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.

Doctoral Programme BioToP Molecular Biotechnology of Proteins BOKU University of Natural Resources and Life Sciences Vienna Vienna Austria

Food Biotechnology Laboratory BOKU University of Natural Resources and Life Sciences Vienna Vienna Austria

Institute of Microbiology of the ASCR v v i Vídeňská 1083 Prague Czech Republic

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Giffhorn F. Fungal pyranose oxidases: occurrence, properties and biotechnical applications in carbohydrate chemistry. Appl Microbiol Biotechnol. 2000;54: 727–740. PubMed

Halada P, Leitner C, Sedmera P, Haltrich D, Volc J. Identification of the covalent flavin adenine dinucleotide-binding region in pyranose 2-oxidase from Trametes multicolor. Anal Biochem. 2003;314: 235–242. PubMed

Hallberg BM, Leitner C, Haltrich D, Divne C. Crystal structure of the 270 kDa homotetrameric lignin-degrading enzyme pyranose 2-oxidase. J Mol Biol. 2004;341: 781–796. PubMed

Leitner C, Haltrich D, Nidetzky B, Prillinger H, Kulbe KD. Production of a novel pyranose 2-oxidase by basidiomycete Trametes multicolor. Appl Biochem Biotechnol. 1998;70–72: 237–248. PubMed

Leitner C, Hess J, Galhaup C, Ludwig R, Nidetzky B, Kulbe KD, et al. Purification and characterization of a laccase from the white-rot fungus Trametes multicolor. Appl Biochem Biotechnol. 2002;98–100: 497–507. PubMed

Ghisla S, Massey V. Mechanisms of flavoprotein-catalyzed reactions. Eur J Biochem. 1989;181: 1–17. PubMed

Prongjit M, Sucharitakul J, Wongnate T, Haltrich D, Chaiyen P. Kinetic mechanism of pyranose 2-oxidase from Trametes multicolor. Biochemistry. 2009;48: 4170–4180. 10.1021/bi802331r PubMed DOI

Brugger D, Krondorfer I, Zahma K, Stoisser T, Bolivar JM, Nidetzky B, et al. Convenient microtiter plate-based, oxygen-independent activity assays for flavin-dependent oxidoreductases based on different redox dyes. Biotechnol J. 2014;9: 474–482. 10.1002/biot.201300336 PubMed DOI PMC

Leitner C, Volc J, Haltrich D. Purification and characterization of pyranose oxidase from the white-rot fungus Trametes multicolor. Appl Environ Microbiol. 2001;67: 3636–3644. PubMed PMC

Pisanelli I, Kujawa M, Spadiut O, Kittl R, Halada P, Volc J, et al. Pyranose 2-oxidase from Phanerochaete chrysosporium—expression in E. coli and biochemical characterization. J Biotechnol. 2009;142: 97–106. 10.1016/j.jbiotec.2009.03.019 PubMed DOI

Kujawa M, Ebner H, Leitner C, Hallberg BM, Prongjit M, Sucharitakul J, et al. Structural basis for substrate binding and regioselective oxidation of monosaccharides at C3 by pyranose 2-oxidase. J Biol Chem. 2006;281: 35104–35115. PubMed

Spadiut O, Tan TC, Pisanelli I, Haltrich D, Divne C. Importance of the gating segment in the substrate-recognition loop of pyranose 2-oxidase. FEBS J. 2010;277: 2892–2909. 10.1111/j.1742-4658.2010.07705.x PubMed DOI

Sucharitakul J, Prongjit M, Haltrich D, Chaiyen P. Detection of a C4a-hydroperoxyflavin intermediate in the reaction of a flavoprotein oxidase. Biochemistry. 2008;47: 8485–8490. 10.1021/bi801039d PubMed DOI

Wongnate T, Chaiyen P. The substrate oxidation mechanism of pyranose 2-oxidase and other related enzymes in the glucose-methanol-choline superfamily. FEBS J. 2013;280: 3009–3027. 10.1111/febs.12280 PubMed DOI

Giffhorn F, Köpper S, Huwig A, Freimund S. Rare sugars and sugar-based synthons by chemo-enzymatic synthesis. Enzyme Microb Technol. 2000;27: 734–742. PubMed

Schneider K, Dorscheid S, Witte K, Giffhorn F, Heinzle E. Controlled feeding of hydrogen peroxide as oxygen source improves production of 5-ketofructose from L-sorbose using engineered pyranose 2-oxidase from Peniophora gigantea. Biotechnol Bioeng. 2012;109: 2941–2945. 10.1002/bit.24572 PubMed DOI

Kwon KY, Kim JY, Youn J, Jeon C, Lee J, Hyeon T, et al. Electrochemical activity studies of glucose oxidase (GOx)-based and pyranose oxidase (POx)-based electrodes in mesoporous carbon: Toward biosensor and biofuel cell applications. Electroanalysis. 2014;26: 2075–2079.

