fructose 6-phosphate amidotransferase from rat liver

The enzyme glutamine:fructose 6-phosphate amidotransferase (L-glutamine:D-fructose-6-phosphate amidotransferase; EC 2.6.1.16, GFAT) catalyzes the formation of glucosamine 6-phosphate from fructose 6-phosphate and glutamine. In view of the important role of GFAT in the hexosamine biosynthetic pathway, we have purified the enzyme from rat liver and characterized its physicochemical properties in comparison to those from the published microbial enzymes. The purified enzyme has a molecular mass of about 75 kDa as determined by sodium dodecyl sulfate polyacrylamide gel electrophoresis. On a Sephacryl S-200 gel filtration column, the purified enzyme eluted in a single peak corresponding to a molecular mass of about 280 kDa, indicating that the active enzyme may be composed of four subunits. The N-terminal amino acid sequence of the purified enzyme was determined as X-G-I-F-A-Y-L-N-Y-H-X-P-R, where X indicates an unidentified residue. The K(M) values of the purified enzyme for fructose 6-phosphate and glutamine were 0.4 and 0.8 mM, respectively. The purified enzyme was inactivated by 4, 4'-dithiodipyridine, and the activity of the inactivated enzyme was restored by dithiothreitol. The inactivation followed pseudo first-order and saturation kinetics with the K(inact) of 5.0 microM. Kinetic studies also indicated that 4,4'-dithiodipyridine is a competitive inhibitor of the enzyme with respect to glutamine. Isolation and analysis of the cysteine-modified peptide indicated that Cys-1 was the modified site. Cys-1 has been suggested to play an important role in enzymatic activity of the Escherichia coli enzyme (M. N. Isupov, G. Obmolova, S. Butterworth, M. Badet-Denisot, B. Badet, I. Polikarpov, J. A. Littlechild, and A. Teplyakov, 1996, Structure 4, 801-810).     

Dehydroquinate synthase in Bacillus subtilis. An enzyme associated with chorismate synthase and flavin reductase.

Dehydroquinate synthase, the enzyme which catalyzes the conversion of 3-deoxy-D-arabino-heptulosonic acid 7-phosphate (DAHP) to 5-dehydroquinate, has been purified from Bacillus subtilis in association with chorismate synthase and NADPH-dependent flavin reductase. The enzyme was only active when associated with chorismate synthase, whereas the flavin reductase could be separated from the complex with retention of dehydroquinate synthase activity. The enzyme requires NAD and either Co2+ or Mn2+ for maximal activity. The activity was completely inhibited by EDTA. The Km of the enzyme for DAHP, NAD, and Co2+ were estimated to be 1.3 X 10(-4), 5.5 X 10(-5), and 5.5 X 10(-5) M, respectively. Enzyme activity was completely inhibited by NADH and the inhibition was not reversed by the addition of NAD, NADPH and NADP were not inhibitory. The enzyme was unstable to heat and lost all activity at 55 degrees C. A protein fraction which did not adsorb to phosphocellulose was found to inhibit the enzyme.     

Isoprenoid biosynthesis in plants - 2C-methyl-D-erythritol-4-phosphate synthase (IspC protein) of Arabidopsis thaliana

The ispC gene of Arabidopsis thaliana was expressed in pseudomature form without the putative plastid-targeting sequence in a recombinant Escherichia coli strain. The recombinant protein was purified by affinity chromatography and was shown to catalyze the formation of 2C-methyl-D-erythritol 4-phosphate from 1-deoxy-D-xylulose 5-phosphate at a rate of 5.6 micromol x min(-1) x mg(-1) (k(cat) 4.4 s(-1)). The Michaelis constants for 1-deoxy-D-xylulose 5-phosphate and the cosubstrate NADPH are 132 and 30 microm, respectively. The enzyme has an absolute requirement for divalent metal ions, preferably Mn2+ and Mg2+, and is inhibited by fosmidomycin with a Ki of 85 nm. The pH optimum is 8.0. NADH can substitute for NADPH, albeit at a low rate (14% as compared to NADPH). The enzyme catalyzes the reverse reaction at a rate of 2.1 micromol x min(-1) x mg(-1).     

