Purification and Characterization of Pyranose Oxidase from the White Rot Fungus Trametes multicolor

Abstract

We purified an intracellular pyranose oxidase from mycelial extracts of the white rot fungus Trametes multicolor by using ammonium sulfate fractionation, hydrophobic interaction, ion-exchange chromatography, and gel filtration. The native enzyme has a molecular mass of 270 kDa as determined by equilibrium ultracentrifugation and is composed of four identical 68-kDa subunits as determined by matrix-assisted laser desorption ionization mass spectrometry. Each subunit contains one covalently bound flavin adenine dinucleotide as its prosthetic group. The enzyme oxidizes several aldopyranoses specifically at position C-2, and its preferred electron donor substrates are D-glucose, D-xylose, and L-sorbose. During this oxidation reaction electrons are transferred to oxygen, yielding hydrogen peroxide. In addition, the enzyme catalyzes the twoelectron reduction of 1,4-benzoquinone, several substituted benzoquinones, and 2,6-dichloroindophenol, as well as the one-electron reduction of the ABTS [2,2-azinobis(3-ethylbenzthiazolinesulfonic acid)] cation radical. As judged by the catalytic efficiencies (k cat /K m ), some of these quinone electron acceptors are much better substrates for pyranose oxidase than oxygen. The optimum pH of the pyranose oxidase-catalyzed reaction depends strongly on the electron acceptor employed and varies from 4 to 8. It has been proposed that the main metabolic function of pyranose oxidase is as a constituent of the ligninolytic system of white rot fungi that provides peroxidases with H 2 O 2 . An additional function could be reduction of quinones, key intermediates that are formed during mineralization of lignin.The enzyme pyranose oxidase (P2O) (pyranose:oxygen 2-oxidoreductase; EC 1.1.3.10), which catalyzes the oxidation of several aldopyranoses at position C-2 to yield the corresponding 2-ketoaldoses (aldos-2-uloses, osones), is widely distributed among wood-degrading basidiomycetes (14,32,44). It has been purified and characterized from several microorganisms, including Phanerochaete chrysosporium (2, 45), Phlebiopsis gigantea (39), Pleurotus ostreatus (40), Polyporus obtusus (28), Trametes (Coriolus) versicolor (35), and unidentified basidiomycete no. 52 (26). The currently available data reveal some general similarities among P2Os from these different fungi. Typically, P2O is a rather large, homotetrameric protein that contains covalently bound flavin adenine dinucleotide (FAD). The in vivo substrates of P2O probably are D-glucose, D-galactose, and D-xylose, which are abundant in lignocellulose and which are oxidized to 2-keto-D-glucose (D-arabino-hexos-2-ulose, 2-dehydro-D-glucose), 2-keto-D-galactose (D-lyxo-hexos-2-ulose, 2-dehydro-D-galactose), and 2-keto-D-xylose (D-threopentos-2-ulose, 2-dehydro-D-xylose), respectively. In addition, P2O also exhibits significant activity with a number of other carbohydrates, including L-sorbose, D-glucono-1,5-lactone, and D-allose (20). The substrate selectivity, however, varies to some extent among P2Os isolated from different fungi. During the oxi...

Engineering of pyranose 2-oxidase for modified oxygen reactivity

“…This reaction catalyzed by POx is typically found in flavoprotein oxidoreductases [11,12] and is of the Ping Pong Bi Bi type [13]. In terms of catalytic efficiency some of the alternative electron acceptors are in fact better substrates for the enzyme than oxygen [1,14], but until now the chemical and structural reasons for reactivity with either electron acceptor are unidentified [15]. Flavoproteins can be classified based on their reactivity with molecular oxygen [11,16,17]: Electron transferases produce significant amounts of O 2 2 [18] and form the neutral flavin semiquinone over the whole range of their pH stability [19], whereas dehydrogenases generate variable, and often very low, amounts of O 2 2 when reacting with O 2 [18].…”

“…N3) of His 167 [5,6]. POx contributes to the ligninolytic system as a producer of H 2 O 2 for lignin-degrading peroxidases [7,8] and is involved in the quinone redox cycling machinery [1]. POx catalyzes the regioselective oxidation of aldopyranoses, preferrentially D-glucose [1], at position C2 and the corresponding 2-ketoaldoses are formed [9].…”

“…The ensuing half reaction reoxidizes the cofactor by reducing molecular oxygen to H 2 O 2 (eq2) [10]. Possible alternative electron acceptors include quinones (eq3), radicals or chelated metal ions [1]. This reaction catalyzed by POx is typically found in flavoprotein oxidoreductases [11,12] and is of the Ping Pong Bi Bi type [13].…”

“…Pyranose 2-oxidase (POx; pyranose:oxygen 2-oxidoreductase; synonym, glucose 2-oxidase; EC1.1.3.10) from Trametes multicolor (TmPOx; synonym, Trametes ochracea [1]) is a member of the glucose-methanol-choline (GMC) family of oxidoreductases [2][3][4]. POx is an intracellular enzyme but is encountered in the culture medium upon prolonged cultivation, probably due to hyphal autolysis [3].…”

“…POx is an intracellular enzyme but is encountered in the culture medium upon prolonged cultivation, probably due to hyphal autolysis [3]. The homotetrameric TmPOx has a molecular mass of 270 kDa [1], and each subunit binds flavin adenine dinucleotide (FAD) covalently as a co-factor via its 8a-methyl group to N e2 (i.e. N3) of His 167 [5,6].…”