Oxalate Oxidase from Ceriporiopsis subvermispora: Biochemical and Cytochemical Studies

Aguilar, Claudio; Urzúa, Ulises; Koenig, Cecilia S.; Vicuña, Rafael

doi:10.1006/abbi.1999.1216

Cited by 67 publications

(49 citation statements)

References 24 publications

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“…Oxalate oxidase was purified from C. subvermispora to homogeneity using a previously published procedure (1). Amino acid sequencing of its N terminus was not possible due to it being blocked.…”

Section: Resultsmentioning

confidence: 99%

“…One unit of enzyme activity was defined as the conversion of 1 mol of substrate to product per min. Oxalate oxidase activity was determined using stopped assays, where the production of hydrogen peroxide was coupled to the oxidation of phenol red using horseradish peroxidase as described previously (1). It was important to subtract control reactions without peroxidase in order to distinguish between the production of hydrogen peroxide and the oxalate-dependent direct oxidation of the dye (37).…”

Section: Methodsmentioning

confidence: 99%

“…An oxalate oxidase (EC 1.2.3.4) was identified in C. subvermispora that could be involved in a pathway leading to the production of hydrogen peroxide (1). This enzyme catalyzes the conversion of oxalate and dioxygen to carbon dioxide and hydrogen peroxide.…”

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confidence: 99%

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Cloning and Sequencing of Two Ceriporiopsis subvermispora Bicupin Oxalate Oxidase Allelic Isoforms: Implications for the Reaction Specificity of Oxalate Oxidases and Decarboxylases

Escutia

Bowater

Edwards

et al. 2005

Appl Environ Microbiol

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Oxalate oxidase is thought to be involved in the production of hydrogen peroxide for lignin degradation by the dikaryotic white rot fungus Ceriporiopsis subvermispora. This enzyme was purified, and after digestion with trypsin, peptide fragments of the enzyme were sequenced using quadrupole time-of-flight mass spectrometry. Starting with degenerate primers based on the peptide sequences, two genes encoding isoforms of the enzyme were cloned, sequenced, and shown to be allelic. Both genes contained 14 introns. The sequences of the isoforms revealed that they were both bicupins that unexpectedly shared the greatest similarity to microbial bicupin oxalate decarboxylases rather than monocupin plant oxalate oxidases (also known as germins). We have shown that both fungal isoforms, one of which was heterologously expressed in Escherichia coli, are indeed oxalate oxidases that possess <0.2% oxalate decarboxylase activity and that the organism is capable of rapidly degrading exogenously supplied oxalate. They are therefore the first bicupin oxalate oxidases to have been described. Heterologous expression of active enzyme was dependent on the addition of manganese salts to the growth medium. Molecular modeling provides new and independent evidence for the identity of the catalytic site and the key amino acid involved in defining the reaction specificities of oxalate oxidases and oxalate decarboxylases.

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Section: Resultsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

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Cloning and Sequencing of Two Ceriporiopsis subvermispora Bicupin Oxalate Oxidase Allelic Isoforms: Implications for the Reaction Specificity of Oxalate Oxidases and Decarboxylases

Escutia

Bowater

Edwards

et al. 2005

Appl Environ Microbiol

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“…This would increase the activity of peroxidases and enhance ligninolysis (3). However, also other ways for the production of H 2 O 2 have been suggested in C. subvermispora, like the MnP/Mn 3ϩ -dependent extracellular oxidation of glyoxylic and oxalic acids (47) or the action of a periplasmic oxalate oxidase (1). Also, the production of MnP in shaking flask cultures starts during the earliest culture stage and gradually decreases thereafter (15), similar to the stability and reactivity of the manganese ion during cultivations (44), whereas CDH was found at a later stage.…”

Section: Discussionmentioning

confidence: 99%

Cellobiose Dehydrogenase from the Ligninolytic Basidiomycete Ceriporiopsis subvermispora

Harreither

Sygmund

Dünhofen

et al. 2009

Appl Environ Microbiol

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Cellobiose dehydrogenase (CDH), an extracellular flavocytochrome produced by several wood-degrading fungi, was detected in cultures of the selective delignifier Ceriporiopsis subvermispora when grown on a cellulose-and yeast extract-based liquid medium. CDH amounted to up to 2.5% of total extracellular protein during latter phases of the cultivation and thus suggested an important function for the fungus under the given conditions. The enzyme was purified 44-fold to apparent homogeneity. It was found to be present in two glycoforms of 98 kDa and 87 kDa with carbohydrate contents of 16 and 4%, respectively. The isoelectric point of both glycoforms is around 3.0, differing by 0.1 units, which is the most acidic value so far reported for a CDH. By using degenerated primers of known CDH sequences, one cdh gene was found in the genomic DNA, cloned, and sequenced. Alignment of the 774-amino-acid protein sequence revealed a high similarity to CDH from other white rot fungi. One notable difference was found in the longer interdomain peptide linker, which might affect the interdomain electron transfer at higher temperatures. The preferred substrate of C. subvermispora CDH is cellobiose, while glucose conversion is strongly discriminated by a 155,000-fold-lower catalytic efficiency. This is a typical feature of a basidiomycete CDH, as are the acidic pH optima for all tested electron acceptors in the range from 2.5 to 4.5.White rot fungi are the most efficient lignocellulose degraders in our ecosystem, and several species, e.g., Phanerochaete chrysosporium, Trametes versicolor, and Ceriporiopsis subvermispora, have been studied in great detail as model organisms for this complex process. The ability to degrade phenolic and nonphenolic lignin structures in wood has made these strains attractive for biotechnological applications mainly in the pulp and paper industry, where C. subvermispora exhibits a substantial advantage over P. chrysosporium and T. versicolor through its ability for selective removal of a large fraction of lignin without attacking the valuable cellulose (16, 38). The lignindegrading system of these fungi is composed of extracellular enzymes together with low-molecular-mass cofactors (21, 46). Typically found ligninolytic enzymes are lignin peroxidase, manganese peroxidase (MnP), and laccase. The secretion pattern of these enzymes varies greatly in white rot fungi (22) and is influenced by culture conditions and medium composition. Whereas P. chrysoporium secretes high lignin and manganese peroxidase activities but no laccase activity (32, 33), C. subvermispora produces several MnP and laccase isoforms but no lignin peroxidase. T. versicolor is the only one of these model organisms known so far to express all three of these ligninolytic enzymes efficiently (5). Together with the cellulolytic enzyme system, these patterns of enzyme activities cause varied degrees of lignin and cellulose breakdown at different cultivation stages. The simultaneous attack of cellulose and lignin is the preferred strategy of T. ...

