Oxidation of Phe454 in the Gating Segment Inactivates Trametes multicolor Pyranose Oxidase during Substrate Turnover.

The flavin-dependent enzyme pyranose oxidase catalyses the oxidation of several pyranose sugars at position C-2. In a second reaction step, oxygen is reduced to hydrogen peroxide. POx is of interest for biocatalytic carbohydrate oxidations, yet it was found that the enzyme is rapidly inactivated under turnover conditions. We studied pyranose oxidase from Trametes multicolor (TmPOx) inactivated either during glucose oxidation or by exogenous hydrogen peroxide using mass spectrometry. MALDI-MS experiments of proteolytic fragments of inactivated TmPOx showed several peptides with a mass increase of 16 or 32 Da indicating oxidation of certain amino acids. Most of these fragments contain at least one methionine residue, which most likely is oxidised by hydrogen peroxide. One peptide fragment that did not contain any amino acid residue that is likely to be oxidised by hydrogen peroxide (DAFSYGAVQQSIDSR) was studied in detail by LC-ESI-MS/MS, which showed a +16 Da mass increase for Phe454. We propose that oxidation of Phe454, which is located at the flexible active-site loop of TmPOx, is the first and main step in the inactivation of TmPOx by hydrogen peroxide. Oxidation of methionine residues might then further contribute to the complete inactivation of the enzyme.

NITRILASE1 regulates the exit from proliferation, genome stability and plant development.

Nitrilases are highly conserved proteins with catabolic activity but much less understood functions in cell division and apoptosis. To elucidate the biological functions of Arabidopsis NITRILASE1, we characterized its molecular forms, cellular localization and involvement in cell proliferation and plant development. We performed biochemical and mass spectrometry analyses of NITRILASE1 complexes, electron microscopy of nitrilase polymers, imaging of developmental and cellular distribution, silencing and overexpression of nitrilases to study their functions. We found that NITRILASE1 has an intrinsic ability to form filaments. GFP-NITRILASE1 was abundant in proliferating cells, distributed in cytoplasm, in the perinuclear area and associated with microtubules. As cells exited proliferation and entered differentiation, GFP-NITRILASE1 became predominantly nuclear. Nitrilase silencing dose-dependently compromised plant growth, led to loss of tissue organization and sustained proliferation. Cytokinesis was frequently aborted, leading to enlarged polyploid cells. In reverse, independently transformed cell lines overexpressing GFP-NITRILASE1 showed slow growth and increased rate of programmed cell death. Altogether, our data suggest that NITRILASE1 homologues regulate the exit from cell cycle and entry into differentiation and simultaneously are required for cytokinesis. These functions are essential to maintain normal ploidy, genome stability and tissue organization.

Characterization of pyranose dehydrogenase from Agaricus meleagris and its application in the C-2 specific conversion of D-galactose.

Pyranose dehydrogenase (PDH) of the mushroom Agaricus meleagris was purified from mycelial culture media to substantial homogeneity using ion-exchange and hydrophobic interaction chromatography. The native enzyme is a monomeric polypeptide with a molecular mass of 66,547Da as determined by matrix-assisted laser desorption/ionisation mass spectrometry containing approximately 7% carbohydrate and covalently bound flavin adenine dinucleotide. The enzyme exhibited a broad sugar substrate tolerance, oxidizing different aldopyranoses to the corresponding C-2 or C-2,3 (di)dehydro sugars. Preferred electron donors with the highest k(cat)/K(m) values were major sugar constituents of cellulose and hemicellulose, namely d-glucose, D-galactose, l-arabinose, D-xylose and cellobiose. This indicates a possible physiological role of the enzyme in lignocellulose breakdown. PDH showed no detectable activity with oxygen, and its reactivity towards electron acceptors was limited to various substituted benzoquinones and complexed metal ions, with the ferricenium ion and the benzoquinone imine 2,6-dichloroindophenole displaying the highest k(cat)/K(m). The enzyme catalyzed in up to 95% yields the regiospecific conversion of D-galactose to 2-dehydro-D-galactose, an intermediate in a possible biotechnologically interesting process for redox isomerization of D-galactose to the prebiotic sugar D-tagatose.

Molecular cloning of three pyranose dehydrogenase-encoding genes from Agaricus meleagris and analysis of their expression by real-time RT-PCR.

