3BG7

Pyranose 2-oxidase from Trametes multicolor, L537G mutant

Summary for 3BG7

• Entry DOI: 10.2210/pdb3bg7/pdb
• Related: 1TT0 2IGK 2IGM 2IGN 2IGO 3BG6 3BLY
• Descriptor: Pyranose oxidase, FLAVIN-ADENINE DINUCLEOTIDE (3 entities in total)
• Functional Keywords: oxidoreductase, gmc oxidoreductase, l537g mutant, rossmanfold, phbh fold, homotetramer, 8-alpha-(n3) histidyl flavinylation
• Biological source: Trametes multicolor (White-rot fungus)
• Total number of polymer chains: 8
• Total formula weight: 561145.90

Authors

Norberg, P.,Tan, T.C.,Divne, C. (deposition date: 2007-11-26, release date: 2008-12-02, Last modification date: 2024-11-06)

Primary citation

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

Experimental method

X-RAY DIFFRACTION (2.1 Å)