Fructosyl Transfer between 1-Kestose and Sucrose in Wheat Leaves

The labeling pattern of the sugar moieties of 1-kestose after in vivo pulse labeling with ¹⁴CO₂ was not the same as that after in vitro labeling with ¹⁴C-sucrose. The two fructosyl residues of 1-kestose had similar specific radioactivities after in vitro synthesis, but after in vivo radiolabeling the specific radioactivity of the terminal fructosyl moiety was significantly less than the internal fructosyl moiety. Evidence is presented that the uneven specific radioactivity of in vivo radiolabeling results from enzymatic transfer of terminal fructosyl residue from 1-kestose to sucrose.     

An enzymatic method for the determination of hemoglobinA<Subscript>1C</Subscript>

Fructosyl peptide oxidase is a flavoenzyme that catalyzes the oxidative deglycation of N-(1-deoxyfructosyl)-Val-His, a model compound of hemoglobin (Hb)A_1C. To develop an enzymatic method for the measurement of HbA_1C, we screened for a proper protease using N-(1-deoxyfructosyl)-hexapeptide as a substrate. Several proteases, including Neutral protease from Bacillus polymyxa, were found to release N-(1-deoxyfructosyl)-Val-His efficiently, however no protease was found to release N-(1-deoxyfructosyl)-Val. Neutral protease also digested HbA_1C to release N-(1-deoxyfructosyl)-Val-His, and then the fructosyl peptide was detected using fructosyl peptide oxidase. The linear relationship was observed between the concentration of HbA_1C and the absorbancy of fructosyl peptide oxidase reaction, hence this new method is a practical means for measuring HbA_1C.     

Active site analysis of fructosyl amine oxidase using homology modeling and site-directed mutagenesis

A three-dimensional structural model of fructosyl amine oxidase from the marine yeast Pichia N1-1 was generated using the crystal structure of monomeric sarcosine oxidase from Bacillus sp. B-0618 as template. The putative active site region was investigated by site-directed mutagenesis, identifying several amino acid residues likely playing important roles in the enzyme reaction. Asn354 was identified as a residue that plays an important role in substrate recognition and that can be substituted in order to change substrate specificity while maintaining high catalytic activity. While the Asn354Ala substitution had no effect on the V _max K _m⁻¹ value for fructosyl valine, the V _max K _m⁻¹ value for fructosyl-^ε N-lysine was decreased 3-fold, thus resulting in a 3-fold improvement in specificity for fructosyl valine over fructosyl-^ε N-lysine.     

Occurrence of Fructosyl-Amino Acid Oxidase-Reactive Compounds in Fungal Cells

Fructosyl-amino acid oxidase (FAOD)-reactive fraction (FRY) was found in commercial yeast extract. FRY showed very hydrophilic property and was adsorbed to phenylboronate silica gel, indicating that it contained the Amadori compound. TLC and amino acid analyses revealed that glucosone, lysine, and arginine were produced from FRY after incubation with FAOD. TOF-MS analysis confirmed that FRY is a mixture of fructosyl lysine and fructosyl arginine. These compounds were also detected in mycelial extract of an FAOD-producer, Aspergillus terreus GP1, grown on the minimum medium, suggesting that a glycation reaction occurs in fungal cells and that FAOD acts toward the resultant Amadori compounds.     

Glucofructosan biosynthesis in Fusarium oxysporum

Low MW glucofructosans have been detected in the medium of Fusarium oxysporum. A 53-fold purification of fructosyl transferase has been achieved by ethanol precipitation, DEAE-cellulose and Sephadex G-100 column chromatography. Maximum fructosyl transferase activity coincided with maximum glucofructosan concentration in the medium. Invertase showed greatest activity in the later stages of growth when glucofructosans were absent. Fructosyl transferase and invertase have been separated by DEAE-cellulose column chromatography. On the basis of kinetic studies and effect of nucleotides on fructosyl transferase in the presence and absence of MgCl₂, a two site active centre linked through a nucleotide bridge is proposed. Fructosyl transferase and invertase are highly phosphorylated.     

