Characterization of Ceriporiopsis subvermispora bicupin oxalate oxidase expressed in Pichia pastoris (original) (raw)
Related papers
Applied and Environmental Microbiology, 2005
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.
Oxalate Oxidase from Ceriporiopsis subvermispora: Biochemical and Cytochemical Studies
Archives of Biochemistry and Biophysics, 1999
The enzyme oxalate oxidase was identified in mycelial extracts of the basidiomycete Ceriporiopsis subvermispora and thereafter purified to homogeneity. The purification procedure included only three steps: Q-Sepharose chromatography, precipitation at pH 3.0, and phosphocellulose chromatography. The enzyme is a 400-kDa homohexamer, as determined by gel permeation in Sephadex G-200 and SDS–polyacrylamide gel electrophoresis. Isoelectrofocusing revealed a pI of 4.2. Optimal activity was obtained at pH 3.5 and at 45°C. The purified enzyme has Km and kcat values of 0.1 mM and 88 s−1, respectively. It is highly specific for oxalate, although it is inhibited at concentrations of this substrate above 2.5 mM. Hystochemistry studies conducted over mycelium slices showed reactions products in both endocellular and periplasmic associated elements. A possible connection between the intracellular metabolism of oxalate and the extracellular ligninolytic activity of the fungus is proposed.
Oxidation reactions catalyzed by manganese peroxidase isoenzymes from Ceriporiopsis subvermispora
Febs Letters, 1995
A total of 11 manganese peroxidase isoenzymes (MnPI-MnPn) with isoelectric points (pIs) in the range of 4.58-3.20 were isolated from liquid-and solid-state cultures of the basidiomycete Ceriporiopsis subvermispora. In the presence of hydrogen peroxide, these isoenzymes showed different requirements for Mn(II) in the oxidation of vanillylacetone, o-dianisidine,p-anisidine and ABTS, whereas oxidation of guaiacol by any isoenzyme did not take place when this metal was omitted. Km values for o-dianisidine and p-anisidine in the absence of Mn(II) are in the range of 0.5-1.0 mM and 4.5-42.0 mM, respectively. Oxalate and citrate, but not tartrate, accelerate the oxidation of o-dianisidine, both in the presence and in the absence of Mn(II).
Hydrogen peroxide inhibition of bicupin oxalate oxidase
PLOS ONE, 2017
Oxalate oxidase is a manganese containing enzyme that catalyzes the oxidation of oxalate to carbon dioxide in a reaction that is coupled with the reduction of oxygen to hydrogen peroxide. Oxalate oxidase from Ceriporiopsis subvermispora (CsOxOx) is the first fungal and bicupin enzyme identified that catalyzes this reaction. Potential applications of oxalate oxidase for use in pancreatic cancer treatment, to prevent scaling in paper pulping, and in biofuel cells have highlighted the need to understand the extent of the hydrogen peroxide inhibition of the CsOxOx catalyzed oxidation of oxalate. We apply a membrane inlet mass spectrometry (MIMS) assay to directly measure initial rates of carbon dioxide formation and oxygen consumption in the presence and absence of hydrogen peroxide. This work demonstrates that hydrogen peroxide is both a reversible noncompetitive inhibitor of the CsOxOx catalyzed oxidation of oxalate and an irreversible inactivator. The build-up of the turnovergenerated hydrogen peroxide product leads to the inactivation of the enzyme. The introduction of catalase to reaction mixtures protects the enzyme from inactivation allowing reactions to proceed to completion. Circular dichroism spectra indicate that no changes in global protein structure take place in the presence of hydrogen peroxide. Additionally, we show that the CsOxOx catalyzed reaction with the three carbon substrate mesoxalate consumes oxygen which is in contrast to previous proposals that it catalyzed a non-oxidative decarboxylation with this substrate.
Applied and Environmental Microbiology, 2001
We expressed cDNAs coding for manganese peroxidases (MnPs) from the basidiomycetes Ceriporiopsis subvermispora (MnP1) and Phanerochaete chrysosporium (H4) under control of the ␣-amylase promoter from Aspergillus oryzae in Aspergillus nidulans. The recombinant proteins (rMnP1 and rH4) were expressed at similar levels and had molecular masses, both before and after deglycosylation, that were the same as those described for the MnPs isolated from the corresponding parental strains. Isoelectric focusing (IEF) analysis of rH4 revealed several isoforms with pIs between 4.83 and 4.06, and one of these pIs coincided with the pI described for H4 isolated from P. chrysosporium (pI 4.6). IEF of rMnP1 resolved four isoenzymes with pIs between 3.45 and 3.15, and the pattern closely resembled the pattern observed with MnPs isolated from C. subvermispora grown in solid-state cultures. We compared the abilities of recombinant MnPs to use various substrates and found that rH4 could oxidize o-dianisidine and p-anisidine without externally added manganese, a property not previously reported for this MnP isoenzyme from P. chrysosporium.
