Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:1.10.3.3 (ascorbate oxidase)
778 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The structural comparison of copper-containing proteins has provided a new dimension to the relationships suggested by sequence similarities. Ryden (1988) summarized the putative relationships, suggesting that a primordial single-domain cupredoxin evolved into the multidomain copper oxidases. The structures have revealed the fact that the differences reside primarily in insertions and deletions at junctions between secondary-structure elements. The mechanism of evolution (e.g., integration of new sequences into regions not essential to the Greek key fold) remains unknown. Which of the properties of a cupredoxin fold are necessary for function is the subject of site-directed mutagenesis studies. Can two of the ligands be interchanged (e.g., the upstream histidine and partially answered by the multidomain copper oxidase structure. The Tyr-Cys-Thr sequence in plastocyanin (in which threonine is a member of the hydrogen-bonding pair) is homologous with the His-Cys-His sequence in ascorbate oxidase. In the latter electron transfer is believed to flow from the type I copper (bound by the cysteine) to the trinuclear cluster, probably via these histidine residues. Hence, one might infer that the tyrosine and threonine have some role in electron transfer. Tyr-83 has been previously implicated in NMR studies as a primary site of electron transfer. The multi-copper protein structures have revealed interesting new features. The extra coppers are bound at domain interfaces, and can be single metals or the novel trinuclear cluster, depending on the availability of liganding histidines. A structural model of ceruloplasmin suggests that it will have at least two type I sites and, possibly, a third type I site such as stellacyanin (no methionine ligand), as well as a binding site for a trinuclear cluster. The similarity of the sequences of N2O reductases and a domain of cytochrome oxidase to the sequences of proteins with known structures suggests that these, too, will have Greek key domains. Galactose oxidase and hemocyanin do not have Greek key folds in their functional domains, although each does have a Greek key domain. The need for a Greek key fold remains obscure. The apoproteins are clearly stable without metals; there are examples other than immunoglobulins of Greek key folds. So far copper seems to be found in a very limited subset of structures; other chapters in this volume show that zinc, for example, has a much wider variety of environments in proteins, as does iron. It may be that the copper-containing Greek key proteins represent a very small evolutionary niche.(ABSTRACT TRUNCATED AT 400 WORDS)
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PMID:Copper protein structures. 179 5

Ascorbate oxidase from the fungus Acremonium sp. HI-25 is a copper-containing glycoprotein that catalyzes the oxidation of ascorbic acid to dehydroascorbic acid. Monosaccharide composition analysis showed that the enzyme contains exclusively N-linked oligosaccharide chains. Following liberation by hydrazinolysis/re-N-acetylation, and fractionation by HPLC on anion exchange. Amide-80 and/or octadecyl silica columns after derivatization with p-aminobenzoic ethyl ester, the structures of the twelve major neutral oligosaccharides were identified by FAB-MS, 400 MHz 1H-NMR, methylation analysis, mild acid hydrolysis, and/or sequential exoglycosidase digestions. Acremonium sp. ascorbate oxidase was found to consist of high-mannose type oligosaccharides (76.3%) having 4 to 9 mannose residues and a series of novel D-galactofuranose-containing high-mannose type oligosaccharides (18.6%) with the following structure.
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PMID:Novel beta-D-galactofuranose-containing high-mannose type oligosaccharides in ascorbate oxidase from Acremonium sp. HI-25. 878 8

