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PubMed1992

Characterization of a chromosomally encoded, non-PTS metabolic pathway for sucrose utilization in Escherichia coli EC3132.

A wild-type isolate, EC3132, of Escherichia coli, that is able to grow on sucrose was isolated and its csc genes (mnemonic for chromosomally coded sucrose genes) transferred to strains of E. coli K12. EC3132 and all sucrose-positive exconjugants and transductants invariably showed a D-serine deaminase (Dsd)-negative phenotype. The csc locus maps adjacent to dsdA, the structural gene for the D-serine deaminase, and contains an inducible regulon, controlled by a sucrose-specific repressor CscR, together with structural genes for a sucrose hydrolase (invertase) CscA, for a D-fructokinase CscK, and for a transport system CscB. Based on DNA sequencing studies, this last codes for a hydrophobic protein of 415 amino acids. CscB is closely related to the beta-galactoside transport system LacY (31.2% identical residues) and a raffinose transport system RafB (32.3% identical residues) of the enteric bacteria, both of the proton symport type. A two-dimensional model common to the three transport proteins, which is based on the integrated consensus sequence, will be discussed.

Amino Acid Sequence↗
PubMed1982

Isoenzyme studies on cercariae from monoinfections and adult worms of Schistosoma mansoni (10 isolates) and S. rodhaini (one isolate) by horizontal polyacrylamide gel electrophoresis and staining of eight enzymes.

The enzyme phosphoglucose isomerase (PGI), phosphoglucomutase (PGM), hexokinase (HK), adenylate kinase (AK), fructokinase (FK), mannose-6-phosphate isomerase (MPI), glucose-6-phosphate dehydrogenase (G-6-PDH) and malate dehydrogenase (MDH) were chosen to study the variation between isolates, cercariae and adults, individuals, and sexes of Schistosoma mansoni and S. rodhaini, using horizontal polyacrylamide gel electrophoresis. The method described allows combinations of six of the eight enzymes to be scored in the homogenate from one adult worm. In adult S. mansoni one phenotype of the eight enzymes was observed in all isolates. In addition, the enzyme PGI showed polymorphism in the isolates from Tala, Kenya and Uganda. PGM in the isolates from Tala, Kenya and South Africa showed polymorphism. The cercarial phenotype differs from the adult phenotype in G-6-PDH, where the cercarial enzyme mobility is slower than that in the adult worm. The low amount of intrastrain variation observed in this species is explained by the limited amount of material used to establish the laboratory stocks, whereas the genetic similarity between geographically widely separated stocks does suggest that only limited geographical variation is likely to occur in S. mansoni. It is suggested that the gene controlling the PGI polymorphism is located on the sex chromosomes of S. mansoni. Mobility differences were observed between S. mansoni and S. rodhaini in the enzyme PGI and PGM, and these characteristics might be useful for a quick identification of schistosome cercariae emerging from Biomphalaria sp. in Africa.U

Adenylate Kinase↗
PubMed2006

Characterization of genes involved in fructose utilization by Lactobacillus fermentum.

The genes encoding phosphoglucose isomerase (fruI) and fructokinase (fruK) of Lactobacillus fermentum NRRL-B-1932 were sequenced. They constituted an operon, which is involved in fructose metabolism of this strain by channeling intracellular fructose into the phosphoketolase pathway. A third open reading frame, unkR, upstream of the operon was identified as homologous to genes of LacI/GalR family repressors. The UnkR repressor's role in transcriptional control of the fruIK operon could, however, not be established by electrophoretic mobility shift assay (EMSA) analysis. Sequence analysis revealed two putative catabolite responsive elements (cre) in the promoter region of fruIK suggesting that the fruIK operon is under negative regulatory control by carbon catabolite repression. Expression and enzyme activity data were compatible with the assumption that the fruIK operon is repressed by glucose. No sugar specific phosphoenolpyruvate sugar transferase system activity for the transport of fructose, glucose, sucrose or mannose could be detected in L. fermentum NRRL-B-1932 cells, which suggest that fructose is taken up by a permease system.

Base Sequence↗
PubMed1996

Co-localization of the ketohexokinase and glucokinase regulator genes to a 500-kb region of chromosome 2p23.

