Structural and Mechanistic Study of Mammalian Thiol Dioxygenases
In mammals, sulfur metabolism is mainly controlled by the action of two enzymes: cysteine dioxygenase (CDO) and cystamine dioxygenase (ADO). The activity of these enzymes is important in controlling metabolic cysteine and taurine concentrations and is associated with autoimmune and neurological conditions, cellular sensing and signaling, energy balance, and fat metabolism. CDO contains a self-processed cofactor, and both enzymes utilize a mononuclear ferrous center to facilitate the incorporation of molecular oxygen into the thiol moiety of their respective substrates. To date, the function of the cofactor of CDO has not been well established, and the mechanism by which thiol dioxygenation takes place is currently under debate. The work proposed here will utilize site-specific incorporation of unnatural amino acids, protein X-ray crystallography, proteomics, electron paramagnetic resonance spectroscopy, and enzymatic activity to establish (1) the mechanism of cofactor biogenesis in CDO, (2) the mechanistic function of the cofactor in thiol deoxygenation, and (3) the structure and enzymatic mechanism of ADO. Since the great oxidative event, life has not only coped with an abundance of molecular oxygen but has indeed tamed the powerful oxidant for use in metabolic processes. Not only is oxygen used an Ã¢â‚¬Ëœelectron sinkÃ¢â‚¬â„¢ to drive aerobic respiration; it is also used as a reactant in myriad reactions from desaturation to dioxygenation. The direct incorporation of molecular oxygen into an organic compound is of particular interest, as such reactions enable the catabolism of otherwise intractable substrates. A common feature among most dioxygenases is the use of at least one iron ion to facilitate electron transfer and the oxidation of carbon. Thiol dioxygenation stands out as unique because an unusual ligand set is used to coordinate a mononuclear iron, carbon is not oxidized during the reaction, and it is the only dioxygenation which adds both oxygens to the same atom. Though other dioxygenases have been well studied, especially 2-oxoglutarate-dependent, catechol, and Rieske dioxygenases, thiol dioxygenation should be expected to proceed via a distinct mechanism. Successful completion of the proposed work will provide clarity into the mechanistic implications of adding two oxygen atoms to one electron-rich sulfur atom rather than two carbon atoms, and the nature of the catalytic contribution provided by the cofactor of CDO will be investigated. Further, the only other known thiol dioxygenase in humans, ADO will be studied to provide common themes in enzymatic sulfur oxidation.