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A biophysical approach to studying N-terminal cysteine oxidase substrate preferences.

Karishma Patel1, Yannasittha Jiramongkol2, Mark D White3

  • 1School of Chemistry, The University of Sydney, Sydney, NSW, Australia; School of Life and Environmental Sciences, The University of Sydney, Sydney, NSW, Australia.

Methods in Enzymology
|August 31, 2025
PubMed
Summary

N-terminal cysteine oxidases (NCOs) act as oxygen sensors regulating protein stability via the N-degron pathway. This study uses surface plasmon resonance (SPR) to analyze the binding of mammalian NCOs to their substrates, clarifying enzyme-substrate interactions.

Keywords:
ADOHypoxiaN-degron pathwayN-terminal cysteine oxidasesOxygenPost-translational modificationProtein terminiSubstrate bindingSurface plasmon resonance

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Area of Science:

  • Biochemistry
  • Molecular Biology
  • Cellular Physiology

Background:

  • N-terminal cysteine oxidases (NCOs) are crucial enzymatic oxygen sensors in animals and plants.
  • They regulate adaptive responses to hypoxia by controlling protein stability through the N-degron pathway.
  • Understanding NCO activity and substrate selectivity is vital but remains incomplete.

Purpose of the Study:

  • To detail optimized protocols for investigating binding interactions between 2-aminoethanethiol (cysteamine) dioxygenase (ADO) and its N-terminal cysteine (Nt-cys) substrates.
  • To utilize surface plasmon resonance (SPR) spectroscopy for analyzing these enzyme-substrate interactions.
  • To provide a biophysical approach for dissecting NCO substrate recognition and regulation mechanisms.

Main Methods:

  • Surface Plasmon Resonance (SPR) spectroscopy was employed to monitor binding events in real-time.
  • Continuous-flow microfluidics systems were utilized within the SPR setup.
  • Optimized protocols were developed for investigating enzyme-ligand and enzyme-substrate interactions.

Main Results:

  • SPR successfully detected rapid and transient binding events between ADO and Nt-cys substrates.
  • The study provides a robust platform for analyzing enzyme-substrate kinetics and affinities.
  • Detailed protocols for investigating NCO interactions were established.

Conclusions:

  • SPR spectroscopy is a versatile tool for studying NCOs and their interactions with Nt-cys substrates.
  • This biophysical approach aids in understanding the molecular mechanisms of NCO substrate recognition.
  • The findings have broad applicability to enzyme systems and post-translational modifications.