The glucokinase regulator (GCKR) is a 65-kDa protein that inhibits glucokinase (hexokinase IV) in liver and pancreatic islet. The role of glucokinase (GCK) as pancreatic beta cell glucose sensor and the finding of GCK mutations in maturity onset diabetes of the young (MODY) suggest GCKR as a further candidate gene for type 2 diabetes. The inhibition of GCK by GCKR is relieved by the binding of fructose-1-phosphate (F-1-P) to GCKR. F-1-P is the end product of ketohexokinase (KHK, fructokinase), which, like GCK and GCKR, is present in both liver and pancreatic islet. KHK is the first enzyme of the specialized pathway that catabolizes dietary fructose. We have isolated genomic clones containing the human GCKR and KHK genes. By fluorescent in situ hybridization (FISH), KHK maps to Chromosome (Chr) 2p23.2-23.3, a new assignment corroborated by somatic cell hybrid analysis. The localization of GCKR, originally reported by others as 2p22.3, has been reassessed by high-resolution FISH, indicating that, like KHK, GCKR maps to 2p23.2-23.3. The proximity of GCKR and KHK was further demonstrated both by two-color interphase FISH, which suggests that the two genes lie within 500 kb of each other, and by analysis of overlapping YAC and P1 clones spanning the interval between GCKR and KHK. A new microsatellite polymorphism was used to place the GCKR-KHK locus between D2S305 and D2S165 on the genetic map. The colocalization of these two metabolically connected genes has implications for the interpretation of linkage or allele association studies in type 2 diabetes. It also raises the possibility of coordinate regulation of GCKR and KHK by common cis-acting regulatory elements.

Base Sequence↗
PubMed2004

Sugar utilization and anoxia tolerance in rice roots acclimated by hypoxic pretreatment.

Although most cereal roots cannot elongate under anoxic conditions, primary roots of three-day-old rice (Oryza sativa L.) seedlings were able to elongate during a 24-h period of anoxia. Hypoxic pretreatment (H-PT) increased the elongation of their roots. Sucrose synthase (EC 2.4.1.13), glucokinase (EC 2.7.1.2), fructokinase (EC 2.7.1.4), pyruvate decarboxylase (EC 4.1.1.1) and alcohol dehydrogenase (EC 1.1.1.1) activities were increased by anoxia in both H-PT and non-pretreated (N-PT) roots. However, these activities were greater in the H-PT roots than in the N-PT roots. The average rate of production of ethanol for the initial 6h after the onset of anoxia was 3.7 and 1.4 micromolg(-1) fresh weight h(-1) for the H-PT and N-PT roots, respectively, suggesting that ethanolic fermentation may increase more quickly in the H-PT roots than in the N-PT roots. Roots of the seedlings lost ATP and total adenine nucleotides in anoxia, however, the H-PT roots maintained higher levels of ATP and total adenine nucleotides compared to the N-PT roots. These results show that rice roots are able to utilize the set of enzymes involved in the metabolism of soluble sugars under anoxia. The ability to maintain an active fermentative metabolism for production of ATP by fueling the glycolytic pathway with fermentable carbohydrate is probably greater in H-PT than in N-PT roots.

Acclimatization↗
PubMed2000

Characterization of isoforms of hexose kinases in rice embryo.

Hexose kinases in rice embryos have been characterized. Six isoforms were detected: i.e. three glucokinases (GK1-3), two hexokinases (HK1 and HK2) and one fructokinase (FK1). Out of these, GK3, HK1 and HK2 were inhibited by mannoheptulose and glucosamine, known inhibitors of hexokinase activity. These inhibitors are also known to be modulators of sugar sensing processes. The results suggest that GK3, HK1 and HK2 may play a role in sensing the cellular sugar status in the rice embryo.

Fructokinases↗
PubMed1998

Structure and function of the genes involved in mannitol, arabitol and glucitol utilization from Pseudomonas fluorescens DSM50106.

A DNA fragment from Pseudomonas fluorescens DSM50106 containing the genes for the uptake and utilization of mannitol, arabitol and glucitol was cloned in Escherichia coli and sequenced. Seven open reading frames (mtlEFGKDYZ) were identified on the 10031 bp fragment. The deduced amino acid sequences of the first four open reading frames (mtlEFGK) revealed significant similarity to the components of the maltose transport system in E. coli and Salmonella typhimurium. The gene mtlD encoding a polyol dehydrogenase was located downstream of mtlK. The deduced proteins of the last two genes on the fragment showed a high similarity to a fructokinase from Vibrio alginolyticus (MtlZ) and a xylulose kinase from Streptomyces rubiginosus (MtlY), respectively. Both genes were expressed in E. coli. MtlZ phosphorylated fructose, glucose and glucitol whereas MtlY was highly specific for xylulose. Upstream of mtlE, a putative promoter/operator region was identified by promoter probe studies which was active in P. fluorescens but not in E. coli.