Construction of engineered fructosyl peptidyl oxidase for enzyme sensor applications under normal atmospheric conditions

Current enzymatic methods for the analysis of glycated proteins use flavoenzymes that catalyze the oxidative deglycation of fructosyl peptides, designated as fructosyl peptidyl oxidases (FPOXs). However, as FPOXs are oxidases, the signals derived from electron mediator-type electrochemical monitoring based on them are affected by dissolved O₂. Improvement of dye-mediated dehydrogenase activity of FPOXs and its application to enzyme electrode construction were therefore undertaken. Saturation mutagenesis study on Asn56 of FPOX from Phaeosphaeria nodorum, produced mutants with marked decreases in the catalytic ability to employ O₂ as the electron acceptor, while showing higher dye-mediated dehydrogenase activity employing artificial electron acceptors than the parental enzyme. Thus constructed virtually fructosyl peptide dehydrogenase, Asn56Ala, was then applied to produce an enzyme electrode for the measurement of fructosyl-^α N-valyl-histidine (f-^αVal-His), the protease-digested product of HbA1c. The enzyme electrode could measure f-^αVal-His in the physiological target range in air.     

Glucofructosan metabolism in Cichorium intybus roots

A 10-fold purification of sucrose sucrose fructosyl transferase from Cichorium intybus roots was achieved by ammonium sulphate fractionation and DEAE-cellulose column chromatography. The energy of activation for this enzyme was ca 48 kJ/mol sucrose. Sucrose sucrose fructosyl transferase and invertase were prominent during early months of growth. Evidence obtained from: (1) the changes in carbohydrate composition at monthly intervals; (2) comparative studies on fructosyl transferase and invertase at different stages of root growth; and (3) incubation studies with [¹⁴C]glucose, [¹⁴C]fructose and [¹⁴C]sucrose revealed that, during the later stages of root growth, fructosan hydrolase is responsible for fructosan hydrolysis. No evidence for the direct transfer of fructose from sucrose to high M_r glucofructosans was obtained.     

Efficient mediator-independent hydrogenation of carbon-carbon double bonds,using the obligate anaerobe,Acetobacterium woodii,with fructose as an electron donor

Fructose and H₂ were compared as electron donors for hydrogenation of carbon-carbon double bonds using Acetobacterium woodii. Caffeate was used as a model substrate. An electron donor was required and both fructose and H₂ were suitable. With fructose as the donor, the K _s for caffeate was 0.5 mM and the V _max was 678 mmol kg_dry weight ⁻¹ h⁻¹.␣Fructose oxidation was coupled very efficiently to caffeate reduction by an alteration in the fructose fermentation so that acetate was no longer produced. Received: 24 June 1996 / Accepted: 1 July 1996     

Substrate inhibition of maize endosperm sucrose synthase by fructose and its interaction with glucose inhibition

Sucrose synthase (EC 2.4.1.13) was purified to homogeneity from developing maize (Zea mays L.) endosperm. Substrate saturation and inhibitor kinetics were examined for the sucrose synthase reaction. The K_m-values for fructose and uridine diphosphate glucose (UDPGlc) were estimated to be 7.8 mM and 76 μM, respectively. Fructose concentrations over 20 mM inhibited sucrose synthase in an uncompetitive manner with respect to UDPGlc. Glucose was also found to be an uncompetitive inhibitor with respect to both fructose and UDPGlc. At inhibitory concentrations of fructose, the apparent K_i for glucose increased linearly with increasing fructose concentration. The results suggest an ordered kinetic mechanism for sucrose synthase where UDPGlc binds first and UDP dissociates last. Fructose and glucose both inhibit by binding to the enzyme-UDP complex. Fructose and glucose, which are present in maize endosperm as the products of invertase, could inhibit sucrose synthase, especially in basal regions of the kernel where hexosesmay accumulate.     

