2ign
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Crystal structure of recombinant pyranose 2-oxidase H167A mutant
OverviewOverview
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 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.
About this StructureAbout this Structure
2IGN is a Single protein structure of sequence from Trametes ochracea with and as ligands. Active as Pyranose oxidase, with EC number 1.1.3.10 Full crystallographic information is available from OCA.
ReferenceReference
Structural basis for substrate binding and regioselective oxidation of monosaccharides at C3 by pyranose 2-oxidase., Kujawa M, Ebner H, Leitner C, Hallberg BM, Prongjit M, Sucharitakul J, Ludwig R, Rudsander U, Peterbauer C, Chaiyen P, Haltrich D, Divne C, J Biol Chem. 2006 Nov 17;281(46):35104-15. Epub 2006 Sep 19. PMID:16984920
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