Form Of Oxidase Research Articles

Characterization of polyphenol oxidase from plants

Polyphenol oxidase (PPO) which can mediate browning reaction is a bifunctional copper-containing enzyme encoded by plant nucleolus gene. It usually leads to excessive browning reaction which reduces the commercial profits of fruits and vegetables. In this paper, PPO genes and enzymes in plants are characterized systematically, and the latest progress is reviewed. Some clonings of PPOs genes are reported; the specific temporal and spatial expression pattern of PPOs genes is described; the model of the structure of the precursor form of catechol oxidase is introduced; the possible functions of PPOs in defending against pathogen, wounding, surrounding stress and other inducing factors are demonstrated; the induction and activation of latent PPOs in some plants is elucidated; the scheme of browning inhibition by L-cysteine is clarified; the mechanism of suicide inhibition of latent PPO and kinetic synergism are established. Furthermore, the area for future study is also discussed.

Involvement of polyamines, diamine oxidase and polyamine oxidase in resistance of barley to Blumeria graminis f. sp. hordei

Polyamine levels and diamine and polyamine oxidase activities have been investigated in the first leaves of barley (Hordeum vulgare L.) in the absence of or following inoculation with conidia of powdery mildew (Blumeria graminis f. sp. hordei). Two cultivars with varying sensitivity to powdery mildew, viz., Chariot (resistant) and Golden Promise (susceptible) were used. The levels of putrescine, spermidine and spermine were found to be higher in the leaves of Chariot than in the leaves of Golden Promise and, with the exception of spermine, were generally higher in both cultivars after inoculation. In inoculated leaves of Chariot, levels of putrescine and spermidine peaked at 9 days and 12 days, respectively. In controls (uninoculated leaves), the activities of these enzymes, and putrescine and spermidine levels also increased but not to the same extent as in inoculated leaves. With Golden Promise, the levels of putrescine and spermidine in the inoculated leaves changed very little over sampling times but were always higher than in the controls. In this cultivar, there was little difference between inoculated leaves and the controls in diamine oxidase activity which reached a maximum value at 9 days post-inoculation. Activity of the bound form of diamine oxidase was low in both the cultivars. Polyamine oxidase was not detected at 3 days after inoculation in either cultivar but activity at fairly low levels was recorded at later times, usually reaching a maximum value at 9 days. The results suggest that polyamine metabolism and diamine oxidase activity in particular may be involved in the mechanism conferring resistance to barley powdery mildew in Chariot.

Peroxynitrite formation from the simultaneous reduction of nitrite and oxygen by xanthine oxidase

One electron reductions of oxygen and nitrite by xanthine oxidase form peroxynitrite. The nitrite and oxygen reducing activities of xanthine oxidase are regulated by oxygen with Koxygen 26 and 100 μM and Knitrite 1.0 and 1.1 mM with xanthine and NADH as donor substrates. Optimal peroxynitrite formation occurs at 70 μM oxygen with purine substrates. Kinetic parameters: Vmax∼50 nmol/min/mg and Km of 22, 36 and 70 μM for hypoxanthine, pterin and nitrite respectively. Peroxynitrite generation is inhibited by allopurinol, superoxide dismutase and diphenylene iodonium. A role for this enzyme activity can be found in the antibacterial activity of milk and circulating xanthine oxidase activity.

Nature of the oxomolybdenum-thiolate pi-bond: implications for Mo-S bonding in sulfite oxidase and xanthine oxidase.