Motif‐based search for a novel fructosyl peptide oxidase from genome databases

The measurement of glycated hemoglobin A1c (HbA1c) has important implications for diagnosis of diabetes and assessment of treatment effectiveness. We proposed specific sequence motifs to identify enzymes that oxidize glycated compounds from genome database searches. The gene encoding a putative fructosyl amino acid oxidase was found in the Phaeosphaeria nodorum SN15 genome and successfully expressed in Escherichia coli. The recombinant protein (XP_001798711) was confirmed to be a novel fructosyl peptide oxidase (FPOX) with high specificity for α‐glycated compounds, such as HbA1c model compounds fructosyl‐^αN‐valine (f‐^αVal) and fructosyl‐^αN‐valyl‐histidine (f‐^αVal‐His). Unlike previously reported FPOXs, the P. nodorum FPOX has a K_m value for f‐^αVal‐His (0.185 mM) that is considerably lower than that for f‐^αVal (0.458 mM). Based on amino acid sequence alignment, three dimensional structural modeling, and site‐directed mutagenesis, Gly60 was found to be a determining residue for the activity towards f‐^αVal‐His. A flexible surface loop region was also found to likely play an important role in accepting f‐^αVal‐His. Biotechnol. Bioeng. 2010; 106: 358–366. © 2010 Wiley Periodicals, Inc.     

Reciprocal regulation of fructose 1,6-bisphosphatase and phosphofructokinase by fructose 2,6-bisphosphate in swine kidney

The effect of fructose 2,6-P₂, AMP and substrates on the coordinate inhibition of FBPase and activation of PFK in swine kidney has been examined. Fructose 2,6-P₂ inhibits the activity of FBPase and stimulates the activity of PFK in the presence of inhibitory concentrations of ATP. Under similar conditions 2.2 μM fructose 2,6-P₂ was required for 50% inhibition of FBPase and 0.04 μM fructose 2,6-P₂ restored 50% of the activity of PFK. Fructose 2,6-P₂ also enhanced the allosteric activation of PFK by AMP and it increased the extent of inhibition of FBPase by AMP. Fructose 2,6-P₂, AMP and fructose 6-P act cooperatively to stimulate the activity of PFK whereas the same latter two effectors and fructose 1,6-P₂ inhibit the activity of FBPase. Taken collectively, these results suggest that an increase in the intracellular level of fructose 2,6-P₂ during gluconeogenesis could effectively overcome the inhibition of PFK by ATP and simulataneously inactivate FBPase. When the level of fructose 2,6-P₂ is low, a glycolytic state would be restored, since under these conditions PFK would be inhibited by ATP and FBPase would be active.     

Engineering of dye-mediated dehydrogenase property of fructosyl amino acid oxidases by site-directed mutagenesis studies of its putative proton relay system

The flavoenzyme fructosyl amino acid oxidase (FAOD) catalyzes the oxidative deglycation of fructosyl amino acids, model compounds of glycated proteins. The high oxygen reactivity of FAODs limits their potential utility in amperometric enzyme sensors employing artificial electron mediators. To alter their electron acceptor availability, site-directed mutagenesis was carried out on conserved residues predicted to be involved in the proton relay system (PRS) of two eukaryotic FAODs, the FAOD from the marine yeast Pichia sp. N1-1 and amadoriase II from the fungus Aspergillus fumigatus. The substitution of a single conserved Asn residue in the putative PRS, Asn47Ala of N1-1 FAOD and Asn52Ala of amadoriase II, resulted in significant loss in the catalytic ability to employ O₂ as the electron acceptor, while having little effect on the dye-mediated dehydrogenase activity employing artificial electron acceptors instead of O₂.     

