No data available that match "Fructokinases"
No data available that match "Fructokinases"
(1/59) Characterization of the divergent sacBK and sacAR operons, involved in sucrose utilization by Lactococcus lactis.
The divergently transcribed sacBK and sacAR operons, which are involved in the utilization of sucrose by Lactococcus lactis NZ9800, were examined by transcriptional and gene inactivation studies. Northern analyses of RNA isolated from cells grown at the expense of different carbon sources revealed three sucrose-inducible transcripts: one of 3.2 kb containing sacB and sacK, a second of 3.4 kb containing sacA and sacR, and a third of 1.8 kb containing only sacR. The inactivation of the sacR gene by replacement recombination resulted in the constitutive transcription of the sacBK and sacAR operons in the presence of different carbon sources, indicating that SacR acts as a repressor of transcription. (+info)
(2/59) The genes controlling sucrose utilization in Clostridium beijerinckii NCIMB 8052 constitute an operon.
The sucrose operon of Clostridium beijerinckii NCIMB 8052 comprises four genes, which encode a sucrose-specific enzyme IIBC(Scr) protein of the phosphotransferase system (ScrA), a transcriptional repressor (ScrR), a sucrose hydrolase (ScrB) and an ATP-dependent fructokinase (ScrK). The scrARBK operon was cloned in Escherichia coli in three stages. Initial isolation was achieved by screening a C. beijerinckii genomic library in E. coli for clones able to utilize sucrose, while the remainder of the operon was isolated by inverse PCR and by plasmid rescue of flanking regions from a scrB mutant constructed by targeted gene disruption. Substrate specificity assays confirmed that the sucrose hydrolase was a beta-fructofuranosidase, able to hydrolyse sucrose and raffinose but not inulin or levans, and that the scrK gene encoded an ATP/Mg2+-dependent fructokinase. Both enzyme activities were induced by sucrose in C. beijerinckii. Disruption of the scr operon of C. beijerinckii by targeted plasmid integration into either the scrR or the scrB gene resulted in strains unable to utilize sucrose, indicating that this was the only inducible sucrose catabolic pathway in this organism. RNA analysis confirmed that the genes of the scr operon were co-transcribed on a 5 kb mRNA transcript and that transcription was induced by sucrose, but not by glucose, fructose, maltose or xylose. Primer extension experiments identified the transcriptional start site as lying 44 bp upstream of the scrA ATG start codon, immediately adjacent to the imperfect pelindrome sequence proposed to be a repressor binding site. Disruption of the scrR gene resulted in constitutive transcription of the upstream scrA gene, suggesting that ScrR encodes a transcriptional repressor which acts at the scrA operator sequence. The scrR gene is therefore itself negatively autoregulated as part of the polycistronic scrARBK mRNA (+info)
(3/59) Molecular analysis of sucrose metabolism of Erwinia amylovora and influence on bacterial virulence.
Sucrose is an important storage and transport sugar of plants and an energy source for many phytopathogenic bacteria. To analyze regulation and biochemistry of sucrose metabolism of the fire blight pathogen Erwinia amylovora, a chromosomal fragment which enabled Escherichia coli to utilize sucrose as sole carbon source was cloned. By transposon mutagenesis, the scr regulon of E. amylovora was tagged, and its nucleotide sequence was determined. Five open reading frames, with the genes scrK, scrY, scrA, scrB, and scrR, had high homology to genes of the scr regulons from Klebsiella pneumoniae and plasmid pUR400. scrB and scrR of E. amylovora were fused to a histidine tag and to the maltose-binding protein (MalE) of E. coli, respectively. ScrB (53 kDa) catalyzed the hydrolysis of sucrose with a K(m) of 125 mM. Binding of a MalE-ScrR fusion protein to an scrYAB promoter fragment was shown by gel mobility shifts. This complex dissociated in the presence of fructose but not after addition of sucrose. Expression of the scr regulon was studied with an scrYAB promoter-green fluorescent protein gene fusion and measured by flow cytometry and spectrofluorometry. The operon was affected by catabolite repression and induced by sucrose or fructose. The level of gene induction correlated to the sucrose concentration in plant tissue, as shown by flow cytometry. Sucrose mutants created by site-directed mutagenesis did not produce significant fire blight symptoms on apple seedlings, indicating the importance of sucrose metabolism for colonization of host plants by E. amylovora. (+info)
(4/59) Overexpression of fructose 2,6-bisphosphatase decreases glycolysis and delays cell cycle progression.