Aldehyde Reductase↗
PubMed1999

Effects of medium containing heparin and theophylline on capacitation and metabolic enzyme activities of ejaculated spermatozoa from dogs with asthenozoospermia.

The percentages of motile sperm (%MO), hyperactivated sperm (%HA), and acrosome-reacted sperm (%AR) of four beagle dogs with asthenozoospermia (AS) and five normal beagle dogs were determined during 7 h of incubation. The metabolic enzyme activities of the sperm was examined after 0 and 4 h of incubation. The sperm were incubated in canine capacitation medium (CCM) and CCM containing either 20 microg ml(-1) heparin (HE), 10 microg ml(-1) theophylline (TH) or 20 microg ml(-1) HE + 10 microg ml(-1) TH in glass tubes at 38 degrees C under 5% CO2 in air. The %HA and %AR were determined by counting the sperm exhibiting star-spin like movement and by the triple stain technique. The spermatozoa in HE + TH CCM were homogenized and centrifuged, and the metabolic activities of hexokinase, fructokinase, glucose-6-phosphodehydrogenase (G6PD), and pyruvate kinase in the sperm cytosol in the supernatant was measured with a spectrophotometer. The mean %MO and %HA values of both AS and normal dogs in the four types of CCM were highest in HE + TH CCM, with a mean %HA in HE + TH CCM of 78 +/- 5% (S.E.) after 7 h of incubation. However, there was little difference in %AR among the four types of CCM. The mean activities of the four enzymes in the sperm of AS dogs before incubation was significantly lower than in the sperm of normal dogs (P < 0.05, 0.01). However, after 4 h of incubation the activities of all enzymes in the sperm of both AS and normal dogs was clearly higher in HE + TH CCM than in the control CCM. These findings indicate that HE and TH in the medium are effective inactivating metabolic enzymes, maintaining longer sperm motility, and efficiently inducing HA even of the sperm of AS dogs.

Acrosome Reaction↗
PubMed1980

Aldehyde-induced adenosine triphosphatase activities of fructose 6-phosphate and fructose kinases.

Chitose-6-P (2,5-anhydromannose-6-P) induces ATPase activity of fructose-6-P kinase with a Vmax 2-3% that of the normal kinase reaction with fructose-6-P or 2,5-anhydromannitol. Chitose (and presumably also chitose-6-P) is 52% hydrated in water while chitose deuterated at C-1 is 60% hydrated because of the equilibrium isotope effect of 0.73 on aldehyde hydration. Deuterated chitose-6-P gave a normal isotope effect on V/K of 1.23, but no effect on Vmax, showing that the free aldehyde is the activator and the hydrated form does not bind appreciably. With fructokinase, chitose can act either as a substrate, being phosphorylated at C-6 when adsorbed with C-6 next to MgATP, or as an inducer of ATPase activity when adsorbed with C-1 next to MgATP. The ATPase has a rate about 25% that of the kinase.

Adenosine Triphosphatases↗
PubMed2005

The crystal structure of Mlc, a global regulator of sugar metabolism in Escherichia coli.

Mlc from Escherichia coli is a transcriptional repressor controlling the expression of a number of genes encoding enzymes of the phosphotransferase system (PTS), including ptsG and manXYZ, the specific enzyme II for glucose and mannose PTS transporters. In addition, Mlc controls the transcription of malT, the gene of the global activator of the mal regulon. The inactivation of Mlc as a repressor is mediated by binding to an actively transporting PtsG (EIICB(Glc)). Here we report the crystal structure of Mlc at 2.7 A resolution representing the first described structure of an ROK (repressors, open reading frames, and kinases) family protein. Mlc forms stable dimers thus explaining its binding affinity to palindromic operator sites. The N-terminal helix-turn-helix domain of Mlc is stabilized by the amphipathic C-terminal helix implicated earlier in EIICB(Glc) binding. Furthermore, the structure revealed a metal-binding site within the cysteine-rich ROK consensus motif that coordinates a structurally important zinc ion. A strongly reduced repressor activity was observed when two of the zinc-coordinating cysteine residues were exchanged against serine or alanine, demonstrating the role of zinc in Mlc-mediated repressor function. The structures of a putative fructokinase from Bacillus subtilis, the glucokinase from Escherichia coli, and a glucomannokinase from Arthrobacter sp. showed high structural homology to the ROK family part of Mlc.

