Unveiling the mechanism of cysteamine dioxygenase: A combined HPLC-MS assay and metal-substitution approach

Methods Enzymol. 2024:703:147-166. doi: 10.1016/bs.mie.2024.05.018. Epub 2024 Jun 22.

Abstract

Mammalian cysteamine dioxygenase (ADO), a mononuclear non-heme Fe(II) enzyme with three histidine ligands, plays a key role in cysteamine catabolism and regulation of the N-degron signaling pathway. Despite its importance, the catalytic mechanism of ADO remains elusive. Here, we describe an HPLC-MS assay for characterizing thiol dioxygenase catalytic activities and a metal-substitution approach for mechanistic investigation using human ADO as a model. Two proposed mechanisms for ADO differ in oxygen activation: one involving a high-valent ferryl-oxo intermediate. We hypothesized that substituting iron with a metal that has a disfavored tendency to form high-valent states would discriminate between mechanisms. This chapter details the expression, purification, preparation, and characterization of cobalt-substituted ADO. The new HPLC-MS assay precisely measures enzymatic activity, revealing retained reactivity in the cobalt-substituted enzyme. The results obtained favor the concurrent dioxygen transfer mechanism in ADO. This combined approach provides a powerful tool for studying other non-heme iron thiol oxidizing enzymes.

Keywords: Biophysical spectroscopy; Electron paramagnetic resonance; Electronic absorption; LC-MS; Metal-substitution; Non-heme iron center; Oxygen activation; Thiol dioxygenase.

MeSH terms

  • Chromatography, High Pressure Liquid / methods
  • Cobalt / chemistry
  • Cobalt / metabolism
  • Dioxygenases / chemistry
  • Dioxygenases / metabolism
  • Enzyme Assays / methods
  • Humans
  • Liquid Chromatography-Mass Spectrometry
  • Mass Spectrometry* / methods
  • Oxidation-Reduction
  • Oxygen / metabolism

Substances

  • cysteamine dioxygenase
  • Cobalt
  • Dioxygenases
  • Oxygen