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“…Oxalate oxidase is widespread in nature and has been found in bacteria (4), fungi (1,5), and various plant tissues (6). It has been detected in barley seedling roots during germination and in the leaves of mature barley plants in response to powdery mildew infection (6,7), suggesting a role in plant signaling and defense.…”

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confidence: 99%

Structural and Spectroscopic Studies Shed Light on the Mechanism of Oxalate Oxidase

Opaleye

Rose

Whittaker

et al. 2006

Journal of Biological Chemistry

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Oxalate oxidase (EC 1.2.3.4) catalyzes the conversion of oxalate and dioxygen to hydrogen peroxide and carbon dioxide. In this study, glycolate was used as a structural analogue of oxalate to investigate substrate binding in the crystalline enzyme. The observed monodentate binding of glycolate to the active site manganese ion of oxalate oxidase is consistent with a mechanism involving C-C bond cleavage driven by superoxide anion attack on a monodentate coordinated substrate. In this mechanism, the metal serves two functions: to organize the substrates (oxalate and dioxygen) and to transiently reduce dioxygen. The observed structure further implies important roles for specific active site residues (two asparagines and one glutamine) in correctly orientating the substrates and reaction intermediates for catalysis. Combined spectroscopic, biochemical, and structural analyses of mutants confirms the importance of the asparagine residues in organizing a functional active site complex.Oxalate oxidase (OXO; EC 1.2.3.4) 6 catalyzes the oxidation of oxalate, reducing dioxygen to hydrogen peroxide and forming 2 mol of carbonOxalate oxidase is widespread in nature and has been found in bacteria (4), fungi (1, 5), and various plant tissues (6). It has been detected in barley seedling roots during germination and in the leaves of mature barley plants in response to powdery mildew infection (6, 7), suggesting a role in plant signaling and defense. The enzyme has been purified to homogeneity from barley seedling roots and its N-terminal sequence determined, allowing the corresponding cDNA to be isolated and the complete primary sequence to be determined (3,8). These developments led to the recognition that the enzyme, OXO, is identical to an important marker of grain development during germination of wheat called germin (3,8). OXO is a member of a functionally diverse protein superfamily known as the cupins (9) or double stranded ␤-helix proteins (10). Barley OXO forms a hexamer that has extreme stability to heat and proteolysis (11).Spectroscopic studies demonstrated that OXO requires manganese for catalysis (12) and subsequent crystallographic studies on the barley enzyme revealed the structure of the hexamer (Fig. 1a) and confirmed the presence of a mononuclear manganese center buried deep within its jellyroll ␤-barrel domain (13). The manganese is bound by the side chains of three histidines and one glutamate residue, as well as two water molecules that occupy adjacent positions in the roughly octahedral metal complex (Fig. 1b). Based on the lack of obvious optical absorption and the presence of a characteristic EPR spectrum, the manganese ion has been assigned as the reduced Mn(II) oxidation state in the resting enzyme (12). Spectroscopic studies using recombinant OXO expressed in Pichia pastoris confirmed the presence of Mn(II) in the resting recombinant enzyme and provided the first spectroscopic evidence for oxalate binding to the manganese (14). The EPR signal of the anaerobic substrate complex, like that of the nativ...

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Oxalate Oxidase from Ceriporiopsis subvermispora: Biochemical and Cytochemical Studies

Cited by 67 publications

References 24 publications

Cloning and Sequencing of Two Ceriporiopsis subvermispora Bicupin Oxalate Oxidase Allelic Isoforms: Implications for the Reaction Specificity of Oxalate Oxidases and Decarboxylases

Cloning and Sequencing of Two Ceriporiopsis subvermispora Bicupin Oxalate Oxidase Allelic Isoforms: Implications for the Reaction Specificity of Oxalate Oxidases and Decarboxylases

Cellobiose Dehydrogenase from the Ligninolytic Basidiomycete Ceriporiopsis subvermispora

Structural and Spectroscopic Studies Shed Light on the Mechanism of Oxalate Oxidase

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