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.     

Structure of oxalate decarboxylase from Bacillus subtilis at 1.75 A resolution

Oxalate decarboxylase is a manganese-dependent enzyme that catalyzes the conversion of oxalate to formate and carbon dioxide. We have determined the structure of oxalate decarboxylase from Bacillus subtilis at 1.75 A resolution in the presence of formate. The structure reveals a hexamer with 32-point symmetry in which each monomer belongs to the cupin family of proteins. Oxalate decarboxylase is further classified as a bicupin because it contains two cupin folds, possibly resulting from gene duplication. Each oxalate decarboxylase cupin domain contains one manganese binding site. Each of the oxalate decarboxylase domains is structurally similar to oxalate oxidase, which catalyzes the manganese-dependent oxidative decarboxylation of oxalate to carbon dioxide and hydrogen peroxide. Amino acid side chains in the two metal binding sites of oxalate decarboxylase and the metal binding site of oxalate oxidase are very similar. Four manganese binding residues (three histidines and one glutamate) are conserved as well as a number of hydrophobic residues. The most notable difference is the presence of Glu333 in the metal binding site of the second cupin domain of oxalate decarboxylase. We postulate that this domain is responsible for the decarboxylase activity and that Glu333 serves as a proton donor in the production of formate. Mutation of Glu333 to alanine reduces the catalytic activity by a factor of 25. The function of the other domain in oxalate decarboxylase is not yet known.     

Real-time monitoring of the oxalate decarboxylase reaction and probing hydron exchange in the product, formate, using fourier transform infrared spectroscopy

Oxalate decarboxylase converts oxalate to formate and carbon dioxide and uses dioxygen as a cofactor despite the reaction involving no net redox change. We have successfully used Fourier transform infrared spectroscopy to monitor in real time both substrate consumption and product formation for the first time. The assignment of the peaks was confirmed using [(13)C]oxalate as the substrate. The K(m) for oxalate determined using this assay was 3.8-fold lower than that estimated from a stopped assay. The infrared assay was also capable of distinguishing between oxalate decarboxylase and oxalate oxidase activity by the lack of formate being produced by the latter. In D(2)O, the product with oxalate decarboxylase was C-deuterio formate rather than formate, showing that the source of the hydron was solvent as expected. Large solvent deuterium kinetic isotope effects were observed on V(max) (7.1 +/- 0.3), K(m) for oxalate (3.9 +/- 0.9), and k(cat)/K(m) (1.8 +/- 0.4) indicative of a proton transfer event during a rate-limiting step. Semiempirical quantum mechanical calculations on the stability of formate-derived species gave an indication of the stability and nature of a likely enzyme-bound formyl radical catalytic intermediate. The capability of the enzyme to bind formate under conditions in which the enzyme is known to be active was determined by electron paramagnetic resonance. However, no enzyme-catalyzed exchange of the C-hydron of formate was observed using the infrared assay, suggesting that a formyl radical intermediate is not accessible in the reverse reaction. This restricts the formation of potentially harmful radical intermediates to the forward reaction.     

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.     

Adsorption of ethylenediaminetetraacetic dianhydride modified oxalate decarboxylase on calcium oxalate

We used ethylenediaminetetraacetic acid dianhydride (EDTAD) to modify oxalate decarboxylase (OXDC) to improve its adsorption on calcium oxalate stones. The modified sites were identified by Ultra performance liquid chromatography-mass spectrometry (UPLC-MS) and the adsorption mechanism of the EDTAD-modified OXDC on calcium oxalate (CaOx) was investigated. We investigated adsorption time, initial enzyme concentration, temperature and solution pH on the adsorption process. Data were analyzed using kinetics, thermodynamics and isotherm adsorption models. UPLC-MS showed that EDTAD was attached to OXDC covalently and suggested that the chemical modification occurred at both the free amino of the side chain and the α-NH₂ of the peptide. The adsorption capacity of the EDTAD-OXDC on calcium oxalate was 53.37% greater than that of OXDC at the initial enzyme concentration of 5 mg/ml, pH = 7.0, at 37° C. The modified enzyme (EDTAD-OXDC) demonstrated improved oxalate degradation activity at pH 4.5?6.0. Kinetic data fitting analysis suggested a pseudo second order kinetic model. Estimates of the thermodynamic parameters including ΔG⁰, ΔH⁰ and ΔS⁰ of the adsorption process showed it to be feasible, spontaneous and endothermic. Isotherm data fitting analysis indicated that the adsorption process is reduced to monolayer adsorption at a low enzyme concentration and to multilayer adsorption at a high enzyme concentration. It may be possible to apply OXDC to degradation of calcium oxalate stones.     

