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.
Heckmann-Pohl
Dorothée M
DM
Institut für Angewandte Mikrobiologie, Universität des Saarlandes, Saarbrücken, Germany. dorothee.heckmann@mx.uni-saarland.de
Bastian
Sabine
S
Altmeier
Silke
S
Antes
Iris
I
D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D001483:Base Sequence; D001487:Basidiomycota / Q000201:enzymology / Q000254:growth & development; D001665:Binding Sites; D002237:Carbohydrate Dehydrogenases / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D002384:Catalysis; D018360:Crystallography, X-Ray; D004795:Enzyme Stability / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics; D005656:Fungal Proteins / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D014644:Genetic Variation; D006639:Histidine / Q000737:chemistry; D006358:Hot Temperature; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008958:Models, Molecular; D008969:Molecular Sequence Data; D010084:Oxidation-Reduction; D010802:Phylogeny; D011485:Protein Binding; D011487:Protein Conformation; D015202:Protein Engineering; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D013329:Structure-Activity Relationship; D013379:Substrate Specificity
D005656:Fungal Proteins; D011994:Recombinant Proteins; D006639:Histidine; D002237:Carbohydrate Dehydrogenases; C022489:pyranose oxidase