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 Angstroms. The sequence is 91% identical to that of a human homolog. The structure reveals that CDO adopts the typical beta-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.

Fig. 1.

CDO reaction scheme showing that O₂ is incorporated into l-cysteine (l-Cys) to yield cysteine sulfinic acid (CSA).

Fig. 2.

The CDO monomer. (Upper) Ribbon illustration showing the CDO fold. The metal atom is shown as a gray sphere, and coordinated water molecules are shown as red spheres. The β-strands are labeled A–M, and the helices are labeled 1–5. Conserved residues are shown as color-coded sticks (blue, His; yellow, Arg; green, Cys; violet, Tyr; orange, Glu). (Lower) Surface illustration showing the entrance to the channel into the active site.

Fig. 3.

CDO active site contoured at 1.2σ. The metal is shown as a gray sphere; His-86, -88, and -140 are the metal ligands. Three additional coordination sites are occupied by water (red spheres). Cys-93 and Tyr-157 are covalently linked, and the hydroxyl group of Tyr-157 is 4.4 Å from the metal. Other conserved active-site residues are also shown.

Fig. 4.

Multiple sequence alignment of CDOs. Conserved residues involved in metal binding are highlighted in red; other conserved residues are highlighted in green.

Fig. 5.

Mechanism for CDO reaction. (A) Resting Fe(II) state. (B) Substrate coordination by sulfur and nitrogen. (C) O₂ coordination, forming a ternary Fe(III)-superoxo complex. (D) The bound sulfur acquires partial cation-radical character, which can be stabilized by the adjacent negative charge on Tyr-157. (E) Combination of bound sulfur and Fe(III)-superoxo to give a cyclic peroxo intermediate. (F) O–O bond breakage to form a sulfoxy cation and metal-bound activated oxygen. (G) Transfer of the metal-bound activated oxygen to form product, cysteine sulfinic acid (CSA).