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Please use this identifier to cite or link to this item: http://repository.li.mahidol.ac.th/dspace/handle/123456789/22949
Title: Structural basis for substrate binding and regioselective oxidation of monosaccharides at C3 by pyranose 2-oxidase
Authors: Magdalena Kujawa
Heidemarie Ebner
Christian Leitner
B. Martin Hallberg
Methinee Prongjit
Jeerus Sucharitakul
Roland Ludwig
Ulla Rudsander
Clemens Peterbauer
Pimchai Chaiyen
Dietmar Haltrich
Christina Divne
AlbaNova University Center
Karolinska Institutet
Mahidol University
Universitat fur Bodenkultur Wien
Chulalongkorn University
Keywords: Biochemistry, Genetics and Molecular Biology
Issue Date: 17-Nov-2006
Citation: Journal of Biological Chemistry. Vol.281, No.46 (2006), 35104-35115
Abstract: Pyranose 2-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 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 kcat, 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 onthedetailsof2-fluoro-2-deoxy-D-glucose binding in position for oxidation at C3, we also outline a probable bindingmodefor 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 Asp452 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 Asp452 and Arg472 as well as the double alanine 452/472 mutant. © 2006 by The American Society for Biochemistry and Molecular Biology, Inc.
URI: https://www.scopus.com/inward/record.uri?partnerID=HzOxMe3b&scp=33845930584&origin=inward
http://repository.li.mahidol.ac.th/dspace/handle/123456789/22949
ISSN: 1083351X
00219258
Appears in Collections:Scopus 2006-2010

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