Reactions of purified hog thyroid peroxidase with H2O2, tyrosine, and methylmercaptoimidazole (goitrogen) in comparison with bovine lactoperoxidase

Stopped flow experiments were carried out with purified hog thyroid peroxidase (A413 nm/A280 nm = 0.42). It reacted with H2O2 to form Compound I with a rate constant of 7.8 X 10(6) M-1 s-1. Compound I was reduced to Compound II by endogeneous donor with a half-life of 0.36 s. Compound I was reduced by tyrosine directly to the ferric enzyme with a rate constant of 7.5 X 10(4) M-1 s-1. Tyrosine could also reduce Compound II to the ferric enzyme with a rate constant of 4.3 X 10(2) M-1 s-1. Methylmercaptoimidazole accelerated the conversion of Compound I to Compound II and reacted with Compound II to form an inactivated form, which was discernible spectrophotometrically. The reactions of thyroid peroxidase with methylmercaptoimidazole quite resembled those of lactoperoxidase, but occurred at higher speeds. The absorption spectra of thyroid peroxidase were similar to those of lactoperoxidase and intestinal peroxidase, but obviously different from those of metmyoglobin, horseradish peroxidase, and chloroperoxidase. Similarity and dissimilarity between thyroid peroxidase and lactoperoxidase are discussed.     

Isolation and characterization of a pyrophosphate-dependent phosphofructokinase from Propionibacterium shermanii.

A pyrophosphate-dependent phosphofructokinase (pyrophosphate; D-fructose-6-phosphate-1-phosphotransferase) has been purified and characterized from extracts of Propionibacterium shermanii. The enzyme catalyzes the transfer of phosphate from pyrophosphate to fructose 6-phosphate to yield fructose-1,6-P2 and phosphate. This unique enzymatic activity was observed initially in Entamoeba histolytica (Reeves, R.E., South, D.J., Blytt, H.G., and Warren, L. G. (1974) J. Biol. Chem. 249, 7734-7741). This is the third pyrophosphate-utilizing enzyme that these two diverse organisms have in common. The others are phosphoenolpyruvate carboxytransphosphorylase and pyruvate phosphate dikinase. The PPi-phosphofructokinase from P. shermanii is specific for fructose-6-P and fructose-1,6-P2, no other phosphorylated sugars were utilized. Phosphate could be replaced by arsenate. The Km values are: phosphate, 6.0 X 10(-4) M; fructose-1, 6-P2, 5.1 X 10(-5) M; pyrophosphate, 6.9 X 10(-5) M; and fructose-6-P, 1.0 X 10(-4) M. The S20w is 5.1 S. The molecular weight of the native enzyme is 95,000. Sodium dodecyl sulfate electrophoresis of the enzyme showed a single band migrating with an Rf corresponding to a molecular weight of 48,000. Extracts of P. shermanii have PPi-phosphofructokinase activity approximately 6 times greater than ATP-phosphofructokinase and 15 to 20 times greater than fructose diphosphatase activities. It is proposed that (a) PPi may replace ATP in the formation of fructose-1-6-P2 when the organism is grown on glucose and (b) when the organism is grown on lactate or glycerol the conversion of fructose-1,6-P2 to fructose-6-P during gluconeogenesis may occur by phosphorolysis rather than hydrolysis.     

