Crystal Structure of Mammalian Cysteine Dioxygenase

Abstract

Cysteine dioxygenase is a mononuclear iron-dependent enzyme responsible for the oxidation of cysteine with molecular oxygen to form cysteine sulfinate. This reaction commits cysteine to either catabolism to sulfate and pyruvate or the taurine biosynthetic pathway. Cysteine dioxygenase is a member of the cupin superfamily of proteins. The crystal structure of recombinant rat cysteine dioxygenase has been determined to 1.5-Å resolution, and these results confirm the canonical cupin ␤-sandwich fold and the rare cysteinyltyrosine intramolecular cross-link (between Cys 93 and Tyr 157 ) seen in the recently reported murine cysteine dioxygenase structure. In contrast to the catalytically inactive mononuclear Ni(II) metallocenter present in the murine structure, crystallization of a catalytically competent preparation of rat cysteine dioxygenase revealed a novel tetrahedrally coordinated mononuclear iron center involving three histidines (His 86 , His 88 , and His 140 ) and a water molecule. Attempts to acquire a structure with bound ligand using either cocrystallization or soaking crystals with cysteine revealed the formation of a mixed disulfide involving Cys 164 near the active site, which may explain previously observed substrate inhibition. This work provides a framework for understanding the molecular mechanisms involved in thiol dioxygenation and sets the stage for exploration of the chemistry of both the novel mononuclear iron center and the catalytic role of the cysteinyl-tyrosine linkage.The cytosolic enzyme cysteine dioxygenase (CDO) 3 (EC 1.13.11.20) catalyzes the irreversible oxidation of cysteine to cysteine sulfinate (Reaction 1). This reaction is required for a variety of critical metabolic pathways (1). CDO initiates the catabolism of cysteine to pyruvate and sulfate, which is essential for the provision of adequate inorganic sulfate and allows pyruvate to enter central pathways of metabolism. Also, the oxidation and excretion of the sulfur of methionine depends on CDO, because the sulfur atoms of methionine and homocysteine are only oxidized after their transfer, via the transsulfuration pathway, to serine to yield cysteine. In addition, CDO activity is essential for the biosynthesis of taurine, which is formed by the decarboxylation of cysteine sulfinate to hypotaurine and further oxidation of hypotaurine to taurine.Clinical evidence indicates that a block in cysteine catabolism, thought to be at CDO, leads to an altered cysteine to sulfate ratio that is associated with sulfate depletion and other adverse effects (1). The prevalence of impaired cysteine catabolism has been reported to be increased in patient populations afflicted with rheumatoid arthritis, liver diseases, Parkinson disease, Alzheimer disease, motor neuron disease, and systemic lupus erythematosus (2-5). These patients frequently exhibit low levels of sulfate in plasma (and in synovial fluid), elevated fasting plasma cysteine concentrations, elevated plasma cysteine to sulfate ratios, and an impaired capacity for sulfation reactions in...

Influence of cysteine 164 on active site structure in rat cysteine dioxygenase

“…The resting state is nearly identical to the WT structure. In X-ray crystallographic models, the iron 13 environment in resting states of CDO is variously modeled, with one [14], two [13,17,21] or three water molecules (when the metal was modeled as a nickel atom [38] pointing towards the active site and a corresponding methionine 179 position.…”

“…[13] A second posttranslational modification forms in mammalian CDO; a disulfide between cysteine 164 and exogenous cysteine. This was initially observed crystallographically [14] and subsequently characterized by us using mass spectrometry. [15] Indeed, the first cysteine bound structure for CDO (PDB ID 2ICI) [16] has been reanalyzed and interpreted as containing such a disulfide.…”

An Insight into the Mechanism of Human Cysteine Dioxygenase

“…1) in Motif 2 are substituted. Recently reported CDO structures from mouse and rat confirmed the cupin fold (18,19). In fact, recent structural studies of cupin proteins clearly demonstrate that the primary sequences of cupin motifs are not highly conserved, as was first believed (20).…”

“…In addition to mammals, CDO was also found in other eukaryotes and prokaryotes by genome analysis (25). Although several mechanisms of CDO have recently been proposed on the basis of structural analysis or x-ray absorption spectroscopy results (18,19,26), there is no direct structural evidence to date of the manner by which substrates bind to CDO. …”

“…In the case of eukaryotic CDOs, the highly conserved glutamic acid in cupin motif 1, mentioned above, is substituted by a cysteine, the thiol sulfur of which forms a thioether bond with the C⑀ of a nearby tyrosine residue (18,19). This rare Tyr-Cys adduct is termed "cross-linked protein derived cofactor" and was first reported in the crystal structure of galactose oxidase in 1991 (27).…”