The electronic structure of cis,trans-(L-N(2)S(2))MoO(X) (where L-N(2)S(2) = N,N'-dimethyl-N,N'-bis(2-mercaptophenyl)ethylenediamine and X = Cl, SCH(2)C(6)H(5), SC(6)H(4)-OCH(3), or SC(6)H(4)CF(3)) has been probed by electronic absorption, magnetic circular dichroism, and resonance Raman spectroscopies to determine the nature of oxomolybdenum-thiolate bonding in complexes possessing three equatorial sulfur ligands. One of the phenyl mercaptide sulfur donors of the tetradentate L-N(2)S(2) chelating ligand, denoted S(180), coordinates to molybdenum in the equatorial plane such that the OMo-S(180)-C(phenyl) dihedral angle is approximately 180 degrees, resulting in a highly covalent pi-bonding interaction between an S(180) p orbital and the molybdenum d(xy) orbital. This highly covalent bonding scheme is the origin of an intense low-energy S --> Mo d(xy) bonding-to-antibonding LMCT transition (E(max) approximately 16000 cm(-)(1), epsilon approximately 4000 M(-)(1) cm(-)(1)). Spectroscopically calibrated bonding calculations performed at the DFT level of theory reveal that S(180) contributes approximately 22% to the HOMO, which is predominantly a pi antibonding molecular orbital between Mo d(xy) and the S(180) p orbital oriented in the same plane. The second sulfur donor of the L-N(2)S(2) ligand is essentially nonbonding with Mo d(xy) due to an OMo-S-C(phenyl) dihedral angle of approximately 90 degrees. Because the formal Mo d(xy) orbital is the electroactive or redox orbital, these Mo d(xy)-S 3p interactions are important with respect to defining key covalency contributions to the reduction potential in monooxomolybdenum thiolates, including the one- and two-electron reduced forms of sulfite oxidase. Interestingly, the highly covalent Mo-S(180) pi bonding interaction observed in these complexes is analogous to the well-known Cu-S(Cys) pi bond in type 1 blue copper proteins, which display electronic absorption and resonance Raman spectra that are remarkably similar to these monooxomolybdenum thiolate complexes. Finally, the presence of a covalent Mo-S pi interaction oriented orthogonal to the MOO bond is discussed with respect to electron-transfer regeneration in sulfite oxidase and Mo=S(sulfido) bonding in xanthine oxidase.

Mobilization of xanthine oxidase from the gastrointestinal tract in acute pancreatitis.

BackgroundXanthine oxidoreductase has been proposed to play a role in the development of local and systemic effects of acute pancreatitis. Under physiologic conditions, the enzyme exists mainly as xanthine dehydrogenase (XDH) but can be converted by proteolytic cleavage to its superoxide-generating form xanthine oxidase (XOD). In addition to its intracellular location XDH/XOD is also associated to the polysaccharide chains of proteoglycans on the external endothelial cell membrane.In the early stages of acute pancreatitis, this enzyme seems to be arising from its mobilization from the gastrointestinal endothelial cell surface. Taking into account the ability of α-amylase to hydrolyze the internal α-1,4 linkages of polysaccharides, we wanted to elucidate the involvement of α-amylase in XDH/XOD mobilization from the gastrointestinal endothelial cell surface and the relevance of the ascitic fluid (AF) as the source of α-amylase in experimental acute pancreatitis.MethodsAcute pancreatitis was induced in male Wistar rats by intraductal administration of 5% sodium taurocholate. In another experimental group 3000 U/Kg α-amylase was i.v. administered. The concentrations of XDH, XOD and α-amylase in plasma and AF and myeloperoxidase (MPO) in lung have been evaluated. In additional experiments, the effect of peritoneal lavage and the absorption of α-amylase present in the AF by an isolated intestine have been determined.ResultsSimilar increase in XDH+XOD activity in plasma was observed after induction of acute pancreatitis and after i.v. administration of α-amylase. Nevertheless, the conversion from XDH to XOD was only observed in the pancreatitis group. Lung inflammation measured as MPO activity was observed only in the pancreatitis group. In addition peritoneal lavage prevented the increase in α-amylase and XDH+XOD in plasma after induction of pancreatitis. Finally, it was observed that α-amylase is absorbed from the AF by the intestine.ConclusionsDuring the early stages of acute pancreatitis, α-amylase absorbed from AF through the gastrointestinal tract could interfere with the binding of XDH/XOD attached to glycoproteins of the endothelial cells. Proteolytic enzymes convert XDH into its oxidase form promoting an increase in circulating XOD that has been reported to be one of the mechanisms involved in the triggering of the systemic inflammatory process.

Rasburicase therapy in acute hyperuricemic renal dysfunction.

