Structure and mechanism of mouse cysteine dioxygenase

Abstract

Cysteine dioxygenase (CDO) catalyzes the oxidation of L-cysteine to cysteine sulfinic acid. Deficiencies in this enzyme have been linked to autoimmune diseases and neurological disorders. The x-ray crystal structure of CDO from Mus musculus was solved to a nominal resolution of 1.75 Å. The sequence is 91% identical to that of a human homolog. The structure reveals that CDO adopts the typical ␤-barrel fold of the cupin superfamily. The NE2 atoms of His-86, -88, and -140 provide the metal binding site. The structure further revealed a covalent linkage between the side chains of Cys-93 and Tyr-157, the cysteine of which is conserved only in eukaryotic proteins. Metal analysis showed that the recombinant enzyme contained a mixture of iron, nickel, and zinc, with increased iron content associated with increased catalytic activity. Details of the predicted active site are used to present and discuss a plausible mechanism of action for the enzyme.cupin ͉ cysteine metabolism ͉ O2-activation M ouse cysteine dioxygenase (CDO) catalyzes the initial step in the biochemical pathway used for oxidation of cysteine to sulfate (1), namely the oxidation of L-cysteine to cysteine sulfinic acid as shown in Fig. 1. The enzyme activity has important medical implications because elevated cysteine levels have been associated with Parkinson's and Alzheimer's diseases (2). High cysteine-to-sulfate ratios have been observed in patients suffering from systemic lupus erthematosus and rheumatoid arthritis (3, 4). Moreover, the Hallervorden-Spatz syndrome, a neurological disorder associated with iron accumulation, has been linked to a decline in CDO activity (5).CDO displays significant sequence identity with some members of the cupin superfamily (6), which have a conserved ␤-barrel fold and share two conserved sequence motifs: G(X) 5 HXH(X) 3,4 E(X) 6 G and G(X) 5 PXG(X) 2 H(X) 3 N (6-8).The two His and Glu residues from the first motif and the His from the second motif coordinate the metal ion in germin, the superfamily archetype (9). The Mus musculus CDO sequence contains the first motif with the exception of the glutamate, which is replaced by cysteine. This substitution is conserved in other eukaryotic CDOs. The second motif is less conserved, and only the His and Asn residues are present in the mouse CDO.CDO does not require an external reductant ( Fig. 1) and incorporates both oxygen atoms from O 2 (10), which justifies the dioxygenase classification, but relatively little else is known about the reaction mechanism. The recombinant enzyme from Rattus norvegicus has been purified and characterized by steadystate kinetics (11); the mouse enzyme investigated here has an identical sequence. Reconstitution of the rat apoenzyme with various transition metals confirmed that iron was required for activity, in accord with the earlier conclusions (1). Moreover, the recombinant rat enzyme was active without a second interacting factor, despite previous reports suggesting that additional components were required (12, 13).Here, we describe the x-ra...

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.…”

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. …”

Mechanism-based inhibition of human persulfide dioxygenase by γ-glutamyl-homocysteinyl-glycine

“…Crystal structures of PDO homologs indicate that only the sulfur atom of the substrate coordinates to the iron, displacing a single water molecule (16,17). This is similar to isopencillin Nsynthase (22) but contrasts with cysteine dioxygenase, in which the substrate serves as a bidentate ligand donating both the sulfur and the α-amino group (20). We speculate that upon diooxygen binding, the water molecule that remains coordinated to iron is used to hydrolyze the GSSO 2 H intermediate (Fig.…”

“…Based on the general mechanism of non-heme iron dioxygenases and, more specifically, cysteine dioxygenase (20), a mechanism was proposed for PDO ( Fig. 2A) in which binding of GSSH to ferrous PDO [1] results in displacement of one of the three coordinated water molecules [2].…”