A secretion signal is present in the Collybia velutipes oxalate decarboxylase gene.

The oxalate decarboxylase (OXDC) gene from Collybia velutipes is overexpressed as an active form in Schizosaccharomyces pombe. The recombinant enzyme shows similar pH optima and stability, while substrate kinetic analysis shows a ninefold decrease in K(m) value with respect to native OXDC. Most of the expressed protein was present in periplasm and remained firmly bound to cell-wall materials. However, 20% of enzyme expressed was secreted out into the medium suggesting the presence of a secretion signal (C. velutipes) in the oxalate decarboxylase gene. This secretion signal is associated with the N-terminal of OXDC as is evident by secretion of nonsecretory genes AmA1 and beta-galactosidase. An expression vector using this signal is constructed for expression and secretion of heterologous proteins in S. pombe.     

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.     

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 ≤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.     

草酸脱羧酶及其应用

草酸脱羧酶是一种含锰的酶,在白腐菌中广泛存在,少数低等真菌和细菌中也能产生。目前,至少10多种草酸脱羧酶得到了分离和纯化。该酶是一种氨基酸残基在379个左右的单体酶,一般都为酸性糖蛋白,含有2个锰离子,形成2个活性区域;表面一些氨基酸被不同程度地糖基化。晶体结构和其它一些波谱学研究解释了其空间结构和可能的电子传递机制。运用PCR技术和cDNA文库技术,越来越多的草酸脱羧酶基因被克隆。已研究的该酶基因中都含有17个左右的内含子,这些内含子在活性域位置上有比较高的保守性。一些特殊氨基酸序列的存在决定了该酶的表达形式为诱导型,菌株的基因调控序列中含有一段受草酸化合物作用的序列。该酶在一些酵母和植物等异源表达系统中有成功表达的报道。该酶的应用主要集中在以下几方面：造纸废水中的草酸盐降解;食品中的草酸降解;草酸生物检测（如,临床诊断）等。     

Oxalate decarboxylase from Agrobacterium tumefaciens C58 is translocated by a twin arginine translocation system

Oxalate decarboxylases (OXDCs) (E.C. 4.1.1.2) are enzymes catalyzing the conversion of oxalate to formate and CO The OXDCs found in fungi and bacteria belong to functionally diverse protein superfamily known as the cupins. Fungi-originated OXDCs are secretory enzymes. However, most bacterial OXDCs are localized in the cytosol, and may be involved in energy metabolism. In Agrobacterium tumefaciens C58, a locus for a putative oxalate decarboxylase is present. In the study reported here, an enzyme was overexpressed in Escherichia coli and showed oxalate carboxylase activity. Computational analysis revealed the A. tumefaciens C58 OXDC contains a signal peptide mediating translocation of the enzyme into the periplasm that was supported by expression of signal-peptideless and full-length versions of the enzyme in A. tumefaciens C58. Further site-directed mutagenesis experiment demonstrated that the A. tumefaciens C58 OXDC is most likely translocated by a twin-arginine translocation (TAT) system.     

Enzymatic oxalate decarboxylation in isolates of Sclerotinia sclerotiorum

Abstract Sclerotinia sclerotiorum isolates B24 (virulent) and SS41 (hypovirulent) possess oxalate decarboxylase. Production was regulated by composition and pH of culture medium and required the presence of oxalate or its precursor, succinic acid, as inducers. Mycelia of both isolates contain equivalent amounts of enzyme.     