Determination of fumonisins in corn: Evaluation of two purification procedures

The performance of two solid phase extraction (SPE) purification procedures, used in the determination of fumonlsin B₁ (FB₁), B₂ (FB₂) and B₃ (FB₂) In corn, was evaluated using both thin-layer chromatography (TLC) and high performance liquid chromatography (HPLC). Fewer interferences were observed In extracts prepared using the strong anion exchange (SAX) media, in contrast to those purified on C₁₈ media, where on occasions, visual discernment of the TLC bands was hampered by the presence of interfering compounds. Precipitate formation, resulting In the blocking of SPE cartridges was also encountered when using the C₁₈ procedure. HPLC analyses of extracts prepared by both media indicated that they gave comparable fumonlsin recoveries from naturally contaminated corn samples. The results suggest that the C₁₈ procedure, originally developed for the TLC analyses of FB₁ in mixed feeds, may also be applied to the determination of FB₂ and FB₂. However, where TLC is used quantitatively for fumonlsin levels <1 μg/g, purification of sample extracts on SAX media is recommended.     

Glucofructosan biosynthesis in Fusarium oxysporum: Regulation and substrate specificity of fructosyl transferase and invertase

Mycelium of Fusarium oxysporum grown on a glucose-containing medium lacked fructosyl transferase and invertase activities. Synthesis of fructosyl t     

Purification and characterization of the enzymes of fructan biosynthesis in tubers of Helianthus tuberosus 'Colombia': I. Fructan: fructan fructosyl transferase

Fructan: fructan fructosyl transferase (FFT), one of the enzymesinvolved in the synthesis of ß-2,1 linked fructosepolymers has been purified 205-fold from tubers of Helianthustuberosus harvested in the accumulation phase. The molecularweight of the native as well as the SDS-denatured protein isapproximately 70 kDa. On IEF, the protein was separated intofive molecular species with pl values between pH 4.5–5.0.The optimum pH for fructosyl transfer activity was between 5.5–7.0.Temperature optimum was in the range of 25-35° C; the Q10value between 25 and 5° C was 1.14. FTT catalysed the self-transferof fructosyl groups with GF₂, GF₃, GF₄ or GF₅ as substrate andacceptor. The rate of elf-transfer with both GF₂ and GF₃ increasedlinearly with substrate concentration up to 100 mol m^–3and was still not saturated at 600 and 300 mol m^–3, respectively.FFT was unable to hydrolyse GF or to catalyse the self-transferwith GF but could mediate the transfer of fructosyl units frominulin on to GF. Key words: Fructan: fructan fructosyl transferase, Helianthus tuberosus, Jerusalem artichoke, purification, kinetics     

Occurrence of fructosyl-amino acid oxidase-reactive compounds in fungal cells

Fructosyl-amino acid oxidase (FAOD)-reactive fraction (FRY) was found in commercial yeast extract. FRY showed very hydrophilic property and was adsorbed to phenylboronate silica gel, indicating that it contained the Amadori compound. TLC and amino acid analyses revealed that glucosone, lysine, and arginine were produced from FRY after incubation with FAOD. TOF-MS analysis confirmed that FRY is a mixture of fructosyl lysine and fructosyl arginine. These compounds were also detected in mycelial extract of an FAOD-producer, Aspergillus terreus GP1, grown on the minimum medium, suggesting that a glycation reaction occurs in fungal cells and that FAOD acts toward the resultant Amadori compounds.     

