Reaction geometry and thermostable variant of pyranose 2-oxidase from the white-rot fungus Peniophora sp.

Pyranose 2-oxidase catalyzes the oxidation of a number of carbohydrates using dioxygen; glucose, for example, is oxidized at carbon 2. The structure of pyranose 2-oxidase with the reaction product 2-keto-beta-d-glucose bound in the active center is reported in a new crystal form at 1.41 A resolution. The binding structure suggests that the alpha-anomer cannot be processed. The binding mode of the oxidized product was used to model other sugars accepted by the enzyme and to explain its specificity and catalytic rates. The reported structure at pH 6.0 shows a drastic conformational change in the loop of residues 454-461 (loop 454-461) at the active center compared to that of a closely homologous enzyme analyzed at pH 4.5 with a bound acetate inhibitor. In our structures, the loop is highly mobile and shifts to make way for the sugar to pass into the active center. Presumably, loop 454-461 functions as a gatekeeper. Apart from the wild-type enzyme, a thermostable variant was analyzed at 1.84 A resolution. In this variant, Glu542 is exchanged for a lysine. The observed stabilization could be a result of the mutated residue changing an ionic contact at a comparatively weak interface of the tetramer.

Improvement of the fungal enzyme pyranose 2-oxidase using protein engineering.

Native pyranose 2-oxidase (P2Ox) was purified from Peniophora sp. and characterized. To improve its catalytic efficiencies and stabilities by protein engineering, we cloned and expressed the P2Ox gene in Escherichia coli and received active, fully flavinylated recombinant P2OxA. Selenomethionine-labeled P2OxA was used for X-ray analysis and the resulting crystal structure enabled the rational design using variant P2OxA1 with the substitution E542K as template. Besides increased thermal and pH stabilities this variant showed improved catalytic efficiencies (k(cat)/K(m)) for the main substrates. A new variant, P2OxA2H, with an additional substitution T158A and a C-terminal His(6)-tag exhibited significantly decreased apparent K(m) values for D-glucose (0.47 mM), l-sorbose (1.79 mM), and D-xylose (1.35 mM). Compared to native P2Ox, the catalytic efficiencies were substantially improved for D-glucose (230-fold), L-sorbose (874-fold), and D-xylose (1751-fold). This P2Ox variant was used for the bioconversion of L-sorbose under O(2)-saturation in a molar scale. The structure-activity relationships of the amino acid substitutions were analyzed by modelling of the mutated P2Ox structures. Molecular docking calculations of various carbohydrates into the crystal structure of P2OxA and the analysis of the protein-ligand interactions in the docked complexes enabled us to explain the substrate specificity of the enzyme by a conserved hydrogen bond pattern which is formed between the protein and all substrates.

Engineering of pyranose 2-oxidase from Peniophora gigantea towards improved thermostability and catalytic efficiency.

To improve the stability and catalytic efficiency of pyranose 2-oxidase (P2Ox) by molecular enzyme evolution, we cloned P2Ox cDNA by RACE-PCR from a cDNA library derived from the basidiomycete Peniophora gigantea. The P2Ox gene was expressed in Escherichia coli BL21(DE3), yielding an intracellular and enzymatically active P2OxB with a volumetric yield of 500 units/l. Site-directed mutagenesis was employed to construct the P2Ox variant E540K (termed P2OxB1), which exhibited increased thermo- and pH-stability compared with the wild type, concomitantly with increased catalytic efficiencies (k(cat)/K(m)) for D-xylose and L-sorbose. P2OxB1 was provided with a C-terminal His(6)-tag (termed P2OxB1H) and subjected to directed evolution using error-prone PCR. Screening based on a chromogenic assay yielded the new P2Ox variant K312E (termed P2OxB2H) that showed significant improvements with respect to k(cat)/K(m) for D-glucose (5.3-fold), methyl-beta-D-glucoside (2.0-fold), D-galactose (4.8-fold), D-xylose (59.9-fold), and L-sorbose (69.0-fold), compared with wild-type P2Ox. The improved catalytic performance of P2OxB2H was demonstrated by bioconversions of L-sorbose that initially was a poor substrate for wild-type P2Ox. This is the first report on the improvement of a pyranose 2-oxidase by a dual approach of site-directed mutagenesis and directed evolution, and the application of the engineered P2Ox in bioconversions.

Crystal structure of pyranose 2-oxidase from the white-rot fungus Peniophora sp.

Pyranose 2-oxidase catalyzes the oxidation of a number of carbohydrates using dioxygen. The enzyme forms a D(2) symmetric homotetramer and contains one covalently bound FAD per subunit. The structure of the enzyme from Peniophora sp. was determined by multiwavelength anomalous diffraction (MAD) based on 96 selenium sites per crystallographic asymmetric unit and subsequently refined to good-quality indices. According to its chain fold, the enzyme belongs to the large glutathione reductase family and, in a more narrow sense, to the glucose-methanol-choline oxidoreductase (GMC) family. The tetramer contains a spacious central cavity from which the substrate enters one of the four active centers by penetrating a mobile barrier. Since this cavity can only be accessed by glucose-sized molecules, the enzyme does not convert sugars that are part of a larger molecule. The geometry of the active center and a comparison with an inhibitor complex of the homologous enzyme cellobiose dehydrogenase allow the modeling of the reaction at a high confidence level.

Screening of sugar converting enzymes using quantitative MALDI-ToF mass spectrometry.

Quantitative matrix-assisted laser desorption/ionization-time-of-flight mass spectrometry (MALDI-ToF MS) was applied for the screening of ten pyranose oxidase variants. Quantitative MALDI-ToF MS using isotopic labeled internal standards and ionic liquid matrices was performed using aliquots of enzyme reaction mixtures without prepurification steps. The results obtained were in good agreement with HPLC measurements. Analysis time was approx. 3.5 min for a five-fold determination. Thus, quantitative MALDI-ToF MS can be used as a tool for screening of sugar converting enzymes.