Competitive inhibition by phenolic compounds of the ascorbic acid oxidation reaction catalyzed by ascorbate oxidase was investigated at pH 7.0 and 23.0 degrees C. Inhibition of p-nitrophenol is pH dependent over the range 5.0-8.0, with inhibitor binding favored at higher pH. Bulky substituents on the phenol nucleus reduce or prevent the inhibitory effect. The presence of phenol affects the binding characteristics of azide to the trinuclear cluster of the enzyme. In particular, binding of azide to type 2 copper is prevented, and the affinity of azide to type 3 copper is reduced. In addition, reduction of type 1 copper is observed upon prolonged incubation of ascorbate oxidase with excess phenol and azide, but not with phenol alone. It is proposed that binding of phenolic inhibitors occurs at or near the site where the substrate (ascorbate) binds. NMR relaxation measurements of the protons of phenols in the presence of ascorbate oxidase show paramagnetic effects due to the proximity of the bound inhibitor to a copper center, likely type 1 copper. Copper-proton distance estimates between this paramagnetic center and p-cresol or p-nitrophenol bound to ascorbate oxidase are between 4.4 and 5.9 A.
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PMID:Inhibition of ascorbate oxidase by phenolic compounds. Enzymatic and spectroscopic studies. 912 5

Five peptides containing (His-X2)-His or (His-X3)-His motifs have been designed and synthesized to coordinate Cu(II). Structural information was obtained by various spectroscopic techniques and was used as constraint to search for local conformational energy minima by molecular mechanics. Thermodynamic stability constants of the Cu(II) chelates was obtained by 19F-NMR. The synthesized Cu(II)-peptide chelates were tested as catalysts of some important red-ox processes occuring in biological systems, in particular oxidation of ascorbate and dismutation of superoxide ion. The catalytic efficiency of the five chelates was much lower than that of ascorbate oxidase. On the contrary, two of them showed kinetic constants for superoxide dismutation about one order of magnitude lower than that of the enzyme Cu,Zn superoxide dismutase. In both cases, the catalytic properties were dependent on the peptide sequence. The relationships between structure and activity are discussed to find the structural parameters crucial for catalytic activity that can be modulated by appropriate design and synthesis of the peptides.
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PMID:Enzyme mimics complexing Cu(II) ion: structure-function relationships. 1060 94

Ascorbic acid (1), a natural antioxidant, was modified by employing transglycosylation activity of Bacillus stearothermophilus maltogenic amylase with maltotriose and acarbose as donor molecules to enhance its oxidative stability. The transglycosylation reaction with maltotriose as donor created mono- and di-glycosyl transfer products with an alpha-(1,6)-glycosidic linkage. In addition, two acarviosine-glucosyl transfer products were generated when transglycosylation was performed with acarbose as a donor. All transfer products were observed by TLC and HPLC, and purified by Q-sepharose anion exchange and Biogel P-2 gel permeation chromatographies. LC/MS and (13)C NMR analyses revealed that the structures of the transfer products were 6-O-alpha-D-glucosyl- (2) and 6-O-alpha-D-maltosyl-ascorbic acids (3) in the reaction of maltotriose, and 6-O-alpha-acarviosine-D-glucosyl- (4) and 2-O-alpha-acarviosine-D-glucosyl ascorbic acids (5) in the reaction of acarbose. The stability of the transglycosylated ascorbic acid derivatives was greatly enhanced against oxidation by Cu(2+) ion and ascorbate oxidase. Among them, compound 3 proved to be the most stable against in vitro oxidation. The antioxidant effects of glycosyl-derivatives of ascorbic acid on the lipid oxidation in cooked chicken breast meat patties indicated that they had antioxidant activities similar to that of ascorbic acid. It is suggested that the transglycosylated ascorbic acids can possibly be applied as effective antioxidants with improved stability in food, cosmetic, and other applications.
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PMID:Modification of ascorbic acid using transglycosylation activity of Bacillus stearothermophilus maltogenic amylase to enhance its oxidative stability. 1201 3

