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Title: Unveiling the mechanism of cysteamine dioxygenase: A combined HPLC-MS assay and metal-substitution approach
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.  more » « less
Award ID(s):
2204225
PAR ID:
10623830
Author(s) / Creator(s):
; ;
Editor(s):
Bridwell-Rabb, Jennifer
Publisher / Repository:
Elsevier
Date Published:
Volume:
703
Issue:
A
ISSN:
0076-6879
ISBN:
978-0-443-31304-2
Page Range / eLocation ID:
147 to 166
Subject(s) / Keyword(s):
Biophysical spectroscopy Electron paramagnetic resonance Electronic absorption LC-MS Metal-substitution Non-heme iron center Oxygen activation Thiol dioxygenase
Format(s):
Medium: X
Sponsoring Org:
National Science Foundation
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