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

Duan, Ran; Li, Jiasong; Liu, Aimin

doi:10.1016/bs.mie.2024.05.018

Cited by 1 publication

(2 citation statements)

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“…The RGS5 peptide substrate was synthesized by Biomatik with the sequence of CKGLAALPHSCLER (>95% purity), corresponding to the first 14 amino acids of the Met-excised N -terminus of RGS5 as described in a previous report . Catalytic assays were conducted using two independent methods: an oxygen consumption assay using Oxygraph and a product assay using an HPLC-MS method, as described elsewhere . Kinetic analysis was conducted using stopped-flow UV–vis experiments on an Applied Photophysics SX20 spectrophotometer, and data were analyzed using SX Pro-Data Viewer analysis software.…”

Section: Methodsmentioning

confidence: 99%

“…ADO crystal structures from human and mouse origins have become available recently. , Despite significant research efforts, the mechanism by which non-heme iron facilitates thiol oxidation in ADO, ,,, and to a large degree CDO, remains an open question for further exploration. ADO and CDO are in the distinct subfamily of proteins within the thiol dioxygenase superfamily and are more closely related to PCO, yet their chemical reaction on the small organic substrate is most similar to that of CDO. These thiol dioxygenases from distinct subfamilies may or may not share the same preference with respect to the mechanistic details in the absence or presence of the cross-linked protein cofactor.…”

Section: Introductionmentioning

confidence: 99%

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Cobalt(II)-Substituted Cysteamine Dioxygenase Oxygenation Proceeds through a Cobalt(III)-Superoxo Complex

Li,

Duan,

Liu

2024

J. Am. Chem. Soc.

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We investigated the metal-substituted catalytic activity of human cysteamine dioxygenase (ADO), an enzyme pivotal in regulating thiol metabolism and contributing to oxygen homeostasis. Our findings demonstrate the catalytic competence of cobalt(II)-and nickel(II)-substituted ADO in cysteamine oxygenation. Notably, Co(II)-ADO exhibited superiority over Ni(II)-ADO despite remaining significantly less active than the natural enzyme. Structural analyses through X-ray crystallography and cobalt K-edge excitation confirmed successful metal substitution with minimal structural perturbations. This provided a robust structural basis, supporting a conserved catalytic mechanism tailored to distinct metal centers. This finding challenges the proposed high-valent ferryl-based mechanism for thiol dioxygenases, suggesting a non-high-valent catalytic pathway in the native enzyme. Further investigation of the cysteamine-bound or a peptide mimic of N-terminus RGS5 bound Co(II)-ADO binary complex revealed the metal center's high-spin (S = 3/2) state. Upon reaction with O 2 , a kinetically and spectroscopically detectable intermediate emerged with a ground spin state of S = 1/2. This intermediate exhibits a characteristic 59 Co hyperfine splitting (A = 67 MHz) structure in the EPR spectrum alongside UV−vis features, consistent with known low-spin Co(III)-superoxo complexes. This observation, unique for protein-bound thiolate-ligated cobalt centers in a protein, unveils the capacities for O 2 activation in such metal environments. These findings provide valuable insights into the nonheme iron-dependent thiol dioxygenase mechanistic landscape, furthering our understanding of thiol metabolism regulation. The exploration of metal-substituted ADO sheds light on the intricate interplay between metal and catalytic activity in this essential enzyme.

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Section: Methodsmentioning

confidence: 99%