Mycelial cultures of 76 strains of lignocellulose-degrading basidiomycete fungi were screened for the activity of pyranose dehydrogenase, a novel sugar oxidoreductase recently detected in Agaricus bisporus. Of these fungi, 37 strains belonging to seven phylogenetically related genera of mostly litter-decomposing Agaricales were positive for the dehydrogenase, based on activity assays towards D-glucose with 1,4-benzoquinone or ferricenium ion as electron acceptors, and on TLC/HPLC analyses of the reaction products. Lack of activity with O(2) as the oxidant, specificity for C-3 of D-glucose, and active extracellular secretion of the enzyme were used as criteria to differentiate pyranose dehydrogenase from pyranose 2-oxidase (EC 1.1.3.10), known to be produced by numerous wood-rotting fungi. Extracellular pyranose dehydrogenase from Macrolepiota rhacodes was heavily glycosylated. The enzyme was characterized as a 78-kDa flavoprotein under denaturing conditions and a 76-kDa native protein using gel filtration. This enzyme had a maximum extracellular activity of 4.1 U ml(-1) in 39-day liquid cultures. It exhibited broad selectivity for sugar substrates and oxidized D-glucose (K(m)=1.82) exclusively at C-3 to 3-dehydro-D-glucose (D-ribo-hexos-3-ulose), in contrast to pyranose dehydrogenases from Agaricus species, which acted at both C-3 and C-2 of D-glucose. The N-terminal sequence, AVVYRHPDEL, showed significant similarity with that reported for A. bisporus.
- MeSH
- Basidiomycota enzymologie růst a vývoj MeSH
- benzochinony metabolismus MeSH
- celulosa metabolismus MeSH
- flavoproteiny analýza chemie metabolismus MeSH
- glukosa metabolismus MeSH
- karbohydrátdehydrogenasy analýza chemie metabolismus MeSH
- kinetika MeSH
- kořeny rostlin mikrobiologie MeSH
- lignin metabolismus MeSH
- metabolismus sacharidů * MeSH
- molekulová hmotnost MeSH
- oxidoreduktasy analýza chemie metabolismus MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- srovnávací studie MeSH
- Názvy látek
- benzochinony MeSH
- celulosa MeSH
- flavoproteiny MeSH
- glukosa MeSH
- karbohydrátdehydrogenasy MeSH
- lignin MeSH
- lignocellulose MeSH Prohlížeč
- oxidoreduktasy MeSH
- pyranose oxidase MeSH Prohlížeč
- quinone MeSH Prohlížeč
Pyranose dehydrogenase purified to homogeneity from the mycelia of the basidiomycete fungus Agaricus bisporus catalyzed the oxidation of D-xylose at C-2 to D-threo-pentos-2-ulose (2-keto-D-xylose) and successively at C-3 to D-glycero-pentos-2,3-diulose (2,3-diketo-D-xylose) using 1,4-benzoquinone as an electron acceptor. The sites of oxidation were deduced from the spectroscopic analysis (MS, NMR) of the N,N-diphenylhydrazone derivatives of the reaction products.
- MeSH
- Agaricus enzymologie MeSH
- hydrazony chemie MeSH
- karbohydrátdehydrogenasy izolace a purifikace metabolismus MeSH
- ketony chemie metabolismus MeSH
- magnetická rezonanční spektroskopie MeSH
- molekulární struktura MeSH
- oxidace-redukce MeSH
- vysokoúčinná kapalinová chromatografie MeSH
- xylosa analogy a deriváty chemie metabolismus MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Názvy látek
- 2,3-diketoxylose MeSH Prohlížeč
- hydrazony MeSH
- karbohydrátdehydrogenasy MeSH
- ketony MeSH
- xylosa MeSH
A novel C-2-specific sugar oxidoreductase, tentatively designated as pyranose 2-dehydrogenase, was purified 68-fold to apparent homogeneity (16.4 U/mg protein) from the mycelia of Agaricus bisporus, which expressed maximum activity of the enzyme during idiophasic growth in liquid media. Using 1,4-benzoquinone as an electron acceptor, pyranose 2-dehydrogenase oxidized d-glucose to d-arabino-2-hexosulose (2-dehydroglucose, 2-ketoglucose), which was identified spectroscopically through its N,N-diphenylhydrazone. The enzyme is highly nonspecific. d-,l-Arabinose, d-ribose, d-xylose, d-galactose, and several oligosaccharides and glycopyranosides were all converted to the corresponding 2-aldoketoses (aldosuloses) as indicated by TLC. d-Glucono-1,5-lactone, d-arabino-2-hexosulose, and l-sorbose were also oxidized at significant rates. UV/VIS spectrum of the native enzyme (lambdamax 274, 362, and 465 nm) was consistent with a flavin prosthetic group. In contrast to oligomeric intracellular pyranose 2-oxidase (EC 1.1.3.10), pyranose 2-dehydrogenase is a monomeric glycoprotein (pI 4.2) incapable of reducing O2 to H2O2 (> 5 x 10(4)-fold lower rate using a standard pyranose oxidase assay); pyranose 2-dehydrogenase is actively secreted into the extracellular fluid (up to 0.5 U/ml culture filtrate). The dehydrogenase has a native molecular mass of approximately 79 kDa as determined by gel filtration; its subunit molecular mass is approximately 75 kDa as estimated by SDS-PAGE. Two pH optima of the enzyme were found, one alkaline at pH 9 (phosphate buffer) and the other acidic at pH 4 (acetate buffer). Ag+, Hg2+, Cu2+, and CN- (10 mM) were inhibitory, while 50 mM acetate had an activating effect.
