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|Title:||Structural basis for substrate binding and regioselective oxidation of monosaccharides at C3 by pyranose 2-oxidase|
B. Martin Hallberg
AlbaNova University Center
Universitat fur Bodenkultur Wien
|Keywords:||Biochemistry, Genetics and Molecular Biology|
|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.|
|Appears in Collections:||Scopus 2006-2010|
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