Spadiut O, Brugger D, Coman V, Haltrich D, Gorton L. Engineered pyranose 2-oxidase: Efficiently turning sugars into electrical energy. Electroanalysis. 2010;22: 813–820.

Tasca F, Timur S, Ludwig R, Haltrich D, Volc J, Antiochia R, et al. Amperometric biosensors for detection of sugars based on the electrical wiring of different pyranose oxidases and pyranose dehydrogenases with osmium redox polymers on graphite electrodes. Electroanalysis. 2007;19: 294–302.

Decamps K, Joye I, Haltrich D, Nicolas J, Courtin C, Delcour J. Biochemical characteristics of Trametes multicolor pyranose oxidase and Aspergillus niger glucose oxidase and implications for their functionality in wheat flour dough. Food Chem. 2012;131: 1485–1492.

Swoboda M, Henig J, Cheng HM, Brugger D, Haltrich D, Plumere N, et al. Enzymatic oxygen scavenging for photostability without pH drop in single-molecule experiments. ACS Nano. 2012;6: 6364–6369. 10.1021/nn301895c PubMed DOI PMC

Leitner C, Neuhauser W, Volc J, Kulbe KD, Nidetzky B, Haltrich D. The Cetus process revisited: a novel enzymatic alternative for the production of aldose-free D-fructose. Biocatal Biotrans. 1998;16: 365–382.

Maria G, Ene MD, Jipa I. Modelling enzymatic oxidation of D-glucose with pyranose 2-oxidase in the presence of catalase. J Mol Catal B-Enzym. 2012;74: 209–218.

Treitz G, Maria G, Giffhorn F, Heinzle E. Kinetic model discrimination via step-by-step experimental and computational procedure in the enzymatic oxidation of D-glucose. J Biotechnol. 2001;85: 271–287. PubMed

Spadiut O, Radakovits K, Pisanelli I, Salaheddin C, Yamabhai M, Tan TC, et al. A thermostable triple mutant of pyranose 2-oxidase from Trametes multicolor with improved properties for biotechnological applications. Biotechnol J. 2009;4: 525–534. 10.1002/biot.200800260 PubMed DOI

Spadiut O, Leitner C, Salaheddin C, Varga B, Vertessy BG, Tan TC, et al. Improving thermostability and catalytic activity of pyranose 2-oxidase from Trametes multicolor by rational and semi-rational design. FEBS J. 2009;276: 776–792. 10.1111/j.1742-4658.2008.06823.x PubMed DOI

Savige WE, Fontana A. Interconversion of methionine and methionine sulfoxide. Methods Enzymol. 1977;47: 453–459. PubMed

Li SH, Schöneich C, Borchardt RT. Chemical instability of protein pharmaceuticals: Mechanisms of oxidation and strategies for stabilization. Biotechnol Bioeng. 1995;48: 490–500. PubMed

Manning MC, Chou DK, Murphy BM, Payne RW, Katayama DS. Stability of protein pharmaceuticals: an update. Pharm Res. 2010;27: 544–575. 10.1007/s11095-009-0045-6 PubMed DOI

Stadtman ER. Oxidation of free amino acids and amino acid residues in proteins by radiolysis and by metal-catalyzed reactions. Ann Rev Biochem. 1993;62: 797–821. PubMed

Stadtman ER, Levine RL. Free radical-mediated oxidation of free amino acids and amino acid residues in proteins. Amino Acids. 2003;25: 207–218. PubMed

Freinbichler W, Colivicchi MA, Stefanini C, Bianchi L, Ballini C, Misini B, et al. Highly reactive oxygen species: detection, formation, and possible functions. Cell Mol Life Sci. 2011;68: 2067–2079. 10.1007/s00018-011-0682-x PubMed DOI PMC

Chaiyen P, Fraaije MW, Mattevi A. The enigmatic reaction of flavins with oxygen. Trends Biochem Sci. 2012;37: 373–380. 10.1016/j.tibs.2012.06.005 PubMed DOI