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

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.

Fig. 1

The reaction, structure, and catalytic mechanism of ADO. (A) Thiol dioxygenase catalyzed reactions. R = H (ADO), −COO⁻ (CDO); ADO and PCO catalyze certain N-terminal Cys-containing peptides and proteins. (B) Active site architecture of human ADO (Wang, Shin, Li, & Liu, 2021); (C) The key reaction intermediates in the two proposed mechanisms. Left: Concurrent dioxygen transfer intermediate. Right: High-valent ferryl-oxo intermediate.

Fig. 2

Comparison of wild-type Co-ADO and C18S/C239S variant by gel filtration chromatography (A) and SDS-PAGE (B). (1) C18S/C239S Fe-ADO; (2) WT Fe-ADO; (3) C18S/C239S Co-ADO; (4) WT Co-ADO; and (5) “Apo-ADO”.

Fig. 3

The optical and EPR spectra of Co-ADO. (A) The optical spectra of 300 μM apo-ADO (black), Co-ADO (red), and their difference spectrum (blue). (B) The EPR spectrum of 100 μM Co-ADO. The EPR data was collected at 30 K with a microwave power of 3.17 mW.

Fig. 4

Iron concentration determination of apo-ADO and Co-ADO produced from M9 media. The black trace shows the standard curve fitted by various concentrations of Fe(NH₄)₂(SO₄)₂. The iron concentration of 1.62 μM in 125 μM apo-ADO indicates that the iron occupancy is 1.30% (blue); The iron concentration of 1.97 μM in 114 μM Co-ADO indicates a 1.73% iron occupancy (red).

Fig. 5

(A) Demonstration of Ni(II)-ADO’s dioxygenase catalytic activity and HPLC profile for ADO activity assay. (1) 50 μM reconstituted Ni(II)-ADO + 10mM cysteamine; (2) 50 μM “apo-ADO” + 10mM cysteamine; (3) HPLC hypotaurine sample; (4) HPLC cysteamine sample; and (5) HPLC blank sample. The details of the reaction setup are described in the text. The negative peak in the HPLC elution profiles emerged due to the difference between the reaction HEPES buffer in the samples and the HPLC solvent. (B) Reaction equation of ADO with cysteamine as a substrate and MS spectrum of the hypotaurine peak in Sample (3 of panel A).