RESUMEN
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 â¼7 Å from the iron center. As such, cross-link formation may not be structurally or functionally significant in ADO.
Asunto(s)
Dominio Catalítico/genética , Dioxigenasas/ultraestructura , Péptidos/química , Conformación Proteica , Animales , Catálisis , Cristalografía por Rayos X , Cisteína/química , Dioxigenasas/química , Dioxigenasas/genética , Humanos , Hierro/química , Ratones , Simulación de Dinámica Molecular , Péptidos/genética , Teoría Cuántica , Especificidad por Sustrato/genética , Tirosina/químicaRESUMEN
A detailed description of the mechanism of C-H hydroxylation by Rieske non-heme iron dioxygenases remains elusive, as the nature of the oxidizing species is not definitively known. DFT calculations on cluster models of nitrobenzene 1,2-dioxygenase were done to explore possible mechanisms arising from oxidation by either the experimentally observed Fe(III)-OOH complex or the putative high-valent HO-Fe(V)=O intermediate formed through a heterolytic O-O bond cleavage. Hydrogen abstraction by HO-Fe(V)=O, followed by oxygen rebound, was found to be consistent with experimental studies. The findings from the quantum mechanical cluster approach were verified by accounting for the effect of the protein environment on transition state geometries and reaction barriers through ONIOM calculations.