Alanine↗
PubMed1993

Inborn errors of fructose metabolism.

A review is presented of genetic defects affecting fructose metabolism in humans. Presently, six conditions have been recognized: fructose malabsorption, fructokinase deficiency, aldolase A and aldolase B deficiency, fructose-1,6-diphosphatase deficiency and D-glyceric aciduria. Clinical presentations of these conditions, enzymatic and/or molecular defects, pathophysiological consequences, and modes of treatments are discussed.

Fructokinases↗
PubMed2003

Response of potato tuber cell division and growth to shade and elevated CO2.

Plants adjust their sink-organ growth rates, development and distribution of dry matter in response to whole-plant photosynthate status. To advance understanding of these processes, potato (Solanum tuberosum L.) plants were subjected to CO(2) and light flux treatments, and early tuber growth was assessed. Atmospheric CO(2) (700 or 350 micro mol mol(-1)) and light flux (shade and control illumination) treatments were imposed at two growth stages: tuber initiation (TI) and tuber bulking (TB). Elevated CO(2) increased accumulation of total net biomass when imposed at both stages, and increased tuber growth rate by about 36 %, but did not increase the number of tubers. Elevated CO(2) increased the number of cells in tubers at both TI and TB stages, whereas shade substantially decreased the number of cells at both stages. Generally, treatments did not affect cell volume or the proportion of nuclei endoreduplicating (repeated nuclear DNA replication in the absence of cell division), but the shade treatment led to a decrease in cell volume at TB and a decrease in endoreduplication at TI. Elevated CO(2) increased, and shade decreased, glucose concentration and soluble invertase activity in the cambial zones at both TI and TB, whereas sucrose concentration and activities of glucokinase, fructokinase, cell-wall-bound invertase and thymidine kinase were unaffected. Modulation of tuber cell division was responsible for much of the growth response to whole-plant photosynthate status, and treatments affected cambial-zone glucose and soluble invertase in a pattern suggesting involvement of a glucose signalling pathway.

Carbohydrate Metabolism↗
PubMed1988

Analysis of sucrose catabolism in Klebsiella pneumoniae and in Scr+ derivatives of Escherichia coli K12.

In contrast to a previous report, strains of Klebsiella pneumoniae were found to take up and phosphorylate the disaccharide sucrose via the phosphoenolpyruvate-dependent carbohydrate phosphotransferase system (PTS). In addition to the two soluble and general components enzymeI and HPr of the PTS, a sucrose-specific enzymeIIScr (gene scrA), together with the enzymeIII, coded for by the gene crr, were needed for the vectorial phosphorylation of sucrose to generate intracellular sucrose 6-phosphate. This sugar phosphate is hydrolysed by a hydrolase (invertase, gene scrB) to generate glucose 6-phosphate and free fructose. The latter is converted to fructose 6-phosphate by an ATP-dependent fructokinase (gene scrK), an enzyme which is part of the sucrose and not of the fructose catabolic pathway. Analysis of different mutants of K. pneumoniae strain 1033, and of Escherichia coli K12 derivatives carrying R'scr plasmids isolated from K. pneumoniae, showed that the genes scrA, B, and K, together with a gene scrR for a repressor, form a genetic unit located on the chromosome of K. pneumoniae. These genes and the corresponding sucrose metabolic pathway are very similar to a previously described scr system encoded on plasmid pUR400 and found in other enteric bacteria.

Chromosome Mapping↗
PubMed2004

Genomic subtraction for the identification of putative new virulence factors of an avian pathogenic Escherichia coli strain of O2 serogroup.