- MeSH
- Agaricus enzymologie MeSH
- arabinosa metabolismus MeSH
- benzochinony farmakologie MeSH
- elektroforéza v polyakrylamidovém gelu MeSH
- galaktosa metabolismus MeSH
- glukonáty metabolismus MeSH
- glukosa analogy a deriváty metabolismus MeSH
- glykosidy metabolismus MeSH
- inhibitory enzymů MeSH
- izoelektrický bod MeSH
- karbohydrátdehydrogenasy antagonisté a inhibitory izolace a purifikace metabolismus MeSH
- ketosy * MeSH
- koncentrace vodíkových iontů MeSH
- kyanidy farmakologie MeSH
- kyslík metabolismus MeSH
- laktony MeSH
- měď farmakologie MeSH
- oligosacharidy metabolismus MeSH
- oxidace-redukce MeSH
- peroxid vodíku metabolismus MeSH
- ribosa metabolismus MeSH
- rtuť farmakologie MeSH
- sorbosa metabolismus MeSH
- stříbro farmakologie MeSH
- xylosa metabolismus MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Názvy látek
- arabinosa MeSH
- benzochinony MeSH
- beta-glucono-1,5-lactone MeSH Prohlížeč
- galaktosa MeSH
- glucosone MeSH Prohlížeč
- glukonáty MeSH
- glukosa MeSH
- glykosidy MeSH
- inhibitory enzymů MeSH
- karbohydrátdehydrogenasy MeSH
- ketosy * MeSH
- kyanidy MeSH
- kyslík MeSH
- laktony MeSH
- měď MeSH
- oligosacharidy MeSH
- peroxid vodíku MeSH
- quinone MeSH Prohlížeč
- ribosa MeSH
- rtuť MeSH
- sorbosa MeSH
- stříbro MeSH
- xylosa MeSH
Two d-glucose-oxidizing enzymes, glucose 1-oxidase (G1O) and pyranose 2-oxidase (P2O, glucose 2-oxidase), have been proposed to play an important role in the ligninolytic system of the white rot fungus Phanerochaete chrysosporium by producing hydrogen peroxide. The possible simultaneous expression and metabolic cooperation of the two oxidases was studied in strains ME-446 (reported as G1O positive) and K-3 (P2O positive) grown in liquid media and under near natural conditions on birch wood blocks. The presence of G1O and P2O in extracts from mycelia and decayed wood was determined by chromatographic, electrophoretic, and immunological methods. Attempts to separate these enzymes and to detect G1O and its reaction product, d-glucono-1,5-lactone, failed. Evidence was obtained only for P2O expression in both strains. Accordingly, P2O, rather than G1O, represents a major source of sugar-derived H2O2 under the culture conditions used.
