Structural basis for substrate binding and regioselective oxidation of monosaccharides at C3 by pyranose 2-oxidase
2006 (English)In: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 281, no 46, 35104-35115 p.Article in journal (Refereed) Published
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.
Place, publisher, year, edition, pages
2006. Vol. 281, no 46, 35104-35115 p.
electron-transfer flavoprotein, p-cresol methylhydroxylase, fungus trametes-multicolor, crystal-structure, phanerochaete-chrysosporium, cellobiose dehydrogenase, alcohol oxidase, covalent flavinylation, catalytic mechanism, lignin degradation
IdentifiersURN: urn:nbn:se:kth:diva-16121DOI: 10.1074/jbc.M604718200ISI: 000241933700044ScopusID: 2-s2.0-33845930584OAI: oai:DiVA.org:kth-16121DiVA: diva2:334163
QC 201005252010-08-052010-08-05Bibliographically approved