Neoplastic disorders may be complicated by acute renal failure (ARF). Different tumors may cause ARF: solid tumors involving the kidney, solid tumors not of hematological origin and not primarily involving the kidney or, more frequently, rapidly developing hematological tumors. The pathogenesis of ARF is different depending on the type of cancer, but the most frequent clinical feature is the acute tumor lysis syndrome, characterized by hyperuricemia, hyperphosphatemia, hyperkalemia, hypocalcemia and acute, frequently oliguric, ARF. The presence of a neoplastic disorder and associated acute illness may sometimes lead to the presence of immunodysfunction, septic complications and multiple organ dysfunction. In these settings patients develop systemic inflammation and diffuse endothelial damage, related to different mediators. Among these substances, in cancer patients, high circulating levels of uric acid are a common finding. Hyperuricemia is caused by the increase of purine metabolism, which is result of the increased cellular turnover or the aggressive cancer chemotherapy regimens that worsen cell lysis and release of purine metabolites. Even if hyperuricemia is not the first insult to the kidney, its development might represent a concomitant factor aggravating other previous or simultaneous insults. The most efficient therapy for lowering uric acid is rasburicase, a recombinant form of urate oxidase, a nonhuman proteolytic enzyme that oxidizes uric acid to allantoin. It is efficacious in reducing serum uric acid levels with associated diuresis more effectively and much faster than allopurinol, and to correct renal dysfunction more rapidly than allopurinol.

13 Chromium, molybdenum, and tungsten

The following provides a selected highlight for each element of the chromium triad in 2003. Chromium: A series of remarkably stable chromium(IV) alkyl and chromium-(III) and -(IV) hydride complexes have been prepared using the triamidoamine tetradentate ligand L1H3. This ligand has also been employed to prepare the first trigonal monopyramidal chromium(II) complex. Molybdenum: Molybdenum(V) coordination complexes have been extensively studied to provide further insights into the active sites found within many molybdoenzymes. A nice example of a model complex that contains the heteroscorpionate ligand L2SH, [(L2S)MoO(bdt)2], has been reported. The complex was structurally characterised and is only the second known model compound that closely approximates the active site structure of reduced forms of sulfite oxidase. Tungsten: The field of molecular magnetism has exploited the use of the [W(CN)8]3− building block in preparing a range of 2-D and 3-D bimetallic assemblies. For example, [Cu(tn)]3[W(CN)8]2·3H2O and [Cu(pn)]3[W(CN)8]2·3H2O have a 2-D layer structure and the magnetic properties of the compounds show typical metamagnetic behaviour. The critical field at which the interlayer antiferromagnetic ground-state switches to a ferromagnetic state is approximately 1.25 T for [Cu(tn)]3[W(CN)8]2·3H2O and 0.35 T for [Cu(pn)]3[W(CN)8]2·3H2O at 1.8 K.

A possible involvement of plasma membrane NAD(P)H oxidase in the switch mechanism of the cell death mode from apoptosis to necrosis in menadione-induced cell injury.

The effects of inhibitors of plasma membrane NADPH oxidase on menadione-induced cell injury processes were studied using human osteosarcoma 143B cells. The intracellular level of superoxide in the cells treated with menadione for 6 h reached a maximum followed by an abrupt decrease. The population of apoptotic cells detected by Annexin V and propidium iodide double staining also reached its maximum at 6 h of menadione-treatment while that of necrotic cells increased continuously reaching 90% of the total population at 9 h of the treatment. Pretreatment of the cells with inhibitors of NADPH oxidase, including diphenyliodonium chloride, apocynin, N-vanillylnonanamide and staurosporine was effective in lowering the menadione-induced elevations of superoxide, and also in the suppression of the switch of the cell death mode from apoptosis to necrosis in menadione-treated cells except for the case of staurosporine. These results strongly suggest that superoxide generated by NADPH oxidase, besides that generated by the mitochondria, may contribute to the remarkable increase in the intracellular level of superoxide in the cells treated with menadione for 6 h resulting in the switch from apoptosis to necrosis, although a direct evidence of the presence of active and inactive forms of NADPH oxidase in control and menadione-treated 143B cells is lacking at present.

Spectroscopic and kinetic properties of recombinant choline oxidase from Arthrobacter globiformis.

