An Insight into the Mechanism of Human Cysteine Dioxygenase

Abstract

Cysteine dioxygenase is a non-heme mononuclear iron metalloenzyme that catalyzes the oxidation of cysteine to cysteine sulfinic acid with addition of molecular dioxygen. This irreversible oxidative catabolism of cysteine initiates several important metabolic pathways related to diverse sulfurate compounds. Cysteine dioxygenase is therefore very important for maintaining the proper hepatic concentration of intracellular free cysteine. Mechanisms for mouse and rat cysteine dioxygenases have recently been reported based on their crystal structures in the absence of substrates, although there is still a lack of direct evidence. Here we report the first crystal structure of human cysteine dioxygenase in complex with its substrate L-cysteine to 2.7 Å , together with enzymatic activity and metal content assays of several single point mutants. Our results provide an insight into a new mechanism of cysteine thiol dioxygenation catalyzed by cysteine dioxygenase, which is tightly associated with a thioether-bonded tyrosine-cysteine cofactor involving Tyr-157 and Cys-93. This cross-linked protein-derived cofactor plays several key roles different from those in galactose oxidase. This report provides a new potential target for therapy of diseases related to human cysteine dioxygenase, including neurodegenerative and autoimmune diseases.Cysteine dioxygenase (CDO, 2 EC 1.13.11.20) is a non-heme mononuclear iron metalloenzyme that catalyzes the irreversible oxidation of cysteine to cysteine sulfinic acid (CSA) with addition of molecular oxygen (1) (Structure 1). This oxidative catabolism of cysteine initiates several important metabolic pathways related to pyruvate and several sulfurate compounds, including sulfate, hypotaurine, and taurine. CDO is expressed at appreciable levels in the brain, kidney, and lung, with extremely high levels in liver tissue (2-5), where CDO plays an important role in maintaining the hepatic concentration of intracellular free cysteine within a proper narrow range (6). When the levels of cysteine decrease below this range, the increase of CDO ubiquitination rate results in rapid degradation of the ubiquitinated portion by the 26 S proteasome system (7,8). However, the precise means by which cysteine regulates CDO ubiquitination remain unknown.Intracellular free cysteine is cytotoxic and neuroexcitotoxic due to oxidative damage via formation of free radicals in the presence of iron (9 -11). Elevated cysteine levels were reported previously in relation to several neurodegenerative diseases, including the well known Parkinson and Alzheimer diseases (12-14), and autoimmune diseases such as systemic lupus erythematosus and rheumatoid arthritis (15, 16). CDO is considered to be involved in these diseases due to its function in regulating free cysteine levels.Sequence alignment classifies CDO as a member of the cupin superfamily (see Fig. 1), whose members possess what may be the most diverse range of functions, encompassing ϳ18 subclasses. Nonetheless, neither of the exact characteristic conserved sequenc...

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

“…This reduction in activity has been shown in both human [16] and rat CDO, [18] but the size of the decrease reported is quite different, depending on substrate concentration. To resolve seemingly conflicting statements about the role of cysteine 164, we re-investigated the C164S variant to explore what changes in activity could be observed and to understand if these could be explained structurally.…”

“…Simmons et al [36] and Ye et al [16] independently of each other, noticed unexpected electron density distal to the Sγ-atom of residue C164 when analyzing crystal structure data from mammalian CDO crystals that had been either co-crystallised with or soaked in cysteine, but neither group was able to unambiguously identify the origin of this feature by refinement of their crystal structure models. Simmons and colleagues, however, found that this electron density was only observed in the structure of CDO in presence of substrate, but not in its absence.…”

“…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. [17] Furthermore, sequence alignment and structural comparison shows that although cysteine is conserved at this position in CDO of multicellular eukaryotes, it is replaced by a catalytically important arginine residue in 3-mercaptopropionate dioxygenases.…”

Discovery and Characterization of a Second Mammalian Thiol Dioxygenase, Cysteamine Dioxygenase

“…Mammalian CDOs contain a similar vicinal proline motif that appears to permit the formation of a loop between two ␤-strands containing important active site residues (13)(14)(15). Future structural analysis of the protein via either x-ray crystallography or nuclear magnetic resonance studies may shed light on the function of the conserved residues in ADO as well as reveal additional insights into the catalytic mechanism of this protein and the related thiol dioxygenase CDO.…”

“…A major advancement in our understanding of CDO structure and function has come from the recent determination of atomic resolution crystal structures of the protein (13)(14)(15). These studies confirmed that CDO is a member of the cupin superfamily, a superfamily that encompasses many functionally diverse proteins including auxin-binding protein, mannose-6-phosphate isomerase, and hydroxyanthranilate dioxygenase.…”