Electronic Journal of Biotechnology, 1998
Ceriporiopsis subvermispora is a white-rot basidiomycete that produces several isoenzymes of manganese peroxidase (MnP). A cDNA of one of them (MnP13-1) has been isolated and sequenced. The deduced aminoacid sequence shows about 60% similarity with the MnPs from Phanerochaete chrysosporium. Based on the crystal structures of MnP and lignin peroxidase (LiP) from P. chrysosporium, and of a peroxidase from Arthromyces ramosus (ARP), we have modeled by homology the three dimensional structure of MnP13-1 using standard modeling procedures. Local molecular mechanics optimization performed in the region corresponding to the binding sites of Ca 2+ and Mn 2+ in MnP13-1 demonstrated that the stereochemistry and the geometry of binding are conserved in both MnPs. A putative aromatic binding site in MnP13-1 is described. We also report structural differences between the two MnPs, arising from the insertion in MnP13-1 of the sequences TGGN between residues S230 and D231 and TDSP at the C-terminal, both of which may have functional significance. The white-rot basidiomycete Ceriporiopsis subvermispora is strongly ligninolytic (Otjen et al. 1987, Blanchette et al. 1992). When growing on wood chips or in agitated liquid cultures, this fungus produces several isoenzymes of manganesedependent peroxidase (MnP) and laccase (Lobos et al. 1994, Salas et al. 1995). We have characterized some isoenzymes of MnP with respect to substrate specificity and requirement of Mn 2 + for activity (Urzúa et al. 1995), and recently we have isolated a cDNA clone of one of them (MnP13-1, GeneBank Access # U60413). The amino acid sequence deduced from the cDNA is over 60% homologous to the published MnP sequences from P. chrysosporium (Pribnow et al. 1989,
Biochimica Et Biophysica Acta-gene Structure and Expression, 2000
Three new genes (Cs-mnp2A, Cs-mnp2B and Cs-mnp3) coding for manganese-dependent peroxidase (MnP) have been identified in the white-rot basidiomycete Ceriporiopsis subvermispora. The mature proteins contain 366 (MnP2A and MnP2B) and 364 (MnP3) amino acids, which are preceded by leader sequences of 21 and 24 amino acids, respectively. Cs-mnp2A and Cs-mnp2B appear to be alleles, since the corresponding protein sequences differ in only five residues. The upstream region of Cs-mnp2B contains a TATA box, AP-1 and AP-2 sites, as well as sites for transcription regulation by metals (two), cAMP (two) and xenobiotics (one). Some of these elements are also found in the regulatory region of Cs-MnP3. Transcription of Cs-mnp2A and Cs-mnp2B, but not that of Cs-mnp3, is activated by manganese. ß
Archives of Biochemistry and Biophysics, 1998
The kinetics of Mn 3+-oxalate formation and decay were investigated in reactions catalyzed by manganese peroxidase (MnP) from the basiomycete Ceriporiopsis subvermispora in the absence of externally added hydrogen peroxide. A characteristic lag observed in the formation of this complex was shortened by glyoxylate or catalytic amounts of Mn 3+ or hydrogen peroxide. MnP titers had a minor effect on this lag and did not influence the decay rate of the complex. In contrast, Mn 2+ and oxalate drastically affected maximal concentrations of the Mn 3+-oxalate complex formed, the decay of which was accelerated at high Mn 2+ levels. The highest concentration of complex was obtained at pH 4.0, whereas an inverse relationship was found between the pH of the reaction and the decay rate of the complex with MnP present. In the absence of MnP, the best fit for the decay kinetics of the complex gave an order of 3/2 at concentrations in the range of 30-100 µM, with a k obs = 0.012 min-1 M-0.5 at pH 4.0. The rate constant increases at lower pH values and decreases at high oxalate concentrations. The physiological relevance of these findings is discaused.
Structure of the Plant Alternative Oxidase
Journal of Biological Chemistry, 2001
All higher plants and many fungi contain an alternative oxidase (AOX), which branches from the cytochrome pathway at the level of the quinone pool. In an attempt, first, to distinguish between two proposed structural models of this di-iron protein, and, second, to examine the roles of two highly conserved tyrosine residues, we have expressed an array of site-specific mutants in Schizosaccharomyces pombe. Mitochondrial respiratory analysis reveals that S. pombe cells expressing AOX proteins in which Glu-217 or Glu-270 were mutated, no longer exhibit antimycin-resistant oxygen uptake, indicating that these residues are essential for AOX activity. Although such data corroborate a model that describes the AOX as an interfacial membrane protein, they are not in full agreement with the most recently proposed ligation sphere of its di-iron center. We furthermore show that upon mutation of Tyr-253 and Tyr-275 to phenylalanines, AOX activity is fully maintained or abolished, respectively. These data are discussed in reference to the importance of both residues in the catalytic cycle of the AOX.