Choline oxidase catalyzes the four-electron oxidation of choline to glycine betaine, with molecular oxygen acting as primary electron acceptor. Recently, the recombinant enzyme expressed in Escherichia coli was purified to homogeneity and shown to contain FAD in a mixture of oxidized and anionic semiquinone redox states [Fan et al. (2003) Arch. Biochem. Biophys., in press]. In this study, methods have been devised to convert the enzyme-bound flavin semiquinone to oxidized FAD and vice versa, allowing characterization of the resulting forms of choline oxidase. The enzyme-bound oxidized flavin showed typical UV-vis absorbance peaks at 359 and 452 nm (with epsilon(452) = 11.4 M(-1) cm(-1)) and emitted light at 530 nm (with lambda(ex) at 452 nm). The affinity of the enzyme for sulfite was high (with a K(d) value of approximately 50 microM at pH 7 and 15 degrees C), suggesting the presence of a positive charge near the N(1)C(2)=O locus of the flavin. The enzyme-bound anionic flavin semiquinone was unusually insensitive to oxygen or ferricyanide at pH 8 and showed absorbance peaks at 372 and 495 nm (with epsilon(372) = 19.95 M(-1) cm(-1)), maximal fluorescence emission at 454 nm (with lambda(ex) at 372 nm), circular dichroic signals at 370 and 406 nm, and an ESR peak-to-peak line width of 13.9 G. Both UV-vis absorbance studies on the enzyme under turnover with choline and steady-state kinetic data with either choline or betaine aldehyde were consistent with the flavin semiquinone being not involved in catalysis. The pH dependence of the kinetic parameters at varying concentrations of both choline and oxygen indicated that a catalytic base is required for choline oxidation but not for oxygen reduction and that the order of the kinetic steps involving substrate binding and product release is not affected by pH.

Dissection of the structural determinants involved in formation of the dimeric form of D-amino acid oxidase from Rhodotorula gracilis: role of the size of the betaF5-betaF6 loop.

The role of the long loop connecting beta-strands F5 and F6 (21 amino acids, Pro302-Leu-Asp-Arg-Thr-Lys-Ser-Pro-Leu-Ser-Leu-Gly-Arg-Gly-Ser-Ala-Arg-Ala-Ala-Lys-Glu322) present in Rhodotorula gracilis d-amino acid oxidase (RgDAAO) was investigated by site-directed mutagenesis. This loop was proposed to play an important role in the 'head-to-tail' monomer-monomer interaction of this dimeric flavoenzyme: in particular, by means of electrostatic interactions between positively charged residues of the betaF5-betaF6 loop of one monomer and negatively charged residues belonging to the alpha-helices I3' and I3" of the other monomer. We produced a mutant of RgDAAO (namely, DAAO-DeltaLOOP2), in which only minor structural perturbations were introduced (only five amino acids were deleted; new sequence of the betaF5-betaF6 loop is Pro302-Leu-Asp-Arg-Thr-Leu-Gly-Arg-Gly-Ser-Ala-Arg-Ala-Ala-Lys-Glu317), and the charge of the betaF5-betaF6 loop not modified. The DeltaLOOP2 mutant is monomeric, has a weaker binding with the FAD cofactor, a decrease of the kinetic efficiency, and slight modifications in its spectral properties. The short version of the loop does not allow a correct monomer-monomer interaction, and its presence in the monomeric DAAO is a destabilizing structural element since the DeltaLOOP2 mutant is highly susceptible to proteolysis. These results, confirming the role of this loop in the subunits interaction and thus in stabilization of the sole dimeric form of RgDAAO, put forward the evidence that even a short deletion of the loop generates a consistent variation of the enzyme structure-function properties.

