3BG7

Pyranose 2-oxidase from Trametes multicolor, L537G mutant

3BG7 の概要

• エントリーDOI: 10.2210/pdb3bg7/pdb
• 関連するPDBエントリー: 1TT0 2IGK 2IGM 2IGN 2IGO 3BG6 3BLY
• 分子名称: Pyranose oxidase, FLAVIN-ADENINE DINUCLEOTIDE (3 entities in total)
• 機能のキーワード: oxidoreductase, gmc oxidoreductase, l537g mutant, rossmanfold, phbh fold, homotetramer, 8-alpha-(n3) histidyl flavinylation
• 由来する生物種: Trametes multicolor (White-rot fungus)
• タンパク質・核酸の鎖数: 8
• 化学式量合計: 561145.90

構造登録者

Norberg, P.,Tan, T.C.,Divne, C. (登録日: 2007-11-26, 公開日: 2008-12-02, 最終更新日: 2024-11-06)

主引用文献

Spadiut, O.,Leitner, C.,Salaheddin, C.,Varga, B.,Vertessy, B.G.,Tan, T.C.,Divne, C.,Haltrich, D.
Improving thermostability and catalytic activity of pyranose 2-oxidase from Trametes multicolor by rational and semi-rational design
Febs J., 276:776-792, 2009

PubMed Abstract: The fungal homotetrameric flavoprotein pyranose 2-oxidase (P2Ox; EC 1.1.3.10) catalyses the oxidation of various sugars at position C2, while, concomitantly, electrons are transferred to oxygen as well as to alternative electron acceptors (e.g. oxidized ferrocenes). These properties make P2Ox an interesting enzyme for various biotechnological applications. Random mutagenesis has previously been used to identify variant E542K, which shows increased thermostability. In the present study, we selected position Leu537 for saturation mutagenesis, and identified variants L537G and L537W, which are characterized by a higher stability and improved catalytic properties. We report detailed studies on both thermodynamic and kinetic stability, as well as the kinetic properties of the mutational variants E542K, E542R, L537G and L537W, and the respective double mutants (L537G/E542K, L537G/E542R, L537W/E542K and L537W/E542R). The selected substitutions at positions Leu537 and Glu542 increase the melting temperature by approximately 10 and 14 degrees C, respectively, relative to the wild-type enzyme. Although both wild-type and single mutants showed first-order inactivation kinetics, thermal unfolding and inactivation was more complex for the double mutants, showing two distinct phases, as revealed by microcalorimetry and CD spectroscopy. Structural information on the variants does not provide a definitive answer with respect to the stabilizing effects or the alteration of the unfolding process. Distinct differences, however, are observed for the P2Ox Leu537 variants at the interfaces between the subunits, which results in tighter association.

PubMed: 19143837
DOI: 10.1111/j.1742-4658.2008.06823.x

実験手法

X-RAY DIFFRACTION (2.1 Å)