The present investigation addresses the problem of the binding mode of phenolic inhibitors and the substrate ascorbate to the active site of ascorbate oxidase. The results from both types of compounds indicate that the binding site is located in a pocket near the type 1 copper center. This information is of general interest for blue multicopper oxidases. Docking calculations performed on the ascorbate oxidase-ascorbate complex show that binding of the substrate occurs in a pocket near type 1 Cu, and is stabilized by at least five hydrogen bonding interactions with protein residues, one of which involves the His512 Cu ligand. Similar docking studies show that the isomeric fluorophenols, which act as competitive inhibitors toward ascorbate, bind to the enzyme in a manner similar to ascorbate. The docking calculations are supported by 19F NMR relaxation measurements performed on fluorophenols in the presence of the enzyme, which show that the bound inhibitors undergo enhanced relaxation by the paramagnetic effect of a nearby Cu center. Unambiguous support to the location of the inhibitor close to type 1 Cu was obtained by comparative relaxation measurements of the fluorophenols in the presence of the ascorbate oxidase derivative where a Zn atom selectively replaces the paramagnetic type 2 Cu. The latter experiments show that contribution to relaxation of the bound inhibitors by the type 2 Cu site is negligible.
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PMID:Probing the location of the substrate binding site of ascorbate oxidase near type 1 copper: an investigation through spectroscopic, inhibition and docking studies. 1500 40

D-Serine causes selective necrosis to the straight portion of the rat renal proximal tubules. The onset is rapid, occurring within 3-4 h and accompanied by proteinuria, glucosuria and aminoaciduria. The metabolism of D-serine by D-amino acid oxidase (D-AAO) may be involved in the mechanism of toxicity. D-AAO is localized within the peroxisomes of renal tubular epithelial cells, which is also the location of D-serine reabsorption. To address the role of D-AAO in D-serine-induced nephrotoxicity, we have examined the effect of sodium benzoate (SB) on the renal injury. SB has been shown to be a potent, competitive inhibitor of kidney D-AAO in vitro. Male Alderley Park rats were exposed to D-serine (500 mg/kg i.p.) 1 h after exposure to SB (125, 250, 500 or 750 mg/kg i.p.). Urine was collected for 0-6 h, then terminal plasma samples and kidneys were taken at 6.5 h. A second group of animals was given SB (500 mg/kg) followed by D-serine (500 mg/kg i.p.; 1 h later) and urine was collected after 0-6, 6-24 and 24-48 h. Terminal plasma samples and kidneys were taken at 48 h. 1H NMR spectroscopic analysis of urine, combined with principal component analysis, demonstrated that SB was able to prevent D-serine-induced perturbations to the urinary profile in a dose dependent manner. This was confirmed by measurement of plasma creatinine and urinary glucose and protein and histopathological examination of the kidneys. Assessment 48 h after D-serine administration revealed that nephrotoxicity was observed in animals pre-treated with SB (500 mg/kg) although the extent of injury was less pronounced than following D-serine alone. These results demonstrate that whilst prior exposure to SB prevents the initial onset of D-serine-induced nephrotoxicity, renal injury is still apparent at later time points. D-AAO activity in the kidney was decreased by 50% 1 h after dosing with SB suggesting that inhibition of this enzyme may be responsible for the observed protection.
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PMID:Sodium benzoate attenuates D-serine induced nephrotoxicity in the rat. 1559 Jan 20

Spectral and catalytic properties of the flavoenzyme AAO (aryl-alcohol oxidase) from Pleurotus eryngii were investigated using recombinant enzyme. Unlike most flavoprotein oxidases, AAO does not thermodynamically stabilize a flavin semiquinone radical and forms no sulphite adduct. AAO catalyses the oxidative dehydrogenation of a wide range of unsaturated primary alcohols with hydrogen peroxide production. This differentiates the enzyme from VAO (vanillyl-alcohol oxidase), which is specific for phenolic compounds. Moreover, AAO is optimally active in the pH range of 5-6, whereas VAO has an optimum at pH 10. Kinetic studies showed that AAO is most active with p-anisyl alcohol and 2,4-hexadien-1-ol. AAO converts m- and p-chlorinated benzyl alcohols at a similar rate as it does benzyl alcohol, but introduction of a p-methoxy substituent in benzyl alcohol increases the reaction rate approx. 5-fold. AAO also exhibits low activity on aromatic aldehydes. 19F NMR analysis showed that fluorinated benzaldehydes are converted into the corresponding benzoic acids. Inhibition studies revealed that the AAO active site can bind a wide range of aromatic ligands, chavicol (4-allylphenol) and p-anisic (4-methoxybenzoic) acid being the best competitive inhibitors. Uncompetitive inhibition was observed with 4-methoxybenzylamine. The properties described above render AAO a unique oxidase. The possible mechanism of AAO binding and oxidation of substrates is discussed in the light of the results of the inhibition and kinetic studies.
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PMID:Spectral and catalytic properties of aryl-alcohol oxidase, a fungal flavoenzyme acting on polyunsaturated alcohols. 1581 2