Identification of the Pseudomonas aeruginosa glmM gene, encoding phosphoglucosamine mutase

A search for a potential algC homologue within the Pseudomonas aeruginosa PAO1 genome database has revealed an open reading frame (ORF) of unknown function, ORF540 in contig 54 (July 1999 Pseudomonas genome release), that theoretically coded for a 445-amino-acid-residue polypeptide (I. M. Tavares, J. H. Leit?o, A. M. Fialho, and I. Sá-Correia, Res. Microbiol. 150:105-116, 1999). The product of this gene is here identified as the phosphoglucosamine mutase (GlmM) which catalyzes the conversion of glucosamine-6-phosphate to glucosamine-1-phosphate, an essential step in the formation of the cell wall precursor UDP-N-acetylglucosamine. The P. aeruginosa gene has been cloned into expression vectors and shown to restore normal peptidoglycan biosynthesis and cell growth of a glmM Escherichia coli mutant strain. The GlmM enzyme from P. aeruginosa has been overproduced to high levels and purified to homogeneity in a six-histidine-tagged form. Beside its phosphoglucosamine mutase activity, the P. aeruginosa enzyme is shown to exhibit phosphomannomutase and phosphoglucomutase activities, which represent about 20 and 2% of its GlmM activity, respectively.     

Studies of soluble rat liver mitochondrial acid ATPases. I. Purification and catalytic properties of ATPase 1.

A method for isolation of a soluble ATPase from rat liver mitochondria after freeze thaw cycling is described. Two enzymatically active fractions were separated by DEAE-cellulose chromatography (ATPase 1 and ATPase 2). ATPase 1 has been purified 300 fold. ATPase 1 was homogenous as judged by polyacrylamide gel electrophoresis. The optimum pH of the enzyme was 5.8-6.0 and the optimum temperature was 45 degrees C. The enzyme follows Michaelis-Menten kinetics: Km (9 X 10(-4) M), Vmax (23,6 mumoles Pi released X min -1 X mg protein -1). The enzyme hydrolysed nucleoside triphosphates, but was inactive upon nucleoside di and monophosphates, glucose 6-phosphate, phosphoserine, pyrophosphate and glycerol 2-phosphate. In contrast to membrane bound ATPase, cations have no effect on the enzyme activity. Nucleoside di and mono phosphates and glycerol 2 phosphate inhibited competitively the enzyme. The enzyme was not affected by oligomycin, but was stimulated by lactate, 2-mercaptoethanol and dithiothreitol.     

Cocoa butter biosynthesis. Purification and characterization of a soluble sn-glycerol-3-phosphate acyltransferase from cocoa seeds

Glycerol-3-phosphate acyltransferase has been purified from the post-microsomal supernatant of cocoa seeds using differential ammonium sulfate solubility along with anion exchange and gel filtration chromatography. Chromatofocusing and isoelectric focusing revealed a series of proteins with acyltransferase activity having isoelectric points close to 5.2. Gel filtration on Sephacryl S-300 in 500 mM NaCl, along with polyacrylamide gel electrophoresis (denaturing and non-denaturing) and immunochemical analysis, gave evidence that the native enzyme has a molecular weight of 2 X 10(5) and consists of an aggregate of 10 Mr 20,000 subunits. The highly purified enzyme carries an acyl donor, probably acyl-CoA, although this is not firmly established. The hydrophobic nature of the purified enzyme was demonstrated by its firm binding to octyl-Sepharose. Mass spectrometric analysis of reaction products revealed the presence of both palmitic and stearic acids. Considering that 1) the fatty acids were derived from the purified enzyme; 2) they were found exclusively in the 1-position of glycerol 3-phosphate; 3) the fatty acid positioning and composition is consistent with that found in cocoa butter, the major storage product of cocoa seeds; and 4) the enzyme is found in the post-microsomal supernatant, it seems reasonable to conclude that the first step in cocoa butter biosynthesis is catalyzed by glycerol-3-phosphate acyltransferase in the cytoplasm of cocoa cotyledon cells.     

