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PubMed2003

Suppression of fructokinase encoded by LeFRK2 in tomato stem inhibits growth and causes wilting of young leaves.

Fructokinases catalyze the key step of fructose phosphorylation in plants. LeFRK2, the major fructokinase-encoding gene in tomato plants, is abundantly expressed in roots, stems, and fruits. To analyze the role of LeFRK2 in plant development, we analyzed transgenic tomato plants with sense and antisense expression of StFRK, the potato homolog of LeFRK2. Increased fructokinase activity had no effect. However, plants in which LeFRK2 was specifically suppressed, either via antisense suppression or via co-suppression, exhibited growth inhibition and wilting of young leaves at daytime. Grafting experiments indicated that a stem interstock of antisense plants was sufficient to inhibit growth and cause leaf wilting. Stem secondary xylem exhibited particular suppression of LeFRK2 and the area of active xylem, estimated by eosin uptake, was significantly smaller in antisense stem compared to that of wild-type plants. These results suggest that LeFRK2 might be required for proper development of xylem that affected growth and wilting.

Antisense Elements (Genetics)↗
PubMed2002

Distinct physiological roles of fructokinase isozymes revealed by gene-specific suppression of Frk1 and Frk2 expression in tomato.

There are two divergent fructokinase isozymes, Frk1 and Frk2 in tomato (Lycopersicon esculentum Mill.) plants. To investigate the physiological functions of each isozyme, the expression of each fructokinase mRNA was independently suppressed in transgenic tomato plants, and the respective phenotypes were evaluated. Suppression of Frk1 expression resulted in delayed flowering at the first inflorescence. Suppression of Frk2 did not effect flowering time but resulted in growth inhibition of stems and roots, reduction of flower and fruit number, and reduction of seed number per fruit. Localization of Frk1 and Frk2 mRNA accumulation by in situ hybridization in wild-type tomato fruit tissue indicated that Frk2 is expressed specifically in early tomato seed development. Fruit hexose and starch content were not effected by the suppression of either Frk gene alone. The results collectively indicate that flowering time is specifically promoted by Frk1 and that Frk2 plays specific roles in contributing to stem and root growth and to seed development. Because Frk1 and Frk2 gene expression was suppressed individually in transgenic plants, other significant metabolic roles of fructokinases may not have been observed if Frk1 and Frk2 play, at least partially, redundant metabolic roles.

Enzyme Activation↗
PubMedDate not supplied

Glucokinase and fructokinase of Trichomonas vaginalis and Tritrichomonas foetus.

Trichomonas vaginalis and Tritrichomonas foetus contain glucokinase and not a hexokinase of broad hexose specificity. Tritrichomonas foetus also contains a specific fructokinase which could be resolved from glucokinase by anion exchange chromatography. Native T. vaginalis glucokinase had a Mr of 76,000, and SDS-PAG electrophoresis showed two equally stained bands corresponding to Mr 40,000 and 38,000. Glucose and ATP were by far the best substrates for both trichomonad glucokinases, with Km values as low as 33-35 microM and 75-83 microM, respectively. Substrate saturation curves for these enzymes were all hyperbolic. Tritrichomonas foetus fructokinase required fructose and ATP, with Km values of 200 microM and 81 microM. None of the activities was affected by a number of potential regulatory metabolites, including glucose-6-phosphate. The only exception was AMP which in supraphysiological concentrations had an inhibitory effect on T. foetus fructokinase. In conclusion, the absence of regulation at the hexose phosphorylation step described here, as well as the presence of an easily reversible PPi: fructose-6-phosphate 1-phosphotransferase described previously (Mertens, E., Van Schaftingen, E. & Müller, M. 1989. Mol. Biochem. Parasitol., 37:183-190), suggest that the rate of the 1st part of glycolysis in trichomonads is controlled only by the intracellular availability of hexoses.

Animals↗
PubMed1980

Biochemical characterization of a fructokinase mutant of Rhizobium meliloti.

