2IGK

Crystal structure of recombinant pyranose 2-oxidase

2IGK の概要

• エントリーDOI: 10.2210/pdb2igk/pdb
• 関連するPDBエントリー: 1TT0
• 分子名称: Pyranose oxidase, FLAVIN-ADENINE DINUCLEOTIDE, 2-(N-MORPHOLINO)-ETHANESULFONIC ACID, ... (4 entities in total)
• 機能のキーワード: oxidoreductase, gmc oxidoreductase, rossmann fold, phbh fold, homotetramer, 8-alpha-(n3) histidyl flavinylation
• 由来する生物種: Trametes ochracea
• タンパク質・核酸の鎖数: 8
• 化学式量合計: 563156.61

構造登録者

Divne, C. (登録日: 2006-09-22, 公開日: 2006-10-10, 最終更新日: 2024-10-16)

主引用文献

Kujawa, M.,Ebner, H.,Leitner, C.,Hallberg, B.M.,Prongjit, M.,Sucharitakul, J.,Ludwig, R.,Rudsander, U.,Peterbauer, C.,Chaiyen, P.,Haltrich, D.,Divne, C.
Structural basis for substrate binding and regioselective oxidation of monosaccharides at c3 by pyranose 2-oxidase.
J.Biol.Chem., 281:35104-35115, 2006

PubMed 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.

PubMed: 16984920
DOI: 10.1074/jbc.M604718200

実験手法

X-RAY DIFFRACTION (1.8 Å)