An angular trinuclear copper(II) complex as a model for the active site of multicopper oxidases

A new angular trinuclear copper(II) complex [Cu 3L 1L 2(1-MeIm)(H 2O)](ClO 4) ( 1), where H 3L 1 and H 2L 2 are penta and tetra-dentate Schiff base ligands, is prepared from a reaction of [CuL 2] and [Cu 2L 1(μ-Br)] in the presence of 1-methylimidazole (1-MeIm) and isolated as a perchlorate salt. The complex which crystallizes in the monoclinic space group P2 1/ n shows the presence of a discrete angular trinuclear copper(II) core in which the monomeric precursor is linked with the dimeric precursor through phenoxo bridges. The core structure resembles well with the active site structure of blue multicopper oxidases. The Cu⋯Cu separations of ca. 3.3 Å in the angular unit is 0.4 Å shorter than those observed in the active site of the oxidized form of ascorbate oxidase. The complex displays a visible band at 620 nm ( ε=500 M −1 cm −1) in DMSO. It shows magnetic moment values of 3.07 and 2.18 μ B at 300 and 18 K, respectively, for the Cu 3 cluster. The complex models the structural and magnetic properties of the trinuclear active site of blue copper oxidases.

Copper(II) complexes with { N, N′, N, N′-bis[(2-hydroxybenzyl) (2-pyridylmethyl)]-1,3-propanediamine}—H 2bbppn: their suitability as models for the inactive form of galactose oxidase

This paper summarizes the results of our studies on the structural, spectral and redox properties of new mononuclear copper(II) complexes which can mimic some structural and spectroscopic features of the enzyme galactose oxidase (GAO) in its inactive form. The crystal structure of the [Cu II(H 2bbppn)](ClO 4) 2 complex 1 (where H 2bbppn={ N, N′, N, N′-bis[(2-hydroxybenzyl) (2-pyridylmethyl)]-1,3-propanediamine}) has been determined by X-ray crystallography. This structure reveals a distorted octahedral geometry around the Cu(II) atom, where the equatorial plane is occupied by alkylamine and pyridine nitrogen atoms (in a cis fashion), and also reveals unusual coordination by two protonated oxygen atoms (from phenol groups) in the axial positions. The long Cu(II)–O phenol distances of 2.474(5) and 2.501(5) Å in complex 1, are significantly similar to the Cu(II)–O(Tyr495) distance (≈2.6 Å) in the GAO. The equatorial Cu–N bonds showing mean values of 2.03 Å, are also in good agreement with the values of ≈2.10 Å for GAO. The addition of base to complex 1 results in the formation of a green complex 2, for which a square-pyramidal geometry is suggested, with one oxygen atom in an equatorial position while the other oxygen atom is not coordinated as indicated by potentiometric titration. Furthermore, the spectroscopy (UV–Vis and EPR) properties of 2 are essentially similar to those observed in the inactive form of GAO.

Protein film voltammetry of Rhodobacter capsulatus xanthine dehydrogenase.

Xanthine dehydrogenase (XDH) from the bacterium Rhodobacter capsulatus catalyzes the hydroxylation of xanthine to uric acid with NAD(+) as the electron acceptor. R. capsulatus XDH forms an (alphabeta)(2) heterotetramer and is highly homologous to homodimeric eukaryotic XDHs. The crystal structures of bovine XDH and R. capsulatus XDH showed that the two proteins have highly similar folds; however, R.capsulatus XDH is at least 5 times more active than bovine XDH and, unlike mammalian XDH, does not undergo the conversion to the oxidase form. Here we demonstrate electrocatalytic activity of the recombinant enzyme, expressed in Escherichia coli, while immobilized on an edge plane pyrolytic graphite working electrode. Furthermore, we have determined all redox potentials of the four cofactors (Mo(VI/V), Mo(V/IV), FAD/FADH, FADH/FADH(2) and two distinct [2Fe-2S](2+/+) clusters) using a combination of potentiometric and voltammetric methods. A novel feature identified in catalytic voltammetry of XDH concerns the potential for the onset of catalysis (ca. 400 mV), which is at least 600 mV more positive than that of the highest potential cofactor. This unusual observation is explained on the basis of a pterin-associated oxidative switch during voltammetry that precedes catalysis.

Novel types of two-domain multi-copper oxidases: possible missing links in the evolution

An analysis of the genome sequence database revealed novel types of two-domain multi-copper oxidases. The two-domain proteins have the conspicuous combination of blue-copper and inter-domain trinuclear copper binding residues, which is common in ceruloplasmin and ascorbate oxidase but not in nitrite reductase, and therefore are considered to retain the characteristics of the plausible ancestral form of ceruloplasmin and ascorbate oxidase. A possible evolutionary relationship of these proteins is proposed.

