2IGO: Crystal Structure Of Pyranose 2-oxidase H167a Mutant With 2-fluoro-2- Deoxy-d-glucose

Citation:
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 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.
PDB ID: 2IGODownload
MMDB ID: 42392
PDB Deposition Date: 2006/9/22
Updated in MMDB: 2006/12
Experimental Method:
x-ray diffraction
Resolution: 1.95  Å
Source Organism:
Similar Structures:
Biological Unit for 2IGO: tetrameric; determined by author and by software (PISA)
Molecular Components in 2IGO
Label Count Molecule
Proteins (4 molecules)
4
Pyranose Oxidase
Molecule annotation
Chemicals (8 molecules)
1
4
2
4
* Click molecule labels to explore molecular sequence information.

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