EPR spectroscopic characterization of the manganese center and a free radical in the oxalate decarboxylase reaction: identification of a tyrosyl radical during turnover

Several molecular mechanisms for cleavage of the oxalate carbon-carbon bond by manganese-dependent oxalate decarboxylase have recently been proposed involving high oxidation states of manganese. We have examined the oxalate decarboxylase from Bacillus subtilis by electron paramagnetic resonance in perpendicular and parallel polarization configurations to test for the presence of such species in the resting state and during enzymatic turnover. Simulation and the position of the half-field Mn(II) line suggest a nearly octahedral metal geometry in the resting state. No spectroscopic signature for Mn(III) or Mn(IV) is seen in parallel mode EPR for samples frozen during turnover, consistent either with a large zero-field splitting in the oxidized metal center or undetectable levels of these putative high-valent intermediates in the steady state. A narrow, featureless g = 2.0 species was also observed in perpendicular mode in the presence of substrate, enzyme, and dioxygen. Additional splittings in the signal envelope became apparent when spectra were taken at higher temperatures. Isotopic editing resulted in an altered line shape only when tyrosine residues of the enzyme were specifically deuterated. Spectral processing confirmed multiple splittings with isotopically neutral enzyme that collapsed to a single prominent splitting in the deuterated enzyme. These results are consistent with formation of an enzyme-based tyrosyl radical upon oxalate exposure. Modestly enhanced relaxation relative to abiological tyrosyl radicals was observed, but site-directed mutagenesis indicated that conserved tyrosine residues in the active site do not host the unpaired spin. Potential roles for manganese and a peripheral tyrosyl radical during steady-state turnover are discussed.     

Oxalate consumption by lactobacilli: evaluation of oxalyl-CoA decarboxylase and formyl-CoA transferase activity in Lactobacillus acidophilus

AIMS: This study was undertaken to evaluate the oxalate-degrading activity in several Lactobacillus species widely used in probiotic dairy and pharmaceutical preparations. Functional characterization of oxalyl-CoA decarboxylase and formyl-CoA transferase in Lactobacillus acidophilus was performed in order to assess the possible contribution of Lactobacillus in regulating the intestinal oxalate homeostasis. METHODS AND RESULTS: In order to determine the oxalate-degrading ability in 60 Lactobacillus strains belonging to 12 species, a screening was carried out by using an enzymatic assay. A high variability in the oxalate-degrading capacity was found in the different species. Strains of Lact. acidophilus and Lactobacillus gasseri showed the highest oxalate-degrading activity. Oxalyl-CoA decarboxylase and formyl-CoA transferase genes from Lact. acidophilus LA14 were cloned and sequenced. The activity of the recombinant enzymes was assessed by capillary electrophoresis. CONCLUSIONS: Strains of Lactobacillus with a high oxalate-degrading activity were identified. The function and significance of Lact. acidophilus LA14 oxalyl-CoA decarboxylase and formyl-CoA transferase in oxalate catabolism were demonstrated. These results suggest the potential use of Lactobacillus strains for the degradation of oxalate in the human gut. SIGNIFICANCE AND IMPACT OF THE STUDY: Identification of probiotic strains with oxalate-degrading activity can offer the opportunity to provide this capacity to individuals suffering from an increased body burden of oxalate and oxalate-associated disorders.     

Distribution of peroxisomes and glycolate metabolism in relation to calcium oxalate formation in Lemna minor L

