Subcellular distribution and kinetic properties of cytosolic and non-cytosolic hexokinases in maize seedling roots: implications for hexose phosphorylation.

Hexose phosphorylation by hexokinases plays an important role in glycolysis, biosynthesis and control of sugar-modulated genes. Several cytosolic hexokinase and fructokinase isoforms have been characterized and organelle-bound hexokinases have also been detected in higher plants. In this study a hexokinase activity is described that is inhibited by ADP (K(i)=30 microM) and mannoheptulose (K(i) congruent with 300 microM) in non-cytosolic fractions (mitochondria, Golgi apparatus and microsomes) obtained from preparations of seedling roots of maize (Zea mays L.). The catalytic efficiency (Vmax/Km) for both ATP and glucose in all non-cytosolic hexokinase fractions is more than one order of magnitude higher than that of cytosolic hexokinase and fructokinases. Low (30%) or no ADP and mannoheptulose inhibition is observed with hexokinase and fructokinase activities derived from the cytosolic compartment obtained after ion exchange and affinity chromatography. The soluble fructokinase (FK) shows fructose cooperativity (Hill n>2). The Vmax/Km ratio is about 3-fold higher for ATP than for other NTPs and no difference for hexose phosphorylation efficiencies is found between cytosolic hexokinase and fructokinase isoforms (FK1, FK2) with ATP as substrate. The K(i) for fructose inhibition is 2 mM for FK1 and 25 mM for FK2. The data indicate that low energy-charge and glucose analogues preferentially inhibit the membrane-bound hexokinases possibly involved in sugar-sensing, but not the cytosolic hexokinases and fructokinases.     

Characterization of two Arabidopsis thaliana fructokinases

Two different fructokinase isoforms of Arabidopsis thaliana have been identified and characterized by non-denaturing electrophoresis followed by activity-staining. The two fructokinases, fructokinase1 (FRK1) and fructokinase2 (FRK2), showed a high specificity for fructose and did not stain when glucose or mannose were used as substrate. Fructose and ATP at high concentrations (above 5 mM) induced a substrate inhibition of the two enzymatic activities. Arabidopsis FRK1 and FRK2 were capable of employing GTP, CTP, UTP and TTP as phosphate donors, although with a significantly lower efficiency than ATP. The two fructokinase activities were also activated by K⁺, at around 10–20 mM, and inhibited by ADP and AMP at concentrations above 10 mM. Finally, FRK1 and FRK2 showed a different expression pattern in the plant, with FRK1 being more abundant in the roots and FRK2 in the shoots. The results demonstrate a simple technique that provides important information about fructokinase activities in the plants and which can be useful for the analysis of Arabidopsis mutants.     

Divergent fructokinase genes are differentially expressed in tomato.

Two cDNA clones (Frk1 and Frk2) encoding fructokinase (EC 2.7.1.4) were isolated from tomato (Lycopersicon esculentum). The Frk2 cDNA encoded a deduced protein of 328 amino acids that was more than 90% identical with a previously characterized potato (Solanum tuberosum) fructokinase. In contrast, the Frk1 cDNA encoded a deduced protein of 347 amino acids that shared only 55% amino acid identity with Frk2. Both deduced proteins possessed and ATP-binding motif and putative substrate recognition site sequences identified in bacterial fructokinases. The Frk1 cDNA was expressed in a mutant yeast (Saccharomyces cerevisiae) line, which lacks the ability to phosphorylate glucose and fructose and is unable to grow on glucose or fructose. Mutant cells expressing Frk1 were complemented to grow on fructose but not glucose, indicating that Frk1 phosphorylates fructose but not glucose, and this activity was verified in extracts of transformed yeast. The mRNA corresponding to Frk2 accumulated to high levels in young, developing tomato fruit, whereas the Frk1 mRNA accumulated to higher levels late in fruit development. The results indicate that fructokinase in tomato is encoded by two divergent genes, which exhibit a differential pattern of expression during fruit development.     

