Oxalate Decarboxylase Activity Research Articles

Enhanced resistance to rice blast and sheath blight in rice (oryza sativa L.) by expressing the oxalate decarboxylase protein Bacisubin from Bacillus subtilis

Oxalate decarboxylase (OxDC), catalyzing the degradation of oxalic acid, is widely distributed in varieties of organisms. In this study, an oxalate decarboxylase gene from Bacillus subtilis strain BS-916, Bacisubin, was transformed into rice variety Nipponbare to generate transgenic rice with increased OxDC activity. Pathogenicity test revealed that the transgenic rice showed enhanced resistance to rice blast and sheath blight. Further RNA-seq analysis between Nipponbare WT (wild type) and transgenic rice identified 1764 DEGs (Differentially expressed genes) including 723 up-regulated unigenes and 1041 down-regulated unigenes. Five GO terms including single-organism process and oxidation-reduction process were significantly enriched in the up-regulated genes. Interestingly, five genes encoding glutaredoxin and one gene encoding MADS box were up- and down-regulated in the transgenic rice, respectively. Collectively, our study advances the understanding of OxDC in resistance to rice disease and its possible mechanisms. Our results also suggest that OxDC would be an effective antifungal protein preventing fungal infection in transgenic rice.

Fungal oxalate decarboxylase activity contributes to Sclerotinia sclerotiorum early infection by affecting both compound appressoria development and function.

Sclerotinia sclerotiorum pathogenesis requires the accumulation of high levels of oxalic acid (OA). To better understand the factors affecting OA accumulation, two putative oxalate decarboxylase (OxDC) genes (Ss-odc1 and Ss-odc2) were characterized. Ss-odc1 transcripts exhibited significant accumulation in vegetative hyphae, apothecia, early stages of compound appressorium development and during plant colonization. Ss-odc2 transcripts, in contrast, accumulated significantly only during mid to late stages of compound appressorium development. Neither gene was induced by low pH or exogenous OA in vegetative hyphae. A loss-of-function mutant for Ss-odc1 (Δss-odc1) showed wild-type growth, morphogenesis and virulence, and was not characterized further. Δss-odc2 mutants hyperaccumulated OA in vitro, were less efficient at compound appressorium differentiation and exhibited a virulence defect which could be fully bypassed by wounding the host plant prior to inoculation. All Δss-odc2 phenotypes were restored to the wild-type by ectopic complementation. An S. sclerotiorum strain overexpressing Ss-odc2 exhibited strong OxDC, but no oxalate oxidase activity. Increasing inoculum nutrient levels increased compound appressorium development, but not penetration efficiency, of Δss-odc2 mutants. Together, these results demonstrate differing roles for S. sclerotiorum OxDCs, with Odc2 playing a significant role in host infection related to compound appressorium formation and function.

Oxalate decarboxylase of the white-rot fungus Dichomitus squalens demonstrates a novel enzyme primary structure and non-induced expression on wood and in liquid cultures

Oxalate decarboxylase (ODC) catalyses the conversion of oxalic acid to formic acid and CO(2) in bacteria and fungi. In wood-decaying fungi the enzyme has been linked to the regulation of intra- and extracellular quantities of oxalic acid, which is one of the key components in biological decomposition of wood. ODC enzymes are biotechnologically interesting for their potential in diagnostics, agriculture and environmental applications, e.g. removal of oxalic acid from industrial wastewaters. We identified a novel ODC in mycelial extracts of two wild-type isolates of Dichomitus squalens, and cloned the corresponding Ds-odc gene. The primary structure of the Ds-ODC protein contains two conserved Mn-binding cupin motifs, but at the N-terminus, a unique, approximately 60 aa alanine-serine-rich region is found. Real-time quantitative RT-PCR analysis confirmed gene expression when the fungus was cultivated on wood and in liquid medium. However, addition of oxalic acid in liquid cultures caused no increase in transcript amounts, thereby indicating a constitutive rather than inducible expression of Ds-odc. The detected stimulation of ODC activity by oxalic acid is more likely due to enzyme activation than to transcriptional upregulation of the Ds-odc gene. Our results support involvement of ODC in primary rather than secondary metabolism in fungi.