Citrate inhibition of rat-kidney cortex phosphofructokinase

The regulatory properties of citrate on the activity of phosphofructokinase (PFK) purified from rat-kidney cortex has been studied. Citrate produces increases in the K_0.5 for Fru-6-P and in the Hill coefficient as well as a decrease in the V_max of the reaction without affecting the kinetic parameters for ATP as substrate. ATP potentiates synergistically the effects of citrate as an inhibitor of the enzyme. Fru-2,6-P₂ and AMP at concentrations equal to K_a were not able to completely prevent citrate inhibition of the enzyme. Physiological concentrations of ATP and citrate produce a strong inhibition of renal PFK suggesting that may participate in the control of glycolysisin vivo.Abbreviations PFK 6-Phosphofructo-1-kinase (EC 2.7.1.11) - Fru-6-P Fructose 6-phosphate - Fru-2,6-P₂ Fructose 2,6-bisphosphate     

Alteration of 1,2-diacylglycerol content in ischemic and reperfused heart

Human term placenta contains an ATP diphosphohydrolase activity which hydrolyses ATP to ADP and inorganic phosphate and ADP to AMP and a second mole of inorganic phosphate. The activity has a pH optimum between 8.0 and 8.5. Magnesium or calcium ions are required for maximum activity. Other nucleoside phosphates, p-nitrophenyl phosphate or sodium pyrophosphate, are not hydrolysed. The activity is not due to ATPases, or to myokinase, as determined by the use of inhibitors. NaF and NaN₃ were found to inhibit strongly the activity thus identifying it as an ATP diphosphohydrolase.A sensitive enzymatic assay for measurement of AMP, one of the products of the reaction, was established, based on the strong inhibition of muscle fructose 1,6-biphosphatase by AMP. The range of the assay was 0.05–0.8 µM AMP. ATP diphosphohydrolase was found to have a rate of AMP production from ADP twice the rate from ATP. Under the same conditions, the assay for Pi release, on the other hand, gave velocities similar to each other for the two substrates.The activity appears to be identical to the ADP-hydrolysing activity in placenta reported by others.Abbreviations Ap₅A P¹ - P⁵-di(adenosine-5) Pentaphosphate - ATP-DPH ATP Diphosphohydrolase - DCCD N,N Dicyclohexycarbodiimide - Fru-P₂ase Fructose 1,6-biphosphatase - SDS Sodium Dodecyl Sulfate - TLC Thin Layer Chromatography     

Polyfructan and Oligofructans Synthesized from Sucrose by Conidia of Aspergillus sydowi IAM 2544

When conidia of Aspergillus sydowi IAM 2544 was incubated with sucrose, polyfructan and oligofructans were synthesized concomitantly in the incubation mixture. The polyfructan isolated from the incubation mixture was shown to have a molecular weight of the order of 20 × 10⁶ which was comparable to those of microbial levans. But it was comprised of chains of 2→1′ linked β-D-fructofuranoside residues as in inulin of higher plants. This polyfructan was considred to be a very unique polysaccharide which differed from any other fructans ever known. Oligofructans were characterized as fructans of 1-kestose series having the general formula of ((1^F-(fructosyl)_n-sucrose)).     

Fructosan metabolism in Cichorium intybus roots

Incorporation of [¹⁴C]sucrose into difructosyl glucose (F₂G), trifructosyl glucose (F₃G) and tetrafructosyl glucose (F₄G) in the presence of various nucleoside triphosphates revealed that formation of F₄G and F₃G is retarded in the presence of ATP, and formation of F₃G and F₂G is significantly enhanced in the presence of CTP, whereas UTP has no effect on the synthesis of these oligosaccharides. Different fructosyl transferases seem to be responsible for the different fructosylation steps and self transfer seems to be the major pathway for fructosan synthesis. Utilization of added glucose, which is formed by sucrose sucrose fructosyl transferase action in vivo, is completely inhibited in acetate buffer whereas in phosphate, citrate and citrate-phosphate buffers glucose is actively utilized. In the presence of fluoride ions both glucose utilization and its conversion to CO₂ is inhibited by ca 50%. CO₂ production from [¹⁴C]glucose is completely inhibited in acetate ions. No evidence for the incorporation of ¹⁴C from [¹⁴C]glucose into [¹⁴C]sucrose is observed. The ratio of bound fructose to bound glucose is the same in the entire length of the root indicating that there is no preferential zone for fructosan synthesis.