Purification and characterization of fructokinase from developing tomato (Lycopersicon esculentum Mill.) fruits

A procedure is described which allows the purification of fructokinase (EC 2.7.1.4) from young tomato fruit. The procedure yielded a 400-fold purification and two isoenzymes designated fructokinase I and II (FKI and FKII) were separated by anion-exchange chromatography. Using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) the molecular mass was estimated to be 35 kDa. Gel filtration on Sepharose-12 indicated that for both fructokinases the functional form is a dimer. Two dimensional isoelectric focusing/SDS-PAGE combined with immunoblotting showed that FKI has two components with isoelectric points (pIs) of 6.42 and 6.55, while four components with pIs from 6.07 to 6.55 were detected for FKII. A mixture of both fructokinases showed that the components of FKI match the more alkaline components of FKII. The activity of both fructokinases increased with increasing pH to around 8.0 and equal activity was observed from 8.0 to 9.5. Both fructokinases were specific for fructose with K _m values for fructose of 0.131 and 0.201 mM for FKI and FKII, respectively. At high concentrations (> 0.5 mM), fructose was also a strong inhibitor with inhibition constants (K _i) of 1.82 and 1.39 mM for FKI and FKII, respectively. The preferred phosphate donor for both isoforms was ATP, and K _m values of 0.11 and 0.15 mM were observed for FKI and FKII. At low concentrations (0.05–0.2 mM), fructose exhibited noncompetitive inhibition with respect to ATP for both fructokinases. This inhibition pattern changed to uncompetitive when higher fructose concentrations (0.5–10 mM) were used. These data indicated that substrate addition is ordered, with ATP adding first. Inhibition by ADP was also affected by the fructose concentrations. At 0.5 mM fructose, FKI showed non-competitive inhibition by ADP with respect to ATP and this inhibition changed to uncompetitive when 3 mM fructose was used. The isoform FKII showed a competitive inhibition pattern for ADP at 0.5 mM fructose which also changed to uncompetitive when 3 mM fructose was used. The features of the regulation of both fructokinases suggest that this enzyme might have a relevant role in carbon metabolism during tomato fruit development.     

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.     

Properties of recombinant ATP-dependent fructokinase from the halotolerant methanotroph <Emphasis Type="Italic">Methylomicrobium alcaliphilum</Emphasis> 20Z

In the cluster of genes for sucrose biosynthesis and cleavage in Methylomicrobium alcaliphilum 20Z, a gene whose encoded sequence showed high similarity to sugar kinases of the ribokinase family was found. By heterologous expression of this gene in Escherichia coli cells and following metal chelate affinity chromatography, the electrophoretically homogenous recombinant enzyme with six histidine residues on the C-end was obtained. The enzyme catalyzes ATP-dependent phosphorylation of fructose into fructose-6-phosphate but is not active with other sugars as phosphoryl acceptors. The fructokinase of M. alcaliphilum 20Z is most active in the presence of Mn²⁺ at pH 9.0 and 60°C, being inhibited by ADP (K _i = 2.50 ± 0.03 mM). The apparent K _m values for fructose and ATP are 0.26 and 1.3 mM, respectively; the maximal activity is 141 U/mg protein. The enzyme shows the highest similarity of translated amino acid sequence with putative fructokinases of methylotrophic and autotrophic proteobacteria whose fruK gene is located in the gene cluster of sucrose biosynthesis. The involvement of fructokinase in sucrose metabolism in M. alcaliphilum 20Z and other methanotrophs and autotrophs is discussed.     

Analyses of enzyme II gene mutants for sugar transport and heterologous expression of fructokinase gene in Corynebacterium glutamicum ATCC 13032

Corynebacterium glutamicum ATCC 13032 has four enzyme II (EII) genes of the phosphotransferase system in its genome encoding transporters for sucrose, glucose, fructose, and an unidentified EII. To analyze the function of these EII genes, they were inactivated via homologous recombination and the resulting mutants characterized for sugar utilization. Whereas the sucrose EII was the only transport system for sucrose in C. glutamicum, fructose and glucose were each transported by a second transporter in addition to their corresponding EII. In addition, the ptsF ptsG double mutant carrying deletions in the EII genes for fructose and glucose accumulated fructose in the culture broth when growing on sucrose. As no fructokinase gene exists in the C. glutamicum genome, the fructokinase gene from Clostridium acetobutylicum was expressed in C. glutamicum and resulted in the direct phosphorylation of fructose without any fructose efflux. Accordingly, since fructokinase could direct fructose flux to the pentose phosphate pathway for the supply of NADPH, fructokinase expression may be a potential strategy for enhancing amino acid production.     