Metal dependence of oxalate decarboxylase activity.

Bacillus subtilis oxalate decarboxylase (OxDC) catalyzes the conversion of oxalate into CO(2) and formate. The enzyme is composed of two cupin domains, each of which contains a Mn(II) ion. Although there is general agreement that Mn(II) in the N-terminal domain mediates OxDC-catalyzed decarboxylation, legitimate questions have been raised concerning the function (if any) of the Mn(II) bound in the C-terminal cupin domain. We have investigated this problem using a series of OxDC mutants in which Mn(II) binding is perturbed by mutagenesis of Glu-101 and Glu-280, which coordinate the metal in the N-terminal and C-terminal domains, respectively. We now demonstrate that decarboxylase activity and total manganese content are sensitive to modifications in either metal-binding glutamate residue. These findings, in combination with EPR measurements, raise the possibility that the C-terminal Mn(II) center can catalyze the decarboxylation reaction. Further support for this conclusion has been provided from a combination of in vivo and in vitro strategies for preparing wild-type OxDC in which Mn(II) is incorporated to a variety of extents. Kinetic characterization of these variants shows that OxDC activity is linearly correlated with manganese content, as might be expected if both sites can catalyze the breakdown of oxalate into formate and CO(2). These studies also represent the first unequivocal demonstration that OxDC activity is uniquely mediated by manganese.

Induction of oxalate decarboxylase by oxalate in a newly isolated Pandoraea sp. OXJ-11 and its ability to protect against Sclerotinia sclerotiorum infection

Pandoraea sp. OXJ-11 has been shown to produce an oxalate decarboxylase. The enzyme could be induced by increasing the oxalate in the medium. An increasing concentration of yeast extract was able to stimulate the cell growth but could not increase the specific oxalate decarboxylase activity. The oxalate decarboxylase was produced maximally at 25-35 degrees C and pH 4.0-9.0, favoring its potential application in protection of host plants from oxalate-producing phytopathogens. The influence of glucose on the induction of oxalate decarboxylase by oxalate was examined, and it was found that glucose inhibited the production of the oxalate decarboxylase. Resistance results showed that Pandoraea sp. OXJ-11 was capable of suppressing Sclerotinia sclerotiorum infection on detached leaflets of Brassica napus plants.

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

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 < or =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.

Assembly of an oxalate decarboxylase produced under sigmaK control into the Bacillus subtilis spore coat.

Over 30 polypeptides are synthesized at various times during sporulation in Bacillus subtilis, and they are assembled at the surface of the developing spore to form a multilayer protein structure called the coat. The coat consists of three main layers, an amorphous undercoat close to the underlying spore cortex peptidoglycan, a lamellar inner layer, and an electron-dense striated outer layer. The product of the B. subtilis oxdD gene was previously shown to have oxalate decarboxylase activity when it was produced in Escherichia coli and to be a spore constituent. In this study, we found that OxdD specifically associates with the spore coat structure, and in this paper we describe regulation of its synthesis and assembly. We found that transcription of oxdD is induced during sporulation as a monocistronic unit under the control of sigma(K) and is negatively regulated by GerE. We also found that localization of a functional OxdD-green fluorescent protein (GFP) at the surface of the developing spore depends on the SafA morphogenetic protein, which localizes at the interface between the spore cortex and coat layers. OxdD-GFP localizes around the developing spore in a cotE mutant, which does not assemble the spore outer coat layer, but it does not persist in spores produced by the mutant. Together, the data suggest that OxdD-GFP is targeted to the interior layers of the coat. Additionally, we found that expression of a multicopy allele of oxdD resulted in production of spores with increased levels of OxdD that were able to degrade oxalate but were sensitive to lysozyme.