To identify putative new virulence factors of avian pathogenic Escherichia coli (APEC) strains, a genomic subtraction was performed between the APEC strain MT512 and the non-pathogenic E. coli strain of avian origin EC79. Seventeen DNA fragments were cloned that were specific for the APEC strain. Among them, nine were identified that were more frequent among pathogenic than non-pathogenic isolates in a collection of 67 avian E. coli. Chromosome or plasmid location, and the nucleotide sequence of these nine fragments were characterized. Four fragments were plasmid-located. The nucleotide sequence of two of them exhibited identity with the sequence of the RepF1B replicon of E. coli plasmids, and the amino-acid deduced sequences from the two other fragments exhibited similarity to the products of genes sitA of Salmonella Typhimurium and iroD of E. coli, which are involved in iron metabolism. Of the five chromosome-located fragments, three were predicted to encode parts of proteins that were significantly homologous to previously described proteins: TktA (transketolase) of Haemophilus influenzae, a FruA (fructokinase) homologue of Listeria innocua and Gp2 (large terminal subunit) of phage 21. The putative products of the two other chromosome-located fragments were homologous to proteins with unknown functions: Z0255 of E. coli strain EDL933 (EHEC) and RatA of Salmonella Typhimurium strain LT2. Both these chromosomal fragments, whose presence is correlated with serogroups O1 and O2 and to the virulence of APEC strains belonging to these serogroups, are good candidates for being part of novel virulence determinants of APEC. Moreover, several fragments were shown to be located close to tRNA selC, asnT or thrW, which suggests they could be part of pathogenicity islands. Six fragments that were shown to be part of whole ORFs present in the APEC strain MT 512 were also present in extra-intestinal pathogenic E. coli (ExPEC) strains of human and animal origin. Thus, the putative novel virulence factors identified in this study could be shared by ExPEC strains of different origins.

Animals↗
PubMed2003

Metabolism of sugars in the endosperm of developing seeds of oilseed rape.

The sugars in the endosperm of a developing seed have many potential roles, including the supply of carbon to the developing embryo and controlling gene expression in it. Our understanding of their metabolism is, however, fragmentary and is confined to a very few species (especially Vicia spp.). To develop a quantitative understanding of the regulation of sugars in seeds of oilseed rape (Brassica napus), we measured relevant enzyme activities, the sizes of the pools of sugars in the liquid endosperm, and the flux of sugars from the endosperm into the embryo. The concentrations of hexose sugars in the liquid endosperm decreased, and sucrose (Suc) increased through development. The overall osmotic potential also fell. The timing of the changes was not precise enough to determine whether they signaled the onset of rapid accumulation of storage products. Changes in endosperm invertase activity were complex and quantitatively do not explain the changes in sugars. The embryo can metabolize hexose sugars in addition to Suc, and possibly at higher rates. Therefore, in addition to invertase, the growing embryo itself has a potential to influence the balance of sugars in the endosperm. The activity of Suc synthase in the embryo was greater than that of invertase during development. This observation and a higher activity of fructokinase than glucokinase in the embryo are both consistent with the embryo using Suc as a carbon source.

Brassica napus↗
PubMed2006

An engineered sorbitol cycle alters sugar composition, not growth, in transformed tobacco.

Many efforts have been made to engineer stress tolerance by accumulating polyols. Transformants that accumulate polyols often show growth inhibition, because polyols are synthesized as a dead-end product in plants that do not naturally accumulate polyols. Here, we show a novel strategy in which a sorbitol cycle was engineered by introducing apple cDNA encoding NAD-dependent sorbitol dehydrogenase (SDH) in addition to sorbitol-6-phosphate dehydrogenase (S6PDH). Tobacco plants transformed only with S6PDH showed growth inhibition, and very few transformants were obtained. In contrast, many transgenic plants with both S6PDH and SDH were easily obtained, and their growth was normal despite their accumulation of sorbitol. Interestingly, the engineered sorbitol cycle enhanced the accumulation of sucrose instead of fructose that was expected to be increased. Sucrose, rather than fructose, was also increased in the immature fruit of tomato plants transformed with an antisense fructokinase gene in which the phosphorylation of fructose was inhibited. A common phenomenon was observed in the metabolic engineering of two different pathways, showing the presence of homeostatic regulation of fructose levels.

Antisense Elements (Genetics)↗
PubMed1980

Partial purification and properties of a mannofructokinase from Streptococcus mutans SL-1.

Fructokinase activity was demonstrated in seven strains of oral streptococci. The enzyme purified from Streptococcus mutans SL-1 was capable of phosphorylating both D-fructose and D-mmannose to their respective 6-phosphates. Phosphorylation of both fructose and mannose was dependent on adenosine 5'-triphosphate and a divalent metal ion. The molecular weight of the purified enzyme was estimated to be 49,000. The apparent Km of the enzyme for fructose was 0.63 mM. This enzyme also utilized mannose as a substrate, with an apparent Km for mannose of 0.37 mM. Since the activities of the enzyme toward mannose and fructose were not separated upon purification of the enzyme and since mannose was a competitive inhibitor of fructose phosphorylation, the purified kinase is a single enzyme, mannofructokinase, with dual specificity for both mannose and fructose. A role for this enzyme in carbohydrate metabolism in S. mutans is postulated.

Adenosine Triphosphate↗
PubMed2002

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.

Adaptation, Physiological↗