Convergent evolution of similar enzymatic function on different protein folds: the hexokinase, ribokinase, and galactokinase families of sugar kinases.

Kinases that catalyze phosphorylation of sugars, called here sugar kinases, can be divided into at least three distinct nonhomologous families. The first is the hexokinase family, which contains many prokaryotic and eukaryotic sugar kinases with diverse specificities, including a new member, rhamnokinase from Salmonella typhimurium. The three-dimensional structure of hexokinase is known and can be used to build models of functionally important regions of other kinases in this family. The second is the ribokinase family, of unknown three-dimensional structure, and comprises pro- and eukaryotic ribokinases, bacterial fructokinases, the minor 6-phosphofructokinase 2 from Escherichia coli, 6-phosphotagatokinase, 1-phosphofructokinase, and, possibly, inosine-guanosine kinase. The third family, also of unknown three-dimensional structure, contains several bacterial and yeast galactokinases and eukaryotic mevalonate and phosphomevalonate kinases and may have a substrate binding region in common with homoserine kinases. Each of the three families of sugar kinases appears to have a distinct three-dimensional fold, since conserved sequence patterns are strikingly different for the three families. Yet each catalyzes chemically equivalent reactions on similar or identical substrates. The enzymatic function of sugar phosphorylation appears to have evolved independently on the three distinct structural frameworks, by convergent evolution. In addition, evolutionary trees reveal that (1) fructokinase specificity has evolved independently in both the hexokinase and ribokinase families and (2) glucose specificity has evolved independently in different branches of the hexokinase family. These are examples of independent Darwinian adaptation of a structure to the same substrate at different evolutionary times. The flexible combination of active sites and three-dimensional folds observed in nature can be exploited by protein engineers in designing and optimizing enzymatic function.     

Mechanism of preventive effects of exendin-4 and des-fluoro-sitagliptin in a murine model of fructose-induced prediabetes

Protective effects of exendin-4 (glucagon-like peptide-1 -GLP-1- receptor agonist) and des-fluoro-sitagliptin (dipeptidyl peptidase-4 inhibitor) on fructose-induced hepatic disturbances were evaluated in prediabetic rats. Complementary, a possible direct effect of exendin-4 in human hepatoblastoma-derived cell line HepG2 incubated with fructose in presence/absence of exendin-9-39 (GLP-1 receptor antagonist) was investigated. In vivo, after 21 days of fructose rich diet, we determined: glycemia, insulinemia, and triglyceridemia; hepatic fructokinase, AMP-deaminase, and G-6-P dehydrogenase (G-6-P DH) activities; carbohydrate-responsive element-binding protein (ChREBP) expression; triglyceride content and lipogenic gene expression (glycerol-3-phosphate acyltransferase -GPAT-, fatty acid synthase -FAS-, sterol regulatory element-binding protein-1c -SREBP-1c); oxidative stress and inflammatory markers expression. In HepG2 cells we measured fructokinase activity and triglyceride content. Hypertriglyceridemia, hyperinsulinemia, enhanced liver fructokinase, AMP-deaminase, and G-6-P DH activities, increased ChREBP and lipogenic genes expression, enhanced triglyceride level, oxidative stress and inflammatory markers recorded in fructose fed animals, were prevented by co-administration of either exendin-4 or des-fluoro-sitagliptin. Exendin-4 prevented fructose-induced increase in fructokinase activity and triglyceride contain in HepG2 cells. These effects were blunted co-incubating with exendin-9-39. The results demonstrated for the first time that exendin-4/des-fluro-sitagliptin prevented fructose-induced endocrine-metabolic oxidative stress and inflammatory changes probably acting on the purine degradation pathway. Exendin 9–39 blunted in vitro protective exendin-4 effects, thereby suggesting a direct effect of this compound on hepatocytes through GLP-1 receptor. Direct effect on fructokinase and AMP-deaminase activities, with a key role in the pathogenesis of liver dysfunction induced by fructose, suggests purine degradation pathway constitute a potential therapeutic objective for GLP-1 receptor agonists.     