A double mutant strain (UR3) of Rhizobium meliloti L5-30 was isolated from a phosphoglucose isomerase mutant (UR1) on the basis of its resistance to fructose inhibition when grown on fructose-rich medium. UR3 lacked both phosphoglucose isomerase and fructokinase activity. A mutant strain (UR4) lacking only the fructokinase activity was derived from UR3; it grew on the same carbon sources as the parent strain, but not on fructose, mannitol, or sorbitol. A spontaneous revertant (UR5) of normal growth phenotype contained fructokinase activity. A fructose transport system was found in L5-30, UR4, and UR5 grown in arabinose-fructose minimal medium. No fructose uptake activity was detected when L5-30 and UR5 were grown on arabinose minimal medium, but this activity was present in strain UR4. Free fructose was concentrated intracellularly by UR4 > 200-fold above the external level. A partial transformation of fructose into mannitol and sorbitol was detected by enzymatic analysis of the uptake products. Polyol dehydrogenase activity was detected in UR4 grown in arabinose-fructose minimal medium. The induction pattern of polyol dehydrogenase activities in this strain might be due to slight intracellular fructose accumulation.

Alcohol Oxidoreductases↗
PubMed1992

Cloning, sequencing, and expression of the Zymomonas mobilis fructokinase gene and structural comparison of the enzyme with other hexose kinases.

The frk gene encoding the enzyme fructokinase (fructose 6-phosphotransferase [EC 2.7.1.4]) from Zymomonas mobilis has been isolated on a partial TaqI digest fragment of the genome and sequenced. An open reading frame of 906 bp corresponding to 302 amino acids was identified on a 3-kbp TaqI fragment. The deduced amino acid sequence corresponds to the first 20 amino acids (including an N-terminal methionine) determined by amino acid sequencing of the purified protein. The 118 bp preceding the methionine codon on this fragment does not appear to contain a promoter sequence. There was weak expression of the active enzyme in the recombinant Escherichia coli clone under control of the lac promoter on the pUC plasmid. Comparison of the amino acid sequence with that of the glucokinase enzyme (EC 2.7.1.2) from Z. mobilis reveals relatively little homology, despite the fact that fructokinase also binds glucose and has kinetic and structural properties similar to those of glucokinase. Also, there is little homology with hexose kinases that have been sequenced from other organisms. Northern (RNA) blot analysis showed that the frk transcript is 1.2 kb long. Fructokinase activity is elevated up to twofold when Z. mobilis was grown on fructose instead of glucose, and there was a parallel increase in frk mRNA levels. Differential mRNA stability was not a factor, since the half-lives of the frk transcript were 6.2 min for glucose-grown cells and 6.6 min for fructose-grown cells.

Amino Acid Sequence↗
PubMed2004

Bifidobacterium longum requires a fructokinase (Frk; ATP:D-fructose 6-phosphotransferase, EC 2.7.1.4) for fructose catabolism.

Although the ability of Bifidobacterium spp. to grow on fructose as a unique carbon source has been demonstrated, the enzyme(s) needed to incorporate fructose into a catabolic pathway has hitherto not been defined. This work demonstrates that intracellular fructose is metabolized via the fructose-6-P phosphoketolase pathway and suggests that a fructokinase (Frk; EC 2.7.1.4) is the enzyme that is necessary and sufficient for the assimilation of fructose into this catabolic route in Bifidobacterium longum. The B. longum A10C fructokinase-encoding gene (frk) was expressed in Escherichia coli from a pET28 vector with an attached N-terminal histidine tag. The expressed enzyme was purified by affinity chromatography on a Co(2+)-based column, and the pH and temperature optima were determined. A biochemical analysis revealed that Frk displays the same affinity for fructose and ATP (Km(fructose) = 0.739 +/- 0.18 mM and Km(ATP) = 0.756 +/- 0.08 mM), is highly specific for D-fructose, and is inhibited by an excess of ATP (>12 mM). It was also found that frk is inducible by fructose and is subject to glucose-mediated repression. Consequently, this work presents the first characterization at the molecular and biochemical level of a fructokinase from a gram-positive bacterium that is highly specific for D-fructose.

Bifidobacterium↗
PubMed1992

Purification and Properties of Fructokinase from Developing Tubers of Potato (Solanum tuberosum L.).

Fructokinase has been purified from developing potato (Solanum tuberosum L.) tubers by a combination of hydrophobic interaction, affinity chromatography, and gel filtration. The protein has a native molecular mass of approximately 70 kD but is apparently a dimer. Ion-exchange chromatography and two-dimensional western blots resolved three major fructokinases, designated FK-I, FK-II, and FK-III in order of their elution from a Mono-Q column. Fructokinase activity proved labile when proteins were purified in the absence of fructose. Kinetically, FKs I, II, and III all have broad pH optima with peaks at about pH 8.5. The enzymes have a high specificity for fructose (K(m) values ranging from 0.041 to 0.128 mm), and can utilize a range of nucleoside triphosphates. Unlike FKs I and II, FK-III is not inhibited by fructose concentrations in excess of 1 mm. MgADP inhibited activity of the three FKs (between 68 and 75% inhibition at 1.0 mm), whereas fructose 6-P caused inhibition at concentrations of 10 mm. There were no regulatory effects observed with a range of other metabolites. K(+) (10 mm) activated FK-I by 4-fold and FKs II and III by only about 50%.

