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Structural basis fo...
Structural basis for substrate binding and regioselective oxidation of monosaccharides at C3 by pyranose 2-oxidase
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Kujawa, Magdalena (author)
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Ebner, Heidemarie (author)
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Leitner, Christian (author)
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- Hallberg, B. Martin (author)
- Karolinska Institutet
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Prongjit, Methinee (author)
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Sucharitakul, Jeerus (author)
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Ludwig, Roland (author)
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Rudsander, Ulla (author)
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Peterbauer, Clemens (author)
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Chaiyen, Pimchai (author)
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Haltrich, Dietmar (author)
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- Divne, Christina (author)
- KTH,Glykovetenskap
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(creator_code:org_t)
- 2006
- 2006
- English.
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In: Journal of Biological Chemistry. - 0021-9258 .- 1083-351X. ; 281:46, s. 35104-35115
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https://urn.kb.se/re...
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https://doi.org/10.1...
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Abstract
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- Pyranose2-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 regios-electivity, 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 Asp(452) 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 Asp(452) and Arg(472) as well as the double alanine 452/472 mutant.
Keyword
- electron-transfer flavoprotein
- p-cresol methylhydroxylase
- fungus trametes-multicolor
- crystal-structure
- phanerochaete-chrysosporium
- cellobiose dehydrogenase
- alcohol oxidase
- covalent flavinylation
- catalytic mechanism
- lignin degradation
Publication and Content Type
- ref (subject category)
- art (subject category)
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- By the author/editor
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Kujawa, Magdalen ...
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Ebner, Heidemari ...
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Leitner, Christi ...
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Hallberg, B. Mar ...
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Prongjit, Methin ...
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Sucharitakul, Je ...
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show more...
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Ludwig, Roland
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Rudsander, Ulla
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Peterbauer, Clem ...
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Chaiyen, Pimchai
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Haltrich, Dietma ...
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Divne, Christina
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show less...
- Articles in the publication
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Journal of Biolo ...
- By the university
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Royal Institute of Technology
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Karolinska Institutet