Structural Basis for Substrate Binding and Regioselective Oxidation of Monosaccharides at C3 by Pyranose 2-Oxidase

Abstract

Pyranose 2-oxidase (P2Ox) participates in fungal lignin degradation by producing the H 2 O 2 needed for lignin-degrading peroxidases. The enzyme oxidizes cellulose-and hemicellulose-derived aldopyranoses at C2 preferentially, but also on C3, to the corresponding ketoaldoses. To investigate the structural determinants of catalysis, covalent flavinylation, substrate binding, and regioselectivity, wild-type and mutant P2Ox enzymes were produced and characterized biochemically and structurally. Removal of the histidyl-FAD linkage resulted in a catalytically competent enzyme containing tightly, but noncovalently bound FAD. This mutant (H167A) is characterized by a 5-fold lower k cat , and a 35-mV lower redox potential, although no significant structural changes were seen in its crystal structure. In previous structures of P2Ox, the substrate loop (residues 452-457) covering the active site has been either disordered or in a conformation incompatible with carbohydrate binding. We present here the crystal structure of H167A in complex with a slow substrate, 2-fluoro-2-deoxy-D-glucose. Pyranose 2-oxidase (P2Ox, 3 pyranose:oxygen 2-oxidoreductase; glucose 2-oxidase; EC 1.1.3.10) is a flavin adenine dinucleotide (FAD)-dependent oxidase present in the hyphal periplasmic space (1) of wood-degrading basidiomycetes (2, 3). These fungi are the only known microorganisms that are capable of fully mineralizing lignin, and P2Ox has a proposed role in the oxidative events (4) of lignin degradation by providing the essential co-substrate, H 2 O 2 , for lignin and manganese peroxidases (5, 6). An alternative hypothesis assigns a role for P2Ox in both H 2 O 2 production and in the reduction of quinones in the periplasm or in the extracellular environment (7). P2Ox from the white-rot fungi Trametes multicolor (Trametes ochracea) and Peniophora gigantea are hitherto the most studied biochemically (7-10) and structurally (11, 12).P2Ox oxidizes a broad range of carbohydrate substrates that are natural constituents of hemicelluloses, allowing most lignocellulose-derived sugars to be utilized. Substrates can be oxidized regioselectively at the C2 position, although some oxidation at C3 can occur as a side reaction (10). For C2 oxidation, D-glucose, D-xylose, and L-sorbose are good or reasonably good substrates, and D-galactose and L-arabinose perform poorly as substrates (7). Based on the catalytic efficiency, k cat /K m , D-glucose (D-Glc) is the best substrate for T. multicolor P2Ox (7). Substrates that are oxidized at C3 were analyzed for P. gigantea P2Ox and include 2-deoxy-D-glucose, 2-keto-D-glucose, and methyl ␤-D-glucosides (13, 10). That oxidation can take place either at C2 or at C3 presupposes two distinct, productive binding modes (referred to here as C2 ox and C3 ox ) for a monosaccharide in the P2Ox active site.P2Ox from T. multicolor is homotetrameric with a molecular mass of 270 kDa (7) where each of the four subunits carries one FAD molecule bound covalently to N ⑀2 (i.e. N3) of His 167 via its 8␣-methyl group (14, 11). The...

Kinetic Mechanism of Pyranose 2-Oxidase from Trametes multicolor

“…In P2O, the residues His548 and Asn593 are conserved as found in most members of the GMC family except glucose oxidase in which only the histidinehistidine pair was found instead (9). The three-dimensional structure of P2O modeled with the substrate D-glucose has shown that the distance from the C2 site of D-glucose to the N(5) of the flavin ring is 3.1 Å , suggesting that the hydride transfer mechanism is possible (10). In addition, the residues His548 and Asn593 can participate in hydrogen bonding of the C2-OH of the sugar (10), indicating that His548 may act as a reactive base in proton abstraction.…”

“…The loop is in the open conformation when a sugar substrate (2-fluoro-2-deoxy-D-glucose) (10) or 2-keto-D-glucose is bound (11). The open conformation is proposed to be relevant to the reductive half-reaction since there is more space at the active site to accommodate the sugar binding; in fact the loop in its closed conformation would interfere with the sugar substrate binding (10). The closed conformation is compatible with the oxidative half-reaction since the active site is more closed off from the bulk solvent, providing a suitable environment for the formation of a C4a-hydroperoxy-FAD intermediate (13,33).…”

“…The reaction catalyzed by P2O can be divided into a reductive half-reaction where the FAD cofactor is reduced by a sugar substrate and an oxidative half-reaction where the reduced FAD is oxidized by molecular oxygen or other electron acceptors such as benzoquinones or metal ions (1). It has been proposed that the enzyme assumes the open conformation during the reductive half-reaction and the closed conformation during the oxidative half-reaction (10). Recent investigation on steady-state kinetics of P2O from T. ochracea shows the difference in the kinetic mechanism at pH below and above 7.0 (12).…”

Engineering of pyranose 2-oxidase for modified oxygen reactivity

“…C-terminal His 6 -tagged POx gene from T. multicolor was expressed in electro-competent E. coli BL21 Star DE3 cells (Thermo Fisher Scientific Biosciences, Waltham, MA) using the pET21d + /POx (pHL2) vector as described in [61]. Cells were grown on LB amp plates (10 g L 21 peptone from casein, 5 g L 21 yeast extract, 10 g L 21 NaCl, 14 g L 21 agar supplemented with 100 mg mL 21 ampicillin) and plasmid pHL2 was used as template for all mutagenic PCRs.…”

Reactivity of sorbose dehydrogenase from Sinorhizobium sp. 97507 for 1,5-anhydro-d-glucitol

“…It is established that pyranose 2-oxidase from Trametes multicolor oxidizes D-glucose at the C-2 position and 2-keto-D-glucose at the C-3 position by rotation of the substrate in the active site. 15) Therefore, the mechanism of action of SiSDH may be elucidated when its X-ray crystal structure is solved. Further study is required to identify the site at which SiSDH acts on 1,5-AG.…”