Journal Article↗
PubMed1978

Fructokinase (Fraction III) of Pea Seeds.

A second fructokinase (EC 2.7.1.4) was obtained from pea seed (Pisum sativum L. var. Progress No. 9) extracts. The enzyme, termed fructokinase (fraction III), was specific for fructose and had little activity with glucose. With fructose concentrations above 0.25 millimolar, there was strong substrate inhibition at the optimum pH (8.0) and also at pH 6.6. The apparent K(m) values at pH 8.0 for fructose and glucose were 0.06 millimolar and 0.14 millimolar, respectively. The apparent K(m) for Mg adenosine 5'-triphosphate (MgATP) was 0.06 millimolar and excess MgATP was inhibitory. Mg(2+) was essential for activity but the enzyme was inhibited by excess Mg(2+) or ATP. Mg adenosine 5'-pyrophosphate was also inhibitory. Activity was stimulated by the addition of monovalent cations: of those tested K(+), Rb(+), and NH(4) (+) were the most effective. The possible role of fructokinase (fraction III) is discussed.

Journal Article↗
PubMed1996

Overexpression, purification, and generation of a thermostable variant of Zymomonas mobilis fructokinase.

The gene encoding fructokinase (EC 2.7.1.4) from Zymomonas mobilis has been expressed at high level in Escherichia coli by modifying the ribosome binding site using the polymerase chain reaction. A simple two-step purification from extracts of the recombinant cells results in highly purified enzyme suitable for use in fructose determination. Using the polymerase chain reaction in mutagenic conditions, a variant of fructokinase was isolated which was more thermostable than the wild type, taking the 30 min half-life from 70.1 to 72.4 degrees C. The purified thermostable variant had the same specific activity as the wild type. Sequencing of the variant indicated that only one amino acid was changed, with Ser 69 becoming Ala. Searches of the mutant libraries for variants that were (a) active with glucose or (b) had reduced inhibition by glucose were unsuccessful.

DNA Primers↗
PubMed1997

Purification and characterization of recombinant tomato fruit (Lycopersicon esculentum Mill.) fructokinase expressed in Escherichia coli.

Fructokinase (FK; ATP:D-fructose 6-phosphotransferase, EC 2.7.1.4) cloned from a tomato fruit cDNA library has been expressed in Escherichia coli. The recombinant protein was purified 159-fold to greater than 99% purity, based on SDS-PAGE analysis. The subunit molecular mass is estimated to be 35 kDa and the nondissociated molecular mass is 72.4 kDa, indicating that the functional form is a dimer. Two-dimensional IEF/SDS-PAGE analyses combined with immunodetection show that both native and recombinant proteins exhibit the same pattern of six closely grouped peptides with pI values ranging from 5.66 to 6.17. Biochemical characterization of the purified recombinant enzyme shows properties essentially identical to those of the native fructokinase purified from young tomato fruit: the pH optimum is 8.0, the K(m) for fructose is 0.22 mM, and severe substrate inhibition is observed when fructose concentration is greater than 0.5 mM (Ki = 3.0 mM). ATP is the preferred phosphate donor (K(m) = 0.13 mM and Vmax/K(m) = 212), followed by GTP (K(m) = 0.45 mM and Vmax/K(m) = 76) and UTP (K(m) = 1.68 mM and Vmax/K(m) = 20), but Vmax values are slightly greater with GTP and UTP. Product inhibition analyses show that the inhibition by ADP with respect to ATP is dependent on fructose concentration [Ki (ADP) = 0.41 mM with 0.5 mM fructose and decreased to 0.12 mM with 3 mM fructose]. Inhibition by fructose 6-P shows weak noncompetitive inhibition with respect to fructose; however, the recombinant protein is slightly more sensitive to fructose 6-P than the native FK.

Cloning, Molecular↗
PubMed2006

Evidence for intracellular spatial separation of hexokinases and fructokinases in tomato plants.

