The Crystal Structure of Cysteamine Dioxygenase Reveals the Origin of the Large Substrate Scope of This Vital Mammalian Enzyme

Abstract

We report the crystal structure of the mammalian non-heme iron enzyme cysteamine dioxygenase (ADO) at 1.9 Å resolution, which shows an Fe and three-histidine (3-His) active site situated at the end of a wide substrate access channel. The open approach to the active site is consistent with the recent discovery that ADO catalyzes not only the conversion of cysteamine to hypotaurine but also the oxidation of N-terminal cysteine (Nt-Cys) peptides to their corresponding sulfinic acids as part of the eukaryotic N-degron pathway. Whole-protein models of ADO in complex with either cysteamine or an Nt-Cys peptide, generated using molecular dynamics and quantum mechanics/molecular mechanics calculations, suggest occlusion of access to the active site by peptide substrate binding. This finding highlights the importance of a small tunnel that leads from the opposite face of the enzyme into the active site, providing a path through which co-substrate O2 could access the Fe center. Intriguingly, the entrance to this tunnel is guarded by two Cys residues that may form a disulfide bond to regulate O2 delivery in response to changes in the intracellular redox potential. Notably, the Cys and tyrosine residues shown to be capable of forming a cross-link in human ADO reside ∼7 Å from the iron center. As such, cross-link formation may not be structurally or functionally significant in ADO.

Charge Maintenance during Catalysis in Nonheme Iron Oxygenases

“…115 The crystal structure of ADO reveals that this enzyme structurally resembles PCOs rather than the expected CDO. 117,118 To date, there is no crystal structure of the ES complexes of ADO nor PCOs, but spectroscopic evidence indicates that cysteamine ligates the ADO metal in a monodentate fashion in both the Fe(II) and Fe(III) states. 119,120 The crystal structure of MDO in complex with a substrate analog, 3-hydroxypropionic acid, shows that the inhibitor binds to the iron in a bidentate fashion by its hydroxyl and carboxyl groups.…”

“…The structures of ADO and PCOs have larger open cavities and dynamic loops, as is seen with HAO, to accommodate peptide-or protein-type substrates. 117,118,123,124 Although CDO has been characterized extensively, its catalytic cycle is less understood. Only one iron-bound persulfenate intermediate has been structurally identified after oxygen binding, and its presence in the cycle is controversial.…”

“…A nearby protein-derived Cys–Tyr cofactor, while nonessential for catalysis, improves the catalytic efficiency. ,− The ternary complex crystal structures of human CDO have been reported using • NO as an O 2 surrogate . The crystal structure of ADO reveals that this enzyme structurally resembles PCOs rather than the expected CDO. , To date, there is no crystal structure of the ES complexes of ADO nor PCOs, but spectroscopic evidence indicates that cysteamine ligates the ADO metal in a monodentate fashion in both the Fe­(II) and Fe­(III) states. , The crystal structure of MDO in complex with a substrate analog, 3-hydroxypropionic acid, shows that the inhibitor binds to the iron in a bidentate fashion by its hydroxyl and carboxyl groups . The difference between the substrate 3-mercaptopropionic acid and the inhibitor 3-hydroxypropionic acid is that the former has a thiol in place of the hydroxyl group.…”

“…Nonetheless, this ligand-bound MDO structure raises an intriguing question as the bidentate mode of substrate binding in MDO would have a negatively charged carboxylate coordinating the iron center in addition to the thiolate, resulting in a different net charge of the iron center in the ES complex compared to that in CDO. The structures of ADO and PCOs have larger open cavities and dynamic loops, as is seen with HAO, to accommodate peptide- or protein-type substrates. ,,, …”

Differences in the Second Coordination Sphere Tailor the Substrate Specificity and Reactivity of Thiol Dioxygenases

“…3,70 A computational study performed using molecular dynamics in conjunction with quantum mechanics/molecular mechanics methods of ADO complexed with 2-AET and a truncated model of an Nt-Cys peptide revealed that the large substrate cavity lends 2-AET significant conformational freedom. 4 In contrast, the larger size of the Nt-Cys peptide allows for interactions with more residues lining the active site cavity, including Asp192 and Tyr198. This prediction is consistent with the markedly lower K M of ADO for Nt-Cys peptide than 2-AET.…”