- MeSH
- Basidiomycota metabolismus MeSH
- glukosaoxidasa metabolismus MeSH
- lignin metabolismus MeSH
- peroxid vodíku metabolismus MeSH
- vysokoúčinná kapalinová chromatografie MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Názvy látek
- glukosaoxidasa MeSH
- lignin MeSH
- peroxid vodíku MeSH
- MeSH
- antibakteriální látky biosyntéza MeSH
- dehydratasy metabolismus MeSH
- enzymy imobilizované MeSH
- glukosa metabolismus MeSH
- karbohydrátdehydrogenasy metabolismus MeSH
- katalasa metabolismus MeSH
- pyrany metabolismus MeSH
- pyrony MeSH
- Publikační typ
- časopisecké články MeSH
- Názvy látek
- antibakteriální látky MeSH
- cortalcerone MeSH Prohlížeč
- dehydratasy MeSH
- enzymy imobilizované MeSH
- glukosa MeSH
- karbohydrátdehydrogenasy MeSH
- katalasa MeSH
- pyranose oxidase MeSH Prohlížeč
- pyranosone dehydratase MeSH Prohlížeč
- pyrany MeSH
- pyrony MeSH
Pyranose oxidase and pyranosone dehydratase (aldos-2-ulose dehydratase), enzymes which convert in coupled reactions D-glucose to beta-pyrone cortalcerone, peaked coincidently during idiophasic growth of Phanerochaete chrysosporium under agitated conditions. The enzymes were purified from mycelial extracts of the fungus and separated from each other by hydrophobic interaction chromatography on Phenyl-Sepharose and Phenyl-Superose. Two pyranosone dehydratase activity peaks, PD I and PD II, were resolved. The major PD I fraction, consisting about 74% of the total dehydratase activity, was further purified by anion exchange chromatography on Mono Q to yield apparently pure enzyme as judged by SDS-PAGE and gel filtration on Superose 12. Isoelectric focusing indicated microheterogeneity of the protein by the presence of at least five protein bands with pI 5.1-5.3. PD II had a pI of 5.75. Overall PD I purification was 60.7-fold with 50% yield. The enzyme acted on several osones (glycosuloses), with the preferred substrate being D-glucosone. D-Xylosone and 6-deoxy-D-glucosone were dehydrated at C-3-C-4 to give the corresponding 5-hydroxy-2,3-dioxoalcanals (4-deoxy-2,3-glycosdiuloses), new enzymatically produced sugar derivatives. The latter labile compounds were trapped as diphenylhydrazine or o-phenylenediamine derivatives and spectroscopically identified. The analogous D-glucosone dehydration product did not accumulate due to its further transformation. pH optimum of PD I activity was 6.0 and its pH stability was optimal at pH 7-11. The enzyme was sensitive to Me2+ chelating agents and some heavy metal ions (Hg2+, Cu2+).
- MeSH
- Basidiomycota enzymologie MeSH
- chelátory farmakologie MeSH
- chromatografie iontoměničová MeSH
- dehydratasy biosyntéza chemie izolace a purifikace metabolismus MeSH
- deoxyglukosa analogy a deriváty biosyntéza metabolismus MeSH
- elektroforéza v polyakrylamidovém gelu MeSH
- isoelektrická fokusace MeSH
- karbohydrátdehydrogenasy biosyntéza MeSH
- ketosy biosyntéza metabolismus MeSH
- koncentrace vodíkových iontů MeSH
- stabilita enzymů MeSH
- substrátová specifita MeSH
- vysoká teplota MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Názvy látek
- 6-deoxyglucosone MeSH Prohlížeč
- chelátory MeSH
- dehydratasy MeSH
- deoxyglukosa MeSH
- karbohydrátdehydrogenasy MeSH
- ketosy MeSH
- pyranose oxidase MeSH Prohlížeč
- pyranosone dehydratase MeSH Prohlížeč
- threo-pentos-2-ulose MeSH Prohlížeč
Several dicarbonyl and tricarbonyl sugars were prepared by the use of fungal enzymes and the antimicrobial effects of their N,N-diphenylhydrazine derivatives were tested. G+ bacteria were more sensitive than G- bacteria especially in the group of disubstituted compounds. Peracetyled derivatives were not active. No inhibition of yeast growth was found.
