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

Abstract

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.

Identification and Characterization of Sclerotinia sclerotiorum NADPH Oxidases

“…Recently, we noted two bicupin proteins in the genome sequence of S. sclerotiorum (SS1G_10796.1, 47% identical to Ceriporiopsis subvermispora Oxo1 [CsOxo1], and SS1G_08814.1, 46% identical to CsOxo2). It has been suggested that cupins function as oxalate oxidases in Ceriporiopsis subvermispora (9). Oxalate oxidase (also known as germin-like in plants) catalyzes the generation of hydrogen peroxide (H 2 O 2 ) from oxalate.…”

Burst Kinetics and Redox Transformations of the Active Site Manganese Ion in Oxalate Oxidase

“…High spin ground states for Mn(II) (3d 5 , S ϭ 5 ⁄ 2), Mn(III) (3d 4 , S ϭ 2), and Mn(IV) (3d 3 , S ϭ 3 ⁄ 2) are all in principal detectable by EPR spectroscopy, although the sensitivity is expected to be much greater for the half-integer spin Mn(II) and Mn(IV) species. The EPR spectrum of the periodate-treated OXO (Fig.…”

“…Oxalate oxidase has a wide phylogenetic distribution and has been found in bacteria (4) and fungi (5) but is most abundant in higher plant tissues, particularly germinating seeds (2,6,7). The barley enzyme, which has been most extensively studied, is a hexameric glycoprotein of the cupin superfamily containing a mononuclear manganese center in each subunit (8,9).…”

Oxalate Efflux Transporter from the Brown Rot Fungus Fomitopsis palustris

“…This oxalate secretion is mainly due to continuous production of oxalate in primary carbon metabolism. Furthermore, the acid secretion could be due to the absence of oxalate degradation activity in F. palustris, in contrast to oxalate-decomposing white rot fungi (9,39,47,49) and several brown rot fungi such as P. placenta (25) and G. trabeum (10). Moreover, we have suggested that F. palustris transports oxalate out of the cells to prevent possible inhibition of intracellular metabolic reactions.…”