Hexose kinases from the plant cytosolic fraction of soybean nodules

The enzymes responsible for the phosphorylation of hexoses in the plant cytosolic fraction of soybean (Glycine max L. Merr cv Williams) nodules have been studied and a hexokinase (ATP:d-hexose 6-phosphotransferase EC 2.7.1.1) and fructokinase (ATP:d-fructose 6-phosphotransferase EC 2.7.1.4) shown to be involved. The plant cytosolic hexokinase had optimum activity from pH 8.2 to 8.9 and the enzyme displayed typical Michaelis-Menten kinetics. Hexokinase had a higher affinity for glucose (K_m 0.075 millimolar) than fructose (K_m 2.5 millimolar) and is likely to phosphorylate mainly glucose in vivo. The plant cytosolic fructokinase had a pH optimum of 8.2 and required K⁺ ions for maximum activity. The enzyme was specific for fructose (apparent K_m 0.077 millimolar) but concentrations of fructose greater than 0.4 millimolar were inhibitory. The native molecular weight of fructokinase was 84,000 ± 5,000. The roles of these enzymes in the metabolism of glucose and fructose in the host cytoplasm of soybean nodules are discussed.     

Fructokinase (Fraction IV) of Pea Seeds

A fructokinase (EC 2.7.1.4) was obtained from pea (Pisum sativum L.) seeds. This enzyme, termed fructokinase (fraction IV), was specific for fructose as substrate and had little activity with glucose or mannose. Excess fructose inhibited the enzyme at the optimum pH (8.2) but not at pH 6.6. MgATP was inhibitory at pH 6.6. The apparent Michaelis-Menten constants at pH 8.2 were 0.057 mm for fructose and 0.10 mm for MgATP. Mg(2+) ions were essential for activity; Mn(2+) could partially replace Mg(2+). Fructokinase (fraction IV) had a requirement for K(+) ions which could be substantially replaced by Rb(+) or NH(4) (+) but not by Na(+). The enzyme was inhibited by MgADP. The possible significance of fructokinase (fraction IV) in plant carbohydrate metabolism is discussed.     

Sequence Analysis of the groESL-cotA Region of the Bacillus subtilis Genome, Containing the Restriction/Modification System Genes

We have determined a 35-kb sequence of the groESL-gutR-cotA(45°–52°) region of the Bacillus subtilis genome.In addition to the groESL, gutRB and cotA genes reported previously,we have newly identified 24 ORFs including gutA and fruC genes,encoding glucitol permease and fructokinase, respectively. Theinherent restriction/modification system genes, hsdMR and hsdMM,were mapped between groESL and gutRB, and we have identifiedtwo open reading frames (ORFs) encoding 5-methylcytosine formingDNA methyl transferase and an operon probably encoding a restrictionenzyme complex. The unusual genome structure of few ORFs andlower GC content around the restriction/modification genes stronglysuggests that the region originated from a bacteriophage integratedduring evolution.     

Distribution of adenosine 5'-triphosphate (ATP)-dependent hexose kinases in microorganisms.

A systematic study of adenosine triphosphate (ATP)-dependent hexose kinases among microorganisms has been undertaken. Sixteen hexose kinases of five major types were partially purified from 12 microorganisms and characterized with respect to specificity for sugar and nucleotide substrates and Michaelis constants for the sugar substrates. Glucokinase activities that phosphorylate glucose and glucosamine are inhibited by N-acetyl-glucosamine and xylose, were found to be present in the non-sulphur photosynthetic bacteria Rhodospirillum rubrum, the blue-green algae Anacystis montana, and the protists Chlorella pyrenoidosa and Chlamydomonas reinhardtii (green algae), Hypochytrium catenoides (Hypochytridiomycete) and Saprolegnia Iitoralis (Oomycete). The myxobacteria Stigmatella aurantiaca contains a glucokinase activity with a different specificity pattern. Anacystis and Chlorella, besides their glucokinase activities, contain highly specific fructokinases, although that from Anacystis can also phosphorylate fructosamine; fructokinase from Anacystis has a molecular weight of 20 000, and exhibits a sigmoidal saturation curve for ATP when the Mg2+/ATP ratio is 2; this curve is transformed to a Michaelian one when under the same conditions an excess of Mg2+ (5 mM) is added. Saprolegnia however, besides the glucokinase, contains a mannofructokinase activity that phosphorylates mannose (Km 0.06 mM) and fructose (1 mM). On the other hand, hexokinase, a low specificity enzyme, was detected in the protist Allomyces arbuscula (Chytridiomycete) and in fungi Mucor hiemalis and Phycomyces blakesleeanus (Zygomycetes), and Schizophyllum commune (Basidiomycete). Schizophyllum contains a glucomannokinase activity together with hexokinase activity. The pattern of distribution of ATP-dependent hexose kinases among microorganisms seems to parallel that reported for biosynthetic pathways for lysine. The correlation with other biochemical parameters is also considered.     