- MeSH
- antibakteriální látky farmakologie MeSH
- Bacillus subtilis účinky léků MeSH
- Escherichia coli účinky léků MeSH
- fenylhydraziny metabolismus farmakologie MeSH
- ketosy metabolismus farmakologie MeSH
- mikrobiální testy citlivosti MeSH
- Publikační typ
- časopisecké články MeSH
- srovnávací studie MeSH
- Názvy látek
- antibakteriální látky MeSH
- fenylhydraziny MeSH
- ketosy MeSH
The ultrastructural distribution of the sugar-oxidizing enzyme pyranose 2-oxidase (POD) in hyphae of Phanerochaete chrysosporium K-3 grown under liquid culture conditions optimal for the enzyme's production was studied by transmission electron microscopy immunocytochemistry. Using the 3-dimethylaminobenzoic acid-3-methyl-2-benzothiazolinone hydrazone hydrochloride H(2)O(2) peroxidase spectrophotometric assay, POD was detected in mycelial extracts from days 7 to 18, with maximum activity recorded on day 12. Onset of POD activity occurred in the secondary phase of hyphal development at a time of stationary growth, glucose limitation, and pH increase. POD was also detected extracellularly in the culture fluid from days 7 to 18, with maximum activity recorded on day 13. At early stages of development (3 to 4 days), using anti-POD antibodies and immunogold labeling, POD was localized in multivesicular and electron-dense bodies and in cell membrane regions. After 10 to 12 days of growth, at maximum POD activity, POD was concentrated within the periplasmic space where it was associated with membrane-bound vesicles and other membrane structures. At later stages of development (17 to 18 days), when the majority of hyphae were lysed, POD was observed associated with residual intracellular membrane systems and vesicles. Transmission electron microscopy immunocytochemical studies also demonstrated an extracellular distribution of the enzyme at the stationary growth phase, showing its association with fungal extracellular slime. In studies of ligninolytic cultures of the same fungus, POD was found to have a similar intracellular and extracellular distribution in slime as that recorded for cultures grown with cornsteep. POD's peripheral cytoplasmic distribution shows similarities to the cellular distribution of that reported previously for H(2)O(2)-dependent lignin and manganese peroxidases in P. chrysosporium.
- Publikační typ
- časopisecké články MeSH
Streptomyces fradiae has two chromatographically distinct forms of glutamate dehydrogenase (GDH): one GDH utilizes NAD as coenzyme, the other uses NADP. The intracellular level of both GDHs is strongly regulated by the nitrogen source in the growth medium. NADP-dependent GDH was purified to homogeneity from crude extracts of S. fradiae. The Mr of the native enzyme was determined to be 200,000 by size-exclusion high-performance liquid chromatography whereas after sodium dodecyl sulphate-polyacrylamide gel electrophoresis one major band of Mr 49,000 was found, suggesting that the enzyme is a tetramer. The enzyme was highly specific for the substrates 2-oxoglutarate and L-glutamate, and required NADP, which could not be replaced by NAD, as a cofactor. The pH optimum was 9.2 for oxidative deamination of glutamate and 8.4 for reductive amination of 2-oxoglutarate. The Michaelis constants (Km) were 28.6 mM for L-glutamate and 0.12 mM for NADP. Km values for reductive amination were 1.54 mM for 2-oxoglutarate, 0.07 mM for NADPH and 30.8 mM for NH+4. The enzyme activity was significantly reduced by adenine nucleotides, particularly ATP.
- MeSH
- adenosindifosfát farmakologie MeSH
- adenosinmonofosfát farmakologie MeSH
- adenosintrifosfát farmakologie MeSH
- elektroforéza v polyakrylamidovém gelu MeSH
- glutamátdehydrogenasa (NADP+) MeSH
- glutamátdehydrogenasa antagonisté a inhibitory izolace a purifikace MeSH
- koenzymy metabolismus MeSH
- molekulová hmotnost MeSH
- Streptomyces enzymologie MeSH
- substrátová specifita MeSH
- Publikační typ
- časopisecké články MeSH
- Názvy látek
- adenosindifosfát MeSH
- adenosinmonofosfát MeSH
- adenosintrifosfát MeSH
- glutamátdehydrogenasa (NADP+) MeSH
- glutamátdehydrogenasa MeSH
- koenzymy MeSH
Homogeneous alanine dehydrogenase isolated from Streptomyces aureofaciens, a producer of tetracycline, was characterized from the point of its molecular and catalytic properties. Using analytical ultracentrifugation the molecular weight of alanine dehydrogenase was found to be 198,000. The enzyme could use as cofactors apart from NAD+ also 1,N6-etheno-NAD+, 3-acetylpyridine-NAD+, deamino-NAD+ and nicotinamide guanine dinucleotide. The enzyme activity in the direction of oxidative deamination was not affected by the addition of nonsubstrate amino acids, however, it was sensitive to inhibitors of SH-groups. Reductive amination of pyruvate was inhibited by L-alanine, L-serine and D-alanine. The inhibition by L-alanine and L-serine was uncompetitive with respect to NADH and noncompetitive with regard to pyruvate and ammonium ions.
