Measuring plant cysteine oxidase interactions with substrates using intrinsic tryptophan fluorescence

Area of Science:

• Biochemistry
• Plant Biology
• Molecular Biology

Background:

• Plant Cysteine Oxidases (PCOs) are oxygen-sensing enzymes crucial for the N-degron pathway.
• PCOs regulate target protein stability, particularly Ethylene Response Factors (ERFVIIs), during hypoxia and submergence.
• Understanding PCO-substrate interactions is key to engineering enhanced plant submergence tolerance.

Purpose of the Study:

• To develop a novel assay for quantifying PCO-substrate binding affinities.
• To investigate the substrate specificity of Arabidopsis thaliana PCOs (AtPCOs).
• To elucidate the interaction dynamics between AtPCOs and their known substrates.

Main Methods:

• Development of a fluorescence-based assay utilizing intrinsic tryptophan fluorescence of AtPCOs.
• Enzyme-substrate complex formation monitored via tryptophan fluorescence quenching under anaerobic conditions.
• Quantification of binding affinities using Ni(II)-substituted enzymes.

Main Results:

• The assay successfully quantified binding affinities between AtPCOs and various substrates.
• AtPCO4 and AtPCO5 showed stronger interactions with ERFVII substrates compared to ZPR2 and VRN2.
• A positive cooperative binding effect was observed for AtPCO4/5 interactions with ERFVIIs and ZPR2.

Conclusions:

• The developed assay is a straightforward and effective tool for studying PCO-substrate interactions.
• ERFVIIs are likely primary targets for AtPCO4 and AtPCO5.
• Findings provide a foundation for enzyme engineering strategies to improve plant submergence tolerance.