Analysis of phloem exudates from the fruit of Cucurbitaceae revealed the presence of several compounds with UV-visible absorption spectra identical to that of l-ascorbic acid. In Cucurbita pepo L. (zucchini), the compounds could be isolated from phloem exudates collected from aerial parts of the plant but were not detected in whole tissue homogenates. The compounds isolated from the phloem exudates of C. pepo fruit were eluted from strong anion exchange resin in the same fraction as l-ascorbic acid and were oxidised by ascorbate oxidase (E.C. 1.10.3.3). The major compound purified from C. pepo fruit exudates demonstrated similar redox properties to l-ascorbic acid and synthetic 6-O-glucosyl-l-ascorbic acid (6-GlcAsA) but differed from those of 2-O-glucosyl-l-ascorbic acid (2-GlcAsA) isolated from the fruit of Lycium barbarum L. Parent and fragment ion masses of the compound were consistent with hexosyl-ascorbate in which the hexose moiety was attached to C5 or C6 of AsA. Acid hydrolysis of the major C. pepo compound resulted in the formation of l-ascorbic acid and glucose. The purified compound yielded a proton NMR spectrum that was almost identical to that of synthetic 6-GlcAsA. A series of l-ascorbic acid conjugates have, therefore, been identified in the phloem of Cucurbitaceae and the most abundant conjugate has been identified as 6-GlcAsA. The potential role of such conjugates in the long-distance transport of l-ascorbic acid is discussed.
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PMID:Ascorbic acid conjugates isolated from the phloem of Cucurbitaceae. 1847 16

Fungal AAO (aryl-alcohol oxidase) provides H2O2 for lignin biodegradation. AAO is active on benzyl alcohols that are oxidized to aldehydes. However, during oxidation of some alcohols, AAO forms more than a stoichiometric number of H2O2 molecules with respect to the amount of aldehyde detected due to a double reaction that involves aryl-aldehyde oxidase activity. The latter reaction was investigated using different benzylic aldehydes, whose oxidation to acids was demonstrated by GC-MS. The steady- and presteady state kinetic constants, together with the chromatographic results, revealed that the presence of substrate electron-withdrawing or electron-donating substituents had a strong influence on activity; the highest activity was with p-nitrobenzaldehyde and halogenated aldehydes and the lowest with methoxylated aldehydes. Moreover, activity was correlated to the aldehyde hydration rates estimated by 1H-NMR. These findings, together with the absence in the AAO active site of a residue able to drive oxidation via an aldehyde thiohemiacetal, suggested that oxidation mainly proceeds via the gem-diol species. The reaction mechanism (with a solvent isotope effect, 2H2Okred, of approx. 1.5) would be analogous to that described for alcohols, the reductive half-reaction involving concerted hydride transfer from the alpha-carbon and proton abstraction from one of the gem-diol hydroxy groups by a base. The existence of two steps of opposite polar requirements (hydration and hydride transfer) explains some aspects of aldehyde oxidation by AAO. Site-directed mutagenesis identified two histidine residues strongly involved in gem-diol oxidation and, unexpectedly, suggested that an active-site tyrosine residue could facilitate the oxidation of some aldehydes that show no detectable hydration. Double alcohol and aldehyde oxidase activities of AAO would contribute to H2O2 supply by the enzyme.
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PMID:Kinetic and chemical characterization of aldehyde oxidation by fungal aryl-alcohol oxidase. 1989 8


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