Engineering pyranose 2-oxidase for modified oxygen reactivity.

Area of Science:

• Biochemistry
• Enzymology
• Protein Engineering

Background:

• Pyranose 2-oxidase (POx) is a flavoprotein catalyzing aldopyranose oxidation.
• Its FAD cofactor is reduced by substrate and reoxidized by electron acceptors, typically oxygen producing H2O2.
• Controlling oxygen reactivity is key for applications like biosensors and biofuel cells to minimize oxidative damage.

Purpose of the Study:

• To engineer POx variants with reduced oxidase activity while maintaining dehydrogenase activity.
• To investigate the structural determinants of oxygen activation and reactivity in POx.

Main Methods:

• Site-saturation mutagenesis of eleven active site amino acids.
• Microtiter plate screening assays using peroxidase/ABTS and DCPIP.
• Characterization of selected variants (T166R, Q448H, L545C, L547R, N593C) using steady-state kinetics with various electron acceptors.

Main Results:

• Identified POx variants with significantly decreased oxidase activity and retained dehydrogenase activity.
• Characterized variants showed altered kinetic constants with oxygen and alternative electron acceptors (DCPIP, 1,4-benzoquinone, ferricenium ion).
• Mutational effects were rationalized based on the enzyme's crystal structure.

Conclusions:

• Site-directed mutagenesis can successfully tune the oxygen reactivity of POx.
• Engineered POx variants offer potential for applications requiring controlled electron transfer and reduced oxidative side reactions.