Partial reactions of d-glucose 6-phosphate–1 l-myoinositol 1-phosphate cyclase

After removal of tightly bound NAD(+) by using charcoal, a preparation of d-glucose 6-phosphate-1 l-myoinositol 1-phosphate cyclase catalysed the reduction of 5-keto-d-glucitol 6-phosphate and 5-keto-d-glucose 6-phosphate by [4-(3)H]NADH to give [5-(3)H]-glucitol 6-phosphate and [5-(3)H]glucose 6-phosphate respectively. The position of the tritium atom in the latter was shown by degradation. Both enzyme-catalysed reductions were strongly inhibited by 2-deoxy-d-glucose 6-phosphate, a powerful competitive inhibitor of inositol cyclase. The charcoal-treated enzyme preparation also converted 5-keto-d-glucose 6-phosphate into [(3)H]myoinositol 1-phosphate in the presence of [4-(3)H]NADH, but less effectively. These partial reactions of inositol cyclase are interpreted as providing strong evidence for the formation of 5-keto-d-glucose 6-phosphate as an enzyme-bound intermediate in the conversion of d-glucose 6-phosphate into 1 l-myoinositol 1-phosphate. The enzyme was partially inactivated by NaBH(4) in the presence of NAD(+). Glucose 6-phosphate did not increase the inactivation, and there was no inactivation in the absence of NAD(+). There was no evidence for Schiff base formation during the cyclization. d-Glucitol 6-phosphate (l-sorbitol 1-phosphate) was a good inhibitor of the overall reaction. It did not inactivate the enzyme. The apparent molecular weight of inositol cyclase as determined by Sephadex chromatography was 2.15x10(5).     

Murine hexose-6-phosphate dehydrogenase: a bifunctional enzyme with broad substrate specificity and 6-phosphogluconolactonase activity

Murine hexose-6-phosphate dehydrogenase has been purified from liver microsomes by affinity chromatography on 2('),5(')-ADP-Sepharose. The purified enzyme has 6-phosphogluconolactonase activity and glucose-6-phosphate dehydrogenase activity and has a native molecular mass of 178 kDa and a subunit molecular mass of 89 kDa. Glucose 6-phosphate, galactose 6-phosphate, 2-deoxyglucose 6-phosphate, glucosamine 6-phosphate, and glucose 6-sulfate are substrates for murine hexose-6-phosphate dehydrogenase, with either NADP or deamino-NADP as coenzyme. This study confirms that hexose-6-phosphate dehydrogenase is a bifunctional enzyme which can catalyze the first two reactions of the pentose phosphate pathway.     

Mechanism of enzymic isomerization and epimerization of D-erythrose 4-phosphate.

The mechanism of the enzymic isomerization and epimerization of D-erythrose 4-phosphate (Ery4P) by an enzyme preparation from bovine liver was investigated with the use of 2H2O. The incorporation of 2H was quantitatively determined by a procedure using gas chromatography-mass spectrometry. About one atom of 2H was incorporated per molecule of the enzymic epimerization reaction product of Ery4P (D-threose 4-phosphate) or that of D-ribulose 5-phosphate. Computer simulation of the Ery4P isomerization reaction indicated that the 2H of 2H2O was not directly incorporated into the enzymic reaction product (D-erythrulose 4-phosphate). Instead, intramolecular transfer of hydrogen atoms had occurred.     

Temperature-dependent presteady state kinetics of lumazine synthase from the hyperthermophilic eubacterium Aquifex aeolicus

6,7-dimethyl-8-ribityllumazine synthase (lumazine synthase) catalyzes the condensation of 5-amino-6-ribitylamino-2,4(1H,3H)-pyrimidinedione and 3,4-dihydroxy-2-butanone 4-phosphate. Presteady state kinetic experiments using the enzyme from the hyperthermophilic bacterium Aquifex aeolicus were monitored by multiwavelength photometry. An early optical transient absorbing around 330 nm is interpreted as a Schiff base intermediate obtained by reaction of the position 5 amino group of the heterocyclic substrate with the carbonyl group of 3,4-dihydroxy-2-butanone 4-phosphate. A second transient with an absorption maximum at 445 nm represents an intermediate resulting from the elimination of orthophosphate from the Schiff base. The rate-determining step is the subsequent formation of the 7-exomethylene type anion of 6,7-dimethyl-8-ribityllumazine. The rate constants for the three partial reactions identified by the stopped flow experiments show linear Arrhenius relations in the temperature range of 15-70 degrees C.     