Four hexokinase (LeHXK1-4) and four fructokinase (LeFRK1-4) genes were identified in tomato plants. Previous GFP fusion studies indicate that the gene product of LeHXK3 is associated with the mitochondria while that of LeHXK4 is located within plastids. In this study we found that the enzyme encoded by the fructokinase gene LeFRK3 is also located within plastids. The presence of LeFrk3 enzyme in plastids raises the question of the origin of fructose in these organelles. The other three FRKs enzymes, LeFrk1&2&4, are located in the cytosol. Unlike LeFrk1&2&4, the two additional HXKs, LeHxk1&2, share a common membrane anchor domain and are associated with the mitochondria similar to LeHxk3. The difference in the locations of the cytoplasmic FRK and HXK isozymes suggests that glucose phosphorylation is confined to defined special intracellular localizations while fructose phosphorylation is less confined.

Amino Acid Sequence↗
PubMed2006

Differential expression of two fructokinases in Oryza sativa seedlings grown under aerobic and anaerobic conditions.

Fructokinases (EC 2.7.1.4) may play an important role in carbohydrate metabolism of Oryza sativa L. (rice) seedlings under anoxia. We present here the molecular and biochemical characterizations of two rice fructokinases, namely OsFK1 and OsFK2. The results show that, at both a transcriptional and a transductional level, OsFK1 is preferentially expressed under aerobic conditions, whereas OsFK2 is induced under anoxia. Substrate inhibition was demonstrated for OsFK1, while OsFK2 appears to be largely unaffected by fructose concentrations up to 10 mM. Sugar modulation of anoxia-induced proteins has been proposed, but our results on rice calli treated with or without glucose (10, 30 or 90 mM) for different time indicate that neither OsFK1 nor OsFK2 are sugar-regulated. We propose that OsFK2 plays a major role in fructose phosphorylation under anoxic conditions.

Aerobiosis↗
PubMed1989

Presence of fructokinase in pancreatic islets.

Homogenates of rat pancreatic islets that had been heated for 5 min at 70 degrees C to inactive hexokinases, catalyzed the ATP-dependent phosphorylation of D-fructose. This reaction was dependent on the presence of K+ and was inhibited by D-tagatose although not by D-glucose or D-glucose 6-phosphate. The phosphorylation product was identified as fructose 1-phosphate through its conversion to a bisphosphate ester by Clostridium difficile fructose 1-phosphate kinase. These findings allowed the conclusion that fructokinase (ketohexokinase) was responsible for this process. Similar results were observed with tumoral insulin-producing cells (RINm5F line). Fructokinase may account for a large share of fructose phosphorylation in intact islets, particularly in the presence of D-glucose.

Animals↗
PubMed2001

Characterization of native and yeast-expressed tomato fruit fructokinase enzymes.

Three fructokinase isozymes (FKI, FKII, FKIII) were separated from both immature and ripe tomato fruit pericarp. All three isozymes were specific for fructose with undetectable activity towards glucose or mannose. The three isozymes could be distinguished from one another with respect to response to fructose, Mg and nucleotide donor concentrations and this allowed the comparison of the fruit enzymes with the gene products of the two known cloned tomato fructokinase genes, LeFRK1 and LeFRK2. FKI was characterized by both substrate (fructose), as well as Mg, inhibition; FKII was inhibited by neither fructose nor Mg; and FKIII was inhibited by fructose but not by Mg. ATP was the preferred nucleotide donor for all three FKs and FKI showed inhibition by CTP and GTP above 1 mM. All three FKs showed competitive inhibition by ADP. During the maturation of the tomato fruit total FK activity decreased dramatically. There were decreases in activity of all three FKs, nevertheless, all were still observed in the ripe fruit. The two tomato LeFRK genes were expressed in yeast and the gene products were characterized with respect to the distinguishing characteristics of fructose, Mg and nucleotide inhibition. Our results indicate that FKI is the gene product of LeFRK2 and FKII is probably the gene product of LeFRK1.

Chromatography, High Pressure Liquid↗
PubMed1985

Determination of the screw sense specificity of bovine liver fructokinase.