Monoamine Oxidase and Head-Twitch Response in Mice: Mechanisms of α-Methylated Substrate Derivatives

It is well known that head-twitch response (HTR) in mice represents hallucinations, since administration of lysergic acid diethylamide (LSD) produces hallucinations in humans, and the HTR in mice is induced by administration of LSD as a hallucinogen. The HTR is produced by excitation of 5-hydroxytryptamine (5-HT) 2A receptors. In this paper, we review the mechanisms of HTR induced by various drugs such as 5-HT precursor, 5-HT receptor agonist, 5-HT releaser, hallucinogenic compounds, benzodiazepins and cannabinoid. The response induced by HTR-inducers is significantly enhanced by combined treatment with a non-selective form of monoamine oxidase (MAO) inhibitor. Thus, the relationship between MAO activity and HTR caused by these drugs (especially, α-methylated analogous compounds which 5-fluoro-α-methyltryptamine, 6-fluoro-α-methyltryptamine and p-hydroxyamphetamine) is presented in detail.

Intramolecular charge transfer and biomimetic reaction kinetics in galactose oxidase model complexes.

One-electron oxidation of two structurally similar CuII-diphenolate complexes, 1 and 2, creates EPR-silent CuII-phenoxyl complexes [1]+ and [2]+ that mimic the oxidized form of the enzyme galactose oxidase (GOase). Both model complexes display novel NIR absorptions assigned to phenolate-phenoxyl charge transfer that resemble a tyrosinate-tyrosyl charge-transfer band observed in the enzymatic system. [1]+ and [2]+ react with benzyl alcohol to form 0.5 equivs of benzaldehyde per complex; biomimetic reduction to CuI-phenol complexes is not observed, but such species may exist transiently. Initial kinetic studies show that [2]+ reacts faster with benzyl alcohol than does [1]+, despite being a significantly weaker oxidant (DeltaE degrees = 370 mV). This acceleration is ascribed to mechanistic differences: [2]+ appears to bind substrate prior to the rate-determining step. Large, nonclassical kinetic isotope effects confirm C-H bond cleavage as the rate-determining step in the reactions of both [1]+ and [2]+ with benzyl alcohol, as is the case for GOase.

Unique amino acids cluster for switching from the dehydrogenase to oxidase form of xanthine oxidoreductase.

In mammals, xanthine oxidoreductase is synthesized as a dehydrogenase (XDH) but can be readily converted to its oxidase form (XO) either by proteolysis or modification of cysteine residues. The crystal structures of bovine milk XDH and XO demonstrated that atoms in the highly charged active-site loop (Gln-423-Lys-433) around the FAD cofactor underwent large dislocations during the conversion, blocking the approach of the NAD+ substrate to FAD in the XO form as well as changing the electrostatic environment around FAD. Here we identify a unique cluster of amino acids that plays a dual role by forming the core of a relay system for the XDH/XO transition and by gating a solvent channel leading toward the FAD ring. A more detailed structural comparison and site-directed mutagenesis analysis experiments showed that Phe-549, Arg-335, Trp-336, and Arg-427 sit at the center of a relay system that transmits modifications of the linker peptide by cysteine oxidation or proteolytic cleavage to the active-site loop (Gln-423-Lys-433). The tight interactions of these residues are crucial in the stabilization of the XDH conformation and for keeping the solvent channel closed. Both oxidative and proteolytic generation of XO effectively leads to the removal of Phe-549 from the cluster causing a reorientation of the bulky side chain of Trp-336, which then in turn forces a dislocation of Arg-427, an amino acid located in the active-site loop. The conformational change also opens the gate for the solvent channel, making it easier for oxygen to reach the reduced FAD in XO.