Spectral studies with lactoperoxidase and thyroid peroxidase: interconversions between native enzyme, compound II, and compound III

Spectral scans in both the visible (650-450 nm) and the Soret (450-380 nm) regions were recorded for the native enzyme, Compound II, and Compound III of lactoperoxidase and thyroid peroxidase. Compound II for each enzyme (1.7 microM) was prepared by adding a slight excess of H2O2 (6 microM), whereas Compound III was prepared by adding a large excess of H2O2 (200 microM). After these compounds had been formed it was observed that they were slowly reconverted to the native enzyme in the absence of exogenous donors. The pathway of Compound III back to the native enzyme involved Compound II as an intermediate. Reconversion of Compound III to native enzyme was accompanied by the disappearance of H2O2 and generation of O2, with approximately 1 mol of O2 formed for each 2 mol of H2O2 that disappeared. A scheme is proposed to explain these observations, involving intermediate formation of the ferrous enzyme. According to the scheme, Compound III participates in a reaction cycle that effectively converts H2O2 to O2. Iodide markedly affected the interconversions between native enzyme, Compound II, and Compound III for lactoperoxidase and thyroid peroxidase. A low concentration of iodide (4 microM) completely blocked the formation of Compound II when lactoperoxidase or thyroid peroxidase was treated with 6 microM H2O2. When the enzymes were treated with 200 microM H2O2, the same low concentration of iodide completely blocked the formation of Compound III and largely prevented the enzyme degradation that otherwise occurred in the absence of iodide. These effects of iodide are readily explained by (i) the two-electron oxidation of iodide to hypoiodite by Compound I, which bypasses Compound II as an intermediate, and (ii) the rapid oxidation of H2O2 to O2 by the hypoiodite formed in the reaction between Compound I and iodide.     

Purification and characterization of a phosphatidylinositol 4-kinase from bovine uteri

Phosphatidylinositol 4-kinase has been purified 10,148-fold to a specific activity of 2.7 mumol/mg/min from bovine uteri. This purification was accomplished by detergent extraction of an acetone powder, ammonium sulfate precipitation, and chromatography on MonoQ, S-Sepharose, MonoP, and hydroxylapatite columns. The purified enzyme has a molecular mass of 55 kDa and appears to be monomeric. Kinetic analyses of the enzymatic activity demonstrated apparent Km values of 18 microM and 22 micrograms/ml (approximately 26 microM) for ATP and phosphatidylinositol, respectively, optimal activity in the pH range of 6.0-7.0, and a sigmoidal dependence of enzymatic activity on [Mg2+]. Ca2+ inhibited the enzyme at nonphysiological concentrations with 50% inhibition observed at a free [Ca2+] of approximately 300 microM. The purified enzyme efficiently utilized both ATP and 2'-deoxy-ATP as phosphoryl donors and specifically phosphorylated phosphatidylinositol on the fourth position. No phosphatidylinositol-4-phosphate 5-kinase activity was observed in the purified enzyme preparations. To our knowledge, this is the first reported purification of a phosphatidylinositol-specific phosphatidylinositol 4-kinase.     

S-adenosylmethionine: protein-arginine methyltransferase. Purification and mechanism of the enzyme.

