2igm
From Proteopedia
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[[Image:2igm.gif|left|200px]] | [[Image:2igm.gif|left|200px]] | ||
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'''Crystal structure of recombinant pyranose 2-oxidase H548N mutant''' | '''Crystal structure of recombinant pyranose 2-oxidase H548N mutant''' | ||
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[[Category: Trametes ochracea]] | [[Category: Trametes ochracea]] | ||
[[Category: Divne, C.]] | [[Category: Divne, C.]] | ||
| - | [[Category: | + | [[Category: Active-site mutant]] |
| - | + | [[Category: Gmc oxidoreductase]] | |
| - | [[Category: | + | [[Category: H548n]] |
| - | [[Category: | + | [[Category: Homotetramer]] |
| - | [[Category: | + | [[Category: Oxidoreductase]] |
| - | [[Category: | + | [[Category: Phbh fold]] |
| - | [[Category: | + | [[Category: Rossman fold]] |
| - | [[Category: | + | ''Page seeded by [http://oca.weizmann.ac.il/oca OCA ] on Sun May 4 07:28:42 2008'' |
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| - | ''Page seeded by [http://oca.weizmann.ac.il/oca OCA ] on | + | |
Revision as of 04:28, 4 May 2008
Crystal structure of recombinant pyranose 2-oxidase H548N mutant
Overview
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 Structure
2IGM is a Single protein structure of sequence from Trametes ochracea. Full crystallographic information is available from OCA.
Reference
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 Page seeded by OCA on Sun May 4 07:28:42 2008