Recombinant Rhodobacter capsulatus Xanthine Dehydrogenase, a Useful Model System for the Characterization of Protein Variants Leading to Xanthinuria I in Humans

Rhodobacter capsulatus xanthine dehydrogenase (XDH) forms an (alphabeta)2 heterotetramer and is highly homologous to homodimeric eukaryotic XDHs. The crystal structures of bovine XDH and R. capsulatus XDH showed that the two proteins have highly similar folds. We have developed an efficient system for the recombinant expression of R. capsulatus XDH in Escherichia coli. The recombinant protein shows spectral features and a range of substrate specificities similar to bovine milk xanthine oxidase. However, R. capsulatus XDH is at least 5 times more active than bovine XDH and, unlike mammalian XDH, does not undergo the conversion to the oxidase form. EPR spectra were obtained for the FeS centers of the enzyme showing an axial signal for FeSI, which is different from that reported for xanthine oxidase. X-ray absorption spectroscopy at the iron and molybdenum K-edge and the tungsten LIII-edge have been used to probe the different metal coordinations of variant forms of the enzyme. Based on a mutation identified in a patient suffering from xanthinuria I, the corresponding arginine 135 was substituted to a cysteine in R. capsulatus XDH, and the protein variant was purified and characterized. Two different forms of XDH-R135C were purified, an active (alphabeta)2 heterotetrameric form and an inactive (alphabeta) heterodimeric form. The active form contains a full complement of redox centers, whereas in the inactive form the FeSI center is likely to be missing.

Alkaline treatment has contrasting effects on the structure of deglycosylated and glycosylated forms of glucose oxidase

X-ray crystallographic studies on glucose oxidase showed a strong interaction between carbohydrate and protein moieties of the glycoprotein. However, experimental studies under physiological conditions reported no influence of carbohydrate moiety on the structural and functional properties of glucose oxidase. In order to demonstrate the role of carbohydrate moiety on the structure and stability, we carried out a detailed comparative study on the pH-induced structural changes in the native and deglycosylated forms of glucose oxidase. Our studies demonstrate that at physiological pH both forms of enzyme have very similar structural and stability properties. Acid denaturation also showed similar structural changes in both forms of the enzyme. However, on alkaline treatment contrasting effects on the structure and stability of the two forms of enzyme were observed. The glycosylated enzyme undergoes partial unfolding with decreased stability at alkaline pH; however, a compaction of native conformation and enhanced stability of enzyme was observed for the deglycosylated enzyme under similar conditions. This is the first experimental demonstration of the influence of carbohydrate moiety on structure and stability of glucose oxidase. The studies also indicate the importance of pH studies in evaluating the effect of carbohydrate moiety on the structural and stability properties of glycoprotein.

Identification and cloning of two forms of liver peroxisomal fatty Acyl CoA Oxidase from the koala ( Phascolarctos cinereus)

In the present study, the cloning, expression and characterization of the rate-limiting enzyme of the peroxisomal β-oxidation spiral, acyl CoA oxidase (AOX), from koala ( Phascolarctos cinereus) liver is described. It has been previously reported that peroxisomal cyanide-insensitive palmitoyl-CoA oxidation activity was absent in koala liver [Comp. Biochem. Physiol. (C) 127 (2000) 327]. This activity is a measure of the overall peroxisomal β-oxidation minus the final step catalysed by thiolase. Two 2039 bp koala liver AOX cDNAs, designated AOX1 and AOX2, were cloned by reverse transcription–polymerase chain reaction and rapid amplification of cDNA ends. The koala AOX cDNAs encode proteins of 662 amino acids. Transfection of the koala AOX cDNAs into Cos-7 cells resulted in the expression of proteins with palmitoyl-CoA oxidase activity. The apparent K m values for AOX1 and AOX2 cDNA-expressed enzymes were 28 and 38 μM, respectively, which are within the range of order of magnitude reported for rat and human purified AOX enzymes (approximately 10 μM). Northern analysis, utilizing the koala AOX1 cDNA as probe, detected a more intense AOX mRNA band in the koala liver as compared to rats and humans. Southern blot analysis of liver genomic DNA samples revealed a single AOX gene fragment of less than 14 kb in koalas, rat and humans, suggesting a single AOX gene. Collectively, the results of this study suggest that the absence of peroxisomal cyanide-insensitive palmitoyl-CoA oxidation activity in the koala liver is possibly due to deficiencies of one or more enzymes downstream of acyl-CoA oxidase and/or deficiencies of mitochondrial β-oxidation enzymes.