Protein methylase I (S-adenosylmethionine: protein-arginine methyltransferase, EC 2.1.1.23) has been purified from calf brain approximately 120-fold with a 14% yield. The final preparation is completely free of any other protein-specific methyltransferases and endogenous substrate protein. The enzyme has an optimum pH of 7.2 and pI value of 5.1. The Km values for S-adenosyl-L-methionine, histone H4, and an ancephalitogenic basic protein are 7.6 X 10(-6), 2.5 X 10(-5), and 7.1 X 10(-5) M, respectively, and the Ki value for S-adenosyl-L-homocysteine is 2.62 X 10(-6) M. The enzyme is highly specific for the arginine residues of protein, and the end products after hydrolysis of the methylated protein are NG,NG-di(asymmetric), NG,N'G-di(symmetric), and NG-monomethylarginine. The ratio of [14C]methyl incorporation into these derivatives by enzyme preparation at varying stages of purification remains unchanged at 40:5:55, strongly indicating that a single enzyme is involved in the synthesis of the three arginine derivatives. The kinetic mechanism of the protein methylase I reaction was studied with the purified enzyme. Initial velocity patterns converging at a point on the extended axis of abscissas were obtained with either histone H4 or S-adenosyl-L-methionine as the varied substrate. Product inhibition by S-adenosyl-L-homocysteine with S-adenosyl-L-methionine as the varied substrate was competitive regardless of whether or not the enzyme was saturated with histone H4. On the other hand, when histone H4 is the variable substrate, noncompetitive inhibition was obtained with S-adenosyl-L-homocysteine under conditions where the enzyme is not saturated with the other substrate, S-adenosyl-L-methionine. These results suggest that the mechanism of the protein methylase I reaction is a Sequential Ordered Bi Bi mechanism with S-adenosyl-L-methionine as the first substrate, histone H4 as the second substrate, methylated histone H4 as the first product, and S-adenosyl-L-homocysteine as the second product released.     

Alternative pathway for the formation of 4,5-dihydroxy-2,3-pentanedione, the proposed precursor of 4-hydroxy-5-methyl-3(2H)-furanone as well as autoinducer-2, and its detection as natural constituent of tomato fruit

The formation of 4-hydroxy-5-methyl-3(2H)-furanone (HMF, norfuraneol) by spinach ribosephosphate isomerase was reinvestigated. Incubation experiments using D-ribose-5-phosphate and D-ribulose-5-phosphate clearly revealed a spontaneous nonenzymatic formation of the hydroxy-furanone from the ketose-phosphate under physiological conditions at 35 degrees C and pH 7.5, whereupon up to 1.3% of D-ribulose-5-phosphate was transformed to HMF within 15 h. 4,5-Dihydroxy-2,3-pentanedione was deduced as ultimate precursor of HMF, since addition of o-phenylenediamine to the incubation mixture led to lower amounts of HMF and to the formation of 3-(1,2-dihydroxyethyl)-2-methylquinoxaline, which was identified by means of high pressure liquid chromatography with diode array detection (HPLC-DAD), HPLC-electrospray ionization-tandem mass spectrometry (HPLC-ESI-MS/MS) and NMR spectroscopy. Additionally, the spontaneous formation of 4,5-dihydroxy-2,3-pentanedione was demontrated by its conversion to the respective alditol acetate using either NaBH(4) or NaBD(4) for the reduction. Comparative gas chromatography-mass spectrometry (GC-MS) analysis revealed the incorporation of two deuterium atoms and confirmed the dicarbonyl structure. Application of 1-13C-D-ribulose-5-phosphate as well as 5-13C-D-ribulose-5-phosphate and analysis of the derived quinoxaline derivatives by HPLC-ESI-MS/MS demonstrated the formation of the methyl-group at C-5 of the carbohydrate phosphate in consequence of a nonenzymatic phosphate elimination. Application of o-phenylenediamine into ripe tomatoes led to the detection of 3-(1,2-dihydroxyethyl)-2-methylquinoxaline by means of HPLC-MS/MS analysis implying the genuine occurrence of 4,5-dihydroxy-2,3-pentanedione in this fruit.