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Gewählte Publikation:

Kujawa, M; Ebner, H; Leitner, C; Hallberg, BM; Prongjit, M; Sucharitakul, J; Ludwig, R; Rudsander, U; Peterbauer, C; Chaiyen, P; Haltrich, D; Divne, C.
(2006): Structural basis for substrate binding and regioselective oxidation of monosaccharides at C3 by pyranose 2-oxidase.
J Biol Chem. 2006; 281(46):35104-35115 FullText FullText_BOKU

Pyranose2-oxidase(P2Ox) participates in fungal lignin degradation by producing the H2O2 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 regios-electivity, 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. Based on the details of 2-fluoro-2-deoxy-D-glucose binding in position for oxidation at C3, we also outline a probable binding mode for D-glucose positioned for regioselective oxidation at C2. The tentative determinant for discriminating between the two binding modes is the position of the O6 hydroxyl group, which in the C2-oxidation mode can make favorable interactions with Asp(452) in the substrate loop and, possibly, a nearby arginine residue (Arg(472)). We also substantiate our hypothesis with steady-state kinetics data for the alanine replacements of Asp(452) and Arg(472) as well as the double alanine 452/472 mutant.
Autor/innen der BOKU Wien:
Haltrich Dietmar
Kujawa Magdalena
Leitner Christian
Ludwig Roland
Peterbauer Clemens Karl
BOKU Gendermonitor:

Find related publications in this database (using NML MeSH Indexing)
Basidiomycota - enzymology
Carbohydrate Conformation - enzymology
Carbohydrate Dehydrogenases - metabolism
Monosaccharides - chemistry
Oxidation-Reduction - chemistry
Protein Binding - chemistry
Substrate Specificity - chemistry

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