Fructokinase from beef liver showed a clear reversal in specificity when the two isomers of ATP beta S were used as substrates with Mg2+ and Cd2+, with the Sp isomer having the higher V/K value with Mg2+ and the Rp isomer the higher value with Cd2+. The delta isomer of MgATP is thus the active form of the substrate. The substitution of sulfur for oxygen in the noncoordinated position of the beta-phosphate caused a 102-fold decrease in V/K over the value seen with MgATP, while substitution in the coordinated position gave a 21-fold decrease over the V/K value seen with CdATP. The Km values were little affected by sulfur substitution, showing that the wrong screw sense isomers were nonproductively bound almost as well as the correct ones. When ADP alpha S was used as a substrate in the reverse reaction, the Sp isomer showed the highest V/K value with both Mg2+ and Cd2+, suggesting that the metal ion is not coordinated to the alpha-phosphate during transphosphorylation. The failure of CrATP to act as a substrate for fructokinase suggests that the enzyme inserts one of its side chains into the inner coordination sphere of the metal ion during the reaction.

Adenosine Triphosphate↗
PubMed1982

Models for the metabolic production of oxalate from xylitol in humans: a role for fructokinase and aldolase.

It has been proposed previously that oxalate precursors may be formed in the transketolase reaction during the metabolism of xylitol. It is shown in this paper that fructokinase and aldolase, purified from human liver, provide an alternative model in that, in coupled sequence, they produce glycolaldehyde, an oxalate precursor, from D-xylulose via D-xylulose 1-phosphate; D-fructose does not give rise to glycolaldehyde. It is concluded that metabolic pathways based on a combination of the transketolase, fructokinase and aldolase reactions can account for the production of glucose, lactate, tetronates (C-threonic and D-erythronic acids) and oxalate (precursors) during the metabolism of xylitol administered parenterally.

Fructokinases↗
PubMed2002

Fructokinase and hexokinase from pollen grains of bell pepper (Capsicum annuum L.): possible role in pollen germination under conditions of high temperature and CO2 enrichment.

The processes of pollen grain development and germination depend on the uptake and metabolism of pollen sugars. In pepper (Capsicum annuum L.), initial sugar metabolism includes sucrose hydrolysis by invertase and subsequent phosphorylation of glucose and fructose by hexose kinases. The main objective of this study was to investigate changes in fructokinase (EC 2.7.1.4) and hexokinase (EC.2.7.1.1) activities in pepper flowers during their development, and to study the possible roles of these enzymes in determining pollen germination capacity under high temperature and under CO(2) enrichment, previously shown to modify sugar concentrations in pepper pollen (Aloni et al., 2001 Physiologia Plantarum 112: 505-512). Fructokinase (FK) activity was predominant in pepper pollen, and increased during pollen maturation. Pollen hexokinase (HK) activity was low and did not change throughout pollen development. High-temperature treatment (day/night, 32/26 degrees C) of pepper plants reduced the percentage of pollen that germinated compared with that under normal temperatures (26/22 degrees C), and concomitantly reduced the activity of FK in mature pollen. High temperature also reduced FK and HK activity in the anther. Under high ambient CO(2) (800 micro l l(-1)) pollen FK activity was enhanced. The results suggest that pollen and anther FK may play a role in the regulation of pollen germination, possibly by providing fructose-6-phosphate for glycolysis, or through conversion to UDP-glucose (UDPG) to support the biosynthesis of cell wall material for pollen tube growth. High temperature stress and CO(2) enrichment may influence pollen germination capacity by affecting these pathways.

Calcium Chloride↗
PubMed1996

Studies on fructose metabolism in cultured astroglial cells and control hepatocytes: lack of fructokinase activity and immunoreactivity in astrocytes.

Astroglia-rich primary cultures derived from the brains of newborn rats can be grown in the presence of sorbitol or fructose. In the present study, evidence was obtained by enzymatic analysis and immunocytochemistry that fructose is further metabolized to fructose-6-phosphate and that fructokinase is lacking in the astrocytes. In contrast, fructose-1-phosphate as well as fructokinase immunoreactivity could be detected in cultured hepatocytes. Considerable amounts of astroglial glycogen were synthesized from fructose. Lactate release in fructose-fed cultures was still 30% that of glucose-fed cells and was abolished in the presence of 2-deoxyglucose. No glycogen was synthesized when sorbitol, which is converted intracellularly to fructose, replaced glucose in the incubation medium. However, lactate release from sorbitol-fed cultures was still significant and was not abolished by 2-deoxyglucose. The results are compatible with the idea of astroglial glycogen being a store of lactate rather than glucose. Furthermore, the results demonstrate that sorbitol is an adequate substrate for astroglial glycolysis but, in contrast to fructose, cannot be utilized for the buildup of glycogen stores.

Animals↗