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Recombinant expression, purification and crystallization of Pyranose 2-Oxidase from Trametes mutlicolor to investigate the structural determinants of oxygen ractivity
KTH, School of Biotechnology (BIO).
2011 (English)Independent thesis Advanced level (degree of Master (Two Years)), 20 credits / 30 HE creditsStudent thesis
Abstract [en]

Background: The molecular basis of enzymatic oxygen reactivity has been investigated for different flavoenzymes but still there are no clearly defined mechanisms for oxygen activation. Pyranose 2-oxidase from Trametes multicolor (P2Ox) and pyranose dehydrogenase from Agaricus meleagris (PDH), share similar catalytic, kinetic and structural features, but still, P2Ox reacts with oxygen whereas PDH does not. Objective: The present study aims to investigate the oxygen reactivity in P2Ox, specifically the importance of Asn593 and surrounding residues. The approach was to target specific active-site residues in P2Ox by directed mutagenesis to disable oxygen reactivity during the oxidative half-reaction without significantly altering the reductive half-reaction. The mutant designs were aided by the structural similarity between P2Ox and PDH. Five mutants were produced in the heterologous host Escherichia coli: N593H, N593Q, N593H/H446E, N593H/H446Q, and N593H/H446N. A preliminary biochemical and kinetic characterization was performed on the mutants, as well as crystallization for crystal-structure determination. Results: Crystals of all variants were obtained at high resolution, except for variant N593H/H446N, which was destabilized and degraded proteolytically. For all mutant structures obtained, the active-site loop is in the closed conformation, implying that the mutants were captured in a conformational state relevant to the oxidative half-reaction. Moreover, N593H shows impaired glucose binding and turnover, and may have impaired C(4a)-adduct formation. N593H/H446E on the other hand, had almost similar glucose affinity as the wild type, but 80-fold lower turnover rate. In addition, the flavin cofactor shows distortion close to the site of mutation. Conclusions: The results pinpoint two mutations, N593H and N593H/H446E that appear particularly interesting for future characterization by stopped-flow kinetics to analyze in detail the effects of the specific mutations on the individual half-reactions.

Place, publisher, year, edition, pages
Keyword [en]
pyranose-oxidizing, pyranose 2-oxidase, oxygen reactivity, mutants, crystal structure, kinetic characterization
National Category
Engineering and Technology
URN: urn:nbn:se:kth:diva-51754OAI: diva2:464930
Subject / course
Educational program
Degree of Master
Available from: 2011-12-14 Created: 2011-12-14 Last updated: 2011-12-14Bibliographically approved

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