Oxalate Decarboxylase Requires Manganese and Dioxygen for Activity: OVEREXPRESSION AND CHARACTERIZATION OF BACILLUS SUBTILIS YvrK AND YoaN

The Bacillus subtilis oxalate decarboxylase (EC ), YvrK, converts oxalate to formate and CO(2). YvrK and the related hypothetical proteins YoaN and YxaG from B. subtilis have been successfully overexpressed in Escherichia coli. Recombinant YvrK and YoaN were found to be soluble enzymes with oxalate decarboxylase activity only when expressed in the presence of manganese salts. No enzyme activity has yet been detected for YxaG, which was expressed as a soluble protein without the requirement for manganese salts. YvrK and YoaN were found to catalyze minor side reactions: oxalate oxidation to produce H(2)O(2); and oxalate-dependent, H(2)O(2)-independent dye oxidations. The oxalate decarboxylase activity of purified YvrK was O(2)-dependent. YvrK was found to contain between 0.86 and 1.14 atoms of manganese/subunit. EPR spectroscopy showed that the metal ion was predominantly but not exclusively in the Mn(II) oxidation state. The hyperfine coupling constant (A = 9.5 millitesla) of the main g = 2 signal was consistent with oxygen and nitrogen ligands with hexacoordinate geometry. The structure of YvrK was modeled on the basis of homology with oxalate oxidase, canavalin, and phaseolin, and its hexameric oligomerization was predicted by analogy with proglycinin and homogentisate 1,2-dioxygenase. Although YvrK possesses two potential active sites, only one could be fully occupied by manganese. The possibility that the C-terminal domain active site has no manganese bound and is buried in an intersubunit interface within the hexameric enzyme is discussed. A mechanism for oxalate decarboxylation is proposed, in which both Mn(II) and O(2) are cofactors that act together as a two-electron sink during catalysis.

Detection and partial characterization of oxalate decarboxylase from Agaricus bisporus

Oxalate decarboxylase activity was present in liquid culture medium and mycelium of Agaricus bisporus. Enzyme activity in the mycelium peaked at two-weekly intervals after primary growth phase and into secondary metabolism, with activity peaks in the medium occurring 7 d later than in the mycelium. The enzyme was partially purified and had two isozymes with pls 3.0 and 3.4. Characterization of the protein by SDS-PAGE and Western blotting against a polyclonal antibody raised to oxalate decarboxylase from Collybia velutipes showed a major protein band of 64 kDa. Oxalate decarboxylase was also detected in the fruit body up to the cup stage of development. The pH optimum for the enzyme was 3.6 and temperature optimum was 35 °C.

Oxalate Decarboxylase from Collybia velutipes: MOLECULAR CLONING AND ITS OVEREXPRESSION TO CONFER RESISTANCE TO FUNGAL INFECTION IN TRANSGENIC TOBACCO AND TOMATO

Oxalic acid is present as nutritional stress in many crop plants like Amaranth and Lathyrus. Oxalic acid has also been found to be involved in the attacking mechanism of several phytopathogenic fungi. A full-length cDNA for oxalate decarboxylase, an oxalate-catabolizing enzyme, was isolated by using 5'-rapid amplification of cDNA ends-polymerase chain reaction of a partial cDNA as cloned earlier from our laboratory (Mehta, A., and Datta, A. (1991) J. Biol. Chem. 266, 23548-23553). By screening a genomic library from Collybia velutipes with this cDNA as a probe, a genomic clone has been isolated. Sequence analyses and comparison of the genomic sequence with the cDNA sequence revealed that the cDNA is interrupted with 17 small introns. The cDNA has been successfully expressed in cytosol and vacuole of transgenic tobacco and tomato plants. The transgenic plants show normal phenotype, and the transferred trait is stably inherited to the next generation. The recombinant enzyme is partially glycosylated and shows oxalate decarboxylase activity in vitro as well as in vivo. Transgenic tobacco and tomato plants expressing oxalate decarboxylase show remarkable resistance to phytopathogenic fungus Sclerotinia sclerotiorum that utilizes oxalic acid during infestation. The result presented in the paper represents a novel approach to develop transgenic plants resistant to fungal infection.

"Active" one-carbon generation in Saccharomyces cerevisiae

A new mutation introducing a one-carbon requirement (e.g., formate) for the glycine-supplemented growth of a serine-glycine auxotroph (ser1) was correlated with a lack of glycine decarboxylase activity. The presence of oxalate decarboxylase activity or glyoxylate decarboxylase activity did not overcome the one-carbon requirement. Another mutation characterized by the absence of oxalate decarboxylase activity did not introduce a one-carbon requirement. The presence and physiological significance of glycine decarboxylase activity in Saccharomyces are thus inferred.