Calcium oxalate formation in Lemna minor L. occurs in structurally specialized cells called crystal idioblasts. Cytochemical and immunocytochemical protocols were employed to study the distribution of peroxisomes and the enzymes glycolate oxidase, glycine decarboxylase and ribulose 1,5-bisphosphate carboxylase-oxygenase (RuBisCO) in relation to synthesis of oxalate used for Ca oxalate formation. These enzymes are necessary for photorespiratory glycolate synthesis and metabolism. Using catalase cytochemistry, microbodies were found to exist in crystal idioblasts but were smaller and fewer than those found in mesophyll cells. Glycolate oxidase, which can oxidize glycolate to oxalate via glyoxylate, could not be found in microbodies of crystal idioblasts at any stage of development. This enzyme increased in amount in microbodies of mesophyll cells as they matured and could even be found in dense amorphous inclusions of mature cell peroxisomes. Glycine decarboxylase and RuBisCO could also be detected in increasing amount in mesophyll cells as they matured but could not be detected in idioblasts or were just detectable. Thus, Lemna idioblasts lack the machinery for synthesis of oxalate from glycolate. Based on these results and other available information, two general models for the generation and accumulation of oxalate used for Ca oxalate formation in crystal idioblasts are proposed. The biochemical specialization of crystal idioblasts indicated by this study is also discussed with respect to differentiation of cellular structure and function.     

The parasporal crystals of Bacillus pumilus strain 15.1: a potential virulence factor?

Bacillus pumilus strain 15.1 was previously found to cause larval mortality in the Med‐fly Ceratitis capitata and was shown to produce crystals in association with the spore. As parasporal crystals are well‐known as invertebrate‐active toxins in entomopathogenic bacteria such as Bacillus thuringiensis (Cry and Cyt toxins) and Lysinibacillus sphaericus (Bin and Cry toxins), the B. pumilus crystals were characterized. The crystals were composed of a 45 kDa protein that was identified as an oxalate decarboxylase by peptide mass fingerprinting, N‐terminal sequencing and by comparison with the genome sequence of strain 15.1. Synthesis of crystals by a plasmid‐cured derivative of strain 15.1 (produced using a novel curing strategy), demonstrated that the oxalate decarboxylase was encoded chromosomally. Crystals spontaneously solubilized when kept at low temperatures, and the protein produced was resistant to trypsin treatment. The insoluble crystals produced by B. pumilus 15.1 did not show significant toxicity when bioassayed against C. capitata larvae, but once the OxdD protein was solubilized, an increase of toxicity was observed. We also demonstrate that the OxdD present in the crystals has oxalate decarboxylate activity as the formation of formate was detected, which suggests a possible mechanism for B. pumilus 15.1 activity. To our knowledge, the characterization of the B. pumilus crystals as oxalate decarboxylase is the first report of the natural production of parasporal inclusions of an enzyme.     

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.     

Observation of superoxide production during catalysis of Bacillus subtilis oxalate decarboxylase at pH 4

This contribution describes the trapping of the hydroperoxyl radical at a pH of 4 during turnover of wild-type oxalate decarboxylase and its T165V mutant using the spin-trap BMPO. Radicals were detected and identified by a combination of EPR and mass spectrometry. Superoxide, or its conjugate acid, the hydroperoxyl radical, is expected as an intermediate in the decarboxylation and oxidation reactions of the oxalate monoanion, both of which are promoted by oxalate decarboxylase. Another intermediate, the carbon dioxide radical anion was also observed. The quantitative yields of superoxide trapping are similar in the wild type and the mutant while it is significantly different for the trapping of the carbon dioxide radical anion. This suggests that the two radicals are released from different sites of the protein.     

EPR spin trapping of an oxalate-derived free radical in the oxalate decarboxylase reaction

EPR spin trapping experiments on bacterial oxalate decarboxylase from Bacillus subtilis under turn-over conditions are described. The use of doubly (13)C-labeled oxalate leads to a characteristic splitting of the observed radical adducts using the spin trap N-tert-butyl-α-phenylnitrone linking them directly to the substrate. The radical was identified as the carbon dioxide radical anion which is a key intermediate in the hypothetical reaction mechanism of both decarboxylase and oxidase activities. X-ray crystallography had identified a flexible loop, SENS161-4, which acts as a lid to the putative active site. Site directed mutagenesis of the hinge amino acids, S161 and T165 was explored and showed increased radical trapping yields compared to the wild type. In particular, T165V shows approximately ten times higher radical yields while at the same time its decarboxylase activity was reduced by about a factor of ten. This mutant lacks a critical H-bond between T165 and R92 resulting in compromised control over its radical chemistry allowing the radical intermediate to leak into the surrounding solution.