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J. Biol. Chem., Vol. 281, Issue 46, 35104-35115, November 17, 2006

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Structural Basis for Substrate Binding and Regioselective Oxidation of Monosaccharides at C3 by Pyranose 2-Oxidase^*

Magdalena Kujawa, Heidemarie Ebner, Christian Leitner, B. Martin Hallberg^¶, Methinee Prongjit^||, Jeerus Sucharitakul^||1, Roland Ludwig, Ulla Rudsander, Clemens Peterbauer, Pimchai Chaiyen^||, Dietmar Haltrich, and Christina Divne²

From the School of Biotechnology, Royal Institute of Technology, Albanova University Center, SE-106 91 Stockholm, Sweden, the ^¶Department of Medical Biochemistry and Biophysics, Karolinska Institute, SE-171 77 Stockholm, Sweden, the ^||Department of Biochemistry and Center for Excellence in Protein Structure and Function, Faculty of Science, Mahidol University, Rama VI Road, Rajthevi, Bangkok 10400, Thailand, and the Department of Food Sciences and Technology, BOKU, University of Natural Resources and Applied Life Sciences, Vienna, A-1190 Vienna, Austria

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 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 Asp⁴⁵² in the substrate loop and, possibly, a nearby arginine residue (Arg⁴⁷²). 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.

Received for publication, May 17, 2006 , and in revised form, September 8, 2006.

The atomic coordinates and structure factors (codes 2IGK, 2IGM, 2IGN, and 2IGO) have been deposited in the Protein Data Bank, Research Collaboratory for Structural Bioinformatics, Rutgers University, New Brunswick, NJ (http://www.rcsb.org/).

^* This work was supported by the Thailand Research Fund (Grant RMU4880028 to P. C. and Grant MRG4980117 to J. S.), by the Royal Golden Jubilee Ph.D. Program (Grant PHD/0151/2547) of the Thailand Research Fund (to M. P.), by the Austrian Research Foundation (Project FWF P15719 [GenBank] to D. H.), and by grants from the Swedish Research Council for Environment, Agricultural Sciences and Spatial Planning, the Swedish Research Council, the CF Lundströms Stiftelse, and the Carl Tryggers Stiftelse (to C. D.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

¹ Present address: Dept. of Biochemistry, Faculty of Dentistry, Chulalongkorn University, Henri-Dunant Rd., Pathumwan, Bangkok 10300, Thailand.

² To whom correspondence should be addressed. Tel.: 46-8-5537-8296; Fax: 46-8-5537-8468; E-mail: divne{at}biotech.kth.se.

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