The ability to overexpress 6-phosphofructo-2-kinase/fructose 2, 6-bisphosphatase (PFK-2)/(FBPase-2) or a truncated form of the enzyme with only the bisphosphatase domain allowed us to analyze the relative role of the kinase and the bisphosphatase activities in regulating fructose 2,6-bisphosphate (Fru-2,6-P(2)) concentration and to elucidate their differential metabolic impact in epithelial Mv1Lu cells. The effect of overexpressing PFK-2/FBPase-2 resulted in a small increase in the kinase activity and in the activity ratio of the bifunctional enzyme, increasing Fru-2,6-P(2) levels, but these changes had no major effects on cell metabolism. In contrast, expression of the bisphosphatase domain increased the bisphosphatase activity, producing a significant decrease in Fru-2,6-P(2) concentration. The fall in the bisphosphorylated metabolite correlated with a decrease in lactate production and ATP concentration, as well as a delay in cell cycle. These results provide support for Fru-2,6-P(2) as a regulator of glycolytic flux and point out the role of glycolysis in cell cycle progression. (+info)
(5/59) 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. (+info)
(6/59) Genetic control of manno(fructo)kinase activity in Escherichia coli.
Mutants of Escherichia coli unable to use fructose by means of the phosphoenolpyruvate/glycose phosphotransferase system mutate further to permit growth on that ketose by derepression of a manno(fructo)kinase (Mak(+) phenotype) present in only trace amounts in the parent organisms (Mak-o phenotype). The mak gene was located at min 8.8 on the E. coli linkage map as an ORF designated yajF, of hitherto unknown function; it specifies a deduced polypeptide of 344 aa. The derepression of Mak activity was associated with a single base change at position 71 (codon 24) of the gene, where GCC (alanine) in Mak-o has been changed to GAC (aspartate) in Mak(+). By cloning selected portions of the total 1,032-bp mak gene into a plasmid that also carried a temperature-sensitive promoter, we showed that the mutation resided in a 117-bp region that does not specify sequences necessary for Mak activity but was located 46 bp upstream of a 915-bp portion that does. Mak(+) and Mak-o strains differ greatly in the heat stability of the enzyme: at 61 degrees C, mak-o cloned into a mak-o recipient loses 50% of its activity in approximately 6 min, whereas it takes over 30 min to achieve a similar reduction in the activity of mak(+) cloned into a mak-o strain. However, the Mak activity of the cloned fragment specifying the enzyme without the regulatory region lost activity with a half-life of 29 min irrespective of whether it was derived from a mak(+) or a mak-o donor, which indicates that the A24D mutation contributes to the high enzyme activity of Mak(+) mutants by serving to protect Mak from denaturation. (+info)
(7/59) 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. (+info)
(8/59) Adaptation of sucrose metabolism in the Escherichia coli wild-type strain EC3132.
Although Escherichia coli strain EC3132 possesses a chromosomally encoded sucrose metabolic pathway, its growth on low sucrose concentrations (5 mM) is unusually slow, with a doubling time of 20 h. In this report we describe the subcloning and further characterization of the corresponding csc genes and adjacent genes. The csc regulon comprises three genes for a sucrose permease, a fructokinase, and a sucrose hydrolase (genes cscB, cscK, and cscA, respectively). The genes are arranged in two operons and are negatively controlled at the transcriptional level by the repressor CscR. Furthermore, csc gene expression was found to be cyclic AMP-CrpA dependent. A comparison of the genomic sequences of the E. coli strains EC3132, K-12, and O157:H7 in addition to Salmonella enterica serovar Typhimurium LT2 revealed that the csc genes are located in a hot spot region for chromosomal rearrangements in enteric bacteria. The comparison further indicated that the csc genes might have been transferred relatively recently to the E. coli wild-type EC3132 at around the time when the different strains of the enteric bacteria diverged. We found evidence that a mobile genetic element, which used the gene argW for site-specific integration into the chromosome, was probably involved in this horizontal gene transfer and that the csc genes are still in the process of optimal adaptation to the new host. Selection for such adaptational mutants growing faster on low sucrose concentrations gave three different classes of mutants. One class comprised cscR(Con) mutations that expressed all csc genes constitutively. The second class constituted a cscKo operator mutation, which became inducible for csc gene expression at low sucrose concentrations. The third class was found to be a mutation in the sucrose permease that caused an increase in transport activity. (+info)