Sugar oxidoreductases such as cellobiose dehydrogenase or pyranose oxidase are widespread enzymes among fungi, whose biological function is largely speculative. We investigated a similar gene family in the mushroom Agaricus meleagris and its expression under various conditions. Three genes (named pdh1, pdh2 and pdh3) putatively encoding pyranose dehydrogenases were isolated. All three genes displayed a conserved structure and organization, and the respective cDNAs contained ORFs translating into polypeptides of 602 or 600 amino acids. The N-terminal sections of all three genes encode putative signal peptides consistent with the enzymes extracellular secretion. We cultivated the fungus on different carbon sources and analyzed the mRNA levels of all three genes over a period of several weeks using real-time RT-PCR. The glyceraldehyde-3-phosphate dehydrogenase gene from A. meleagris was also isolated and served as reference gene. pdh2 and pdh3 are essentially transcribed constitutively, whereas pdh1 expression is upregulated upon exhaustion of the carbon source; pdh1 appears to be additionally regulated under conditions of oxygen limitation. These data are consistent with an assumed role in lignocellulose degradation.

Characteristics of Gloeophyllum trabeum alcohol oxidase, an extracellular source of H2O2 in brown rot decay of wood.

A novel alcohol oxidase (AOX) has been purified from mycelial pellets of the wood-degrading basidiomycete Gloeophyllum trabeum and characterized as a homooctameric nonglycosylated protein with native and subunit molecular masses of 628 and 72.4 kDa, containing noncovalently bonded flavin adenine dinucleotide. The isolated AOX cDNA contained an open reading frame of 1,953 bp translating into a polypeptide of 651 amino acids displaying 51 to 53% identity with other published fungal AOX amino acid sequences. The enzyme catalyzed the oxidation of short-chain primary aliphatic alcohols with a preference for methanol (K(m) = 2.3 mM, k(cat) = 15.6 s(-1)). Using polyclonal antibodies and immunofluorescence staining, AOX was localized on liquid culture hyphae and extracellular slime in sections from degraded wood and on cotton fibers. Transmission electron microscopy immunogold labeling localized the enzyme in the hyphal periplasmic space and wall and on extracellular tripartite membranes and slime, while there was no labeling of hyphal peroxisomes. AOX was further shown to be associated with membranous or slime structures secreted by hyphae in wood fiber lumina and within the secondary cell walls of degraded wood fibers. The differences in AOX targeting compared to the known yeast peroxisomal localization were traced to a unique C-terminal sequence of the G. trabeum oxidase, which is apparently responsible for the protein's different translocation. The extracellular distribution and the enzyme's abundance and preference for methanol, potentially available from the demethylation of lignin, all point to a possible role for AOX as a major source of H(2)O(2), a component of Fenton's reagent implicated in the generally accepted mechanisms for brown rot through the production of highly destructive hydroxyl radicals.

Properties of pyranose dehydrogenase purified from the litter-degrading fungus Agaricus xanthoderma.

We purified an extracellular pyranose dehydrogenase (PDH) from the basidiomycete fungus Agaricus xanthoderma using ammonium sulfate fractionation and ion-exchange and hydrophobic interaction chromatography. The native enzyme is a monomeric glycoprotein (5% carbohydrate) containing a covalently bound FAD as its prosthetic group. The PDH polypeptide consists of 575 amino acids and has a molecular mass of 65 400 Da as determined by MALDI MS. On the basis of the primary structure of the mature protein, PDH is a member of the glucose-methanol-choline oxidoreductase family. We constructed a homology model of PDH using the 3D structure of glucose oxidase from Aspergillus niger as a template. This model suggests a novel type of bi-covalent flavinylation in PDH, 9-S-cysteinyl, 8-alpha-N3-histidyl FAD. The enzyme exhibits a broad sugar substrate tolerance, oxidizing structurally different aldopyranoses including monosaccharides and oligosaccharides as well as glycosides. Its preferred electron donor substrates are D-glucose, D-galactose, L-arabinose, and D-xylose. As shown by in situ NMR analysis, D-glucose and D-galactose are both oxidized at positions C2 and C3, yielding the corresponding didehydroaldoses (diketoaldoses) as the final reaction products. PDH shows no detectable activity with oxygen, and its reactivity towards electron acceptors is rather limited, reducing various substituted benzoquinones and complexed metal ions. The azino-bis-(3-ethylbenzthiazolin-6-sulfonic acid) cation radical and the ferricenium ion are the best electron acceptors, as judged by the catalytic efficiencies (k(cat)/K(m)). The enzyme may play a role in lignocellulose degradation.