Substrate specificity and product inhibition of different forms of fructokinases and hexokinases in developing potato tubers

The substrate dependence and product inhibition of three different fructokinases and three different hexokinases from growing potato (Solanum tuberosum L.) tubers was investigated. The tubers contained three specific fructokinases (FK1, FK2, FK3) which had a high affinity for fructose K _m=64, 90 and 100 (M) and effectively no activity with glucose or other hexose sugars. The affinity for ATP (K _m=26, 25 and 240 M) was at least tenfold higher than for other nucleoside triphosphates. All three fructokinases showed product inhibition by high fructose (K _i=5.7, 6.0 and 21 mM) and were also inhibited by ADP competitively to ATP. Sensitivity to ADP was increased in the presence of high fructose, or fructose-6-phosphate. In certain conditions, the K _i (ADP) was about threefold below the K _m (ATP). All three fructokinase were also inhibited by fructose-6-phosphate acting non-competitively to fructose (K _i=1.3 mM for FK2). FK1 and FK2 showed very similar kinetic properties whereas FK3, which is only present at low activities in the tuber but high activities in the leaf, had a generally lower affinity for ATP, and lower sensitivity to inhibition by ADP and fructose. The tuber also contained three hexokinases (HK1, HK2, HK3) which had a high affinity for glucose (K _m=41, 130 and 35 M) and mannose but a poor affinity for fructose (K _m=11, 22 and 9 mM). All three hexokinases had a tenfold higher affinity for ATP (K _m=90, 280 and 560 M) than for other nucleoside triphosphates. HK1 and HK2 were both inhibited by ADP (K _i=40 and 108 M) acting competitively to ATP. HK1, but not HK2, was inhibited by glucose-6-phosphate, which acted non-competitively to glucose (K _i=4.1 mM). HK1 and HK2 differed, in that HK1 had a narrower pH optimum, a higher affinity for its substrate, and showed inhibition by glucose-6-phosphate. The relevance of these properties for the regulation of hexose metabolism in vivo is discussed.Abbreviations FK fructokinase - Fru6P fructose-6-phosphate - Glc6P glucose-6-phosphate - HK hexokinase - NTP nucleoside triphosphate - P_i inorganic phosphate - UDPGlc uridine-5-diphosphoglucose This work was supported by the Deutsche Froschungsgemeinschaft (SFB 137). We are grateful to Professor E. Beck (Lehrstuhl für Pflanzenphysiologie, Universität Bayreuth, FRG) for providing laboratory facilities.     

Estimation of fructokinase in crude tissue preparations

An assay for fructokinase activity is described that permits an accurate estimation of specific fructokinase in crude tissue preparations without interference of hexokinase activity. It utilizes two properties of hexokinases which differentiate hexokinase from fructokinase: (1) hexokinase activity is more labile to [H⁺] than is fructokinase, and (2) hexokinase activity is markedly inhibited by N-acetylglucosamine while fructokinase activity is relatively unaffected.     

Nucleotide sequence and analysis of the Vibrio alginolyticus sucrose uptake-encoding region

The nucleotide sequence of the Vibrio alginolyticus sucrose uptake-encoding region was determined, and contained two genes, scrA and scrK. The scrA gene encodes an enzyme IISucrose (EIIScr) protein of the phosphoenolpyruvate dependent phosphotransferase system and the scrK gene encodes a fructokinase. The deduced amino acid (aa) sequence for the V. alginolyticus EIIScr protein was homologous with the EIIScr proteins from Streptococcus mutans, Salmonella typhimurium (pUR400 system) and Bacillus subtilis. The deduced aa sequence for the V. alginolyticus fructokinase was homologous with the Escherichia coli enzymes, 6-phosphofructokinase (isoenzyme 2) and ribokinase. Transposon phoA mutagenesis experiments indicated that the EIIScr protein was a membrane-bound protein with a region that extended into the periplasm.