3K4M

Pyranose 2-oxidase Y456W mutant in complex with 2FG

3K4M の概要

• エントリーDOI: 10.2210/pdb3k4m/pdb
• 関連するPDBエントリー: 1TT0 2IGK 2IGM 2IGN 2IGO 3BG6
• 分子名称: Pyranose 2-oxidase, FLAVIN-ADENINE DINUCLEOTIDE, 2-deoxy-2-fluoro-beta-D-glucopyranose, ... (5 entities in total)
• 機能のキーワード: oxidoreductase, gmc oxidoreductase, y456w mutant, rossmann fold, phbh fold, homotetramer, 8-alpha-(n3) histidyl flavinylation
• 由来する生物種: Trametes ochracea (White-rot fungus)
• タンパク質・核酸の鎖数: 8
• 化学式量合計: 564798.10

構造登録者

Divne, C.,Tan, T.C. (登録日: 2009-10-05, 公開日: 2010-05-12, 最終更新日: 2024-10-30)

主引用文献

Spadiut, O.,Tan, T.C.,Pisanelli, I.,Haltrich, D.,Divne, C.
Importance of the gating segment in the substrate-recognition loop of pyranose 2-oxidase.
Febs J., 277:2892-2909, 2010

PubMed Abstract: Pyranose 2-oxidase from Trametes multicolor is a 270 kDa homotetrameric enzyme that participates in lignocellulose degradation by wood-rotting fungi and oxidizes a variety of aldopyranoses present in lignocellulose to 2-ketoaldoses. The active site in pyranose 2-oxidase is gated by a highly conserved, conformationally degenerate loop (residues 450-461), with a conformer ensemble that can accommodate efficient binding of both electron-donor substrate (sugar) and electron-acceptor substrate (oxygen or quinone compounds) relevant to the sequential reductive and oxidative half-reactions, respectively. To investigate the importance of individual residues in this loop, a systematic mutagenesis approach was used, including alanine-scanning, site-saturation and deletion mutagenesis, and selected variants were characterized by biochemical and crystal-structure analyses. We show that the gating segment ((454)FSY(456)) of this loop is particularly important for substrate specificity, discrimination of sugar substrates, turnover half-life and resistance to thermal unfolding, and that three conserved residues (Asp(452), Phe(454) and Tyr(456)) are essentially intolerant to substitution. We furthermore propose that the gating segment is of specific importance for the oxidative half-reaction of pyranose 2-oxidase when oxygen is the electron acceptor. Although the position and orientation of the slow substrate 2-deoxy-2-fluoro-glucose when bound in the active site of pyranose 2-oxidase variants is identical to that observed earlier, the substrate-recognition loop in F454N and Y456W displays a high degree of conformational disorder. The present study also lends support to the hypothesis that 1,4-benzoquinone is a physiologically relevant alternative electron acceptor in the oxidative half-reaction.

PubMed: 20528921
DOI: 10.1111/j.1742-4658.2010.07705.x

実験手法

X-RAY DIFFRACTION (2.2 Å)