Thiol dioxygenases are important enzymes for human health; they are involved in the detoxification and catabolism of toxic thiol-containing natural products such as cysteine. As such, these enzymes have relevance to the development of Alzheimer\'s and Parkinson\'s diseases in the brain. Recent crystal structure coordinates of cysteine and 3-mercaptopropionate dioxygenase (CDO and MDO) showed major differences in the second-coordination spheres of the two enzymes. To understand the difference in activity between these two analogous enzymes, we created large, active-site cluster models. We show that CDO and MDO have different iron(III)-superoxo-bound structures due to differences in ligand coordination. Furthermore, our studies show that the differences in the second-coordination sphere and particularly the position of a positively charged Arg residue results in changes in substrate positioning, mobility and enzymatic turnover. Furthermore, the substrate scope of MDO is explored with cysteinate and 2-mercaptosuccinic acid and their reactivity is predicted.

Cysteine dioxygenase (CDO) is a nonheme mononuclear iron enzyme, which catalyzes the oxidation of cysteine to cysteine sulfinic acid. Crystal structure studies of mammalian CDO showed that there is a cross-linked cysteine-tyrosine (Cys-Tyr) cofactor in its active site. Moreover, the formation of the Cys-Tyr cofactor requires the metal cofactor (Fe2+) and O2, and it was previously considered to substantially enhance the catalytic efficiency and half-life of CDO. Recently, a pure human CDO (F2-CDO) without including the Cys-Tyr cofactor was crystalized by the site-directed mutagenesis approach in the anaerobic condition. In this work, to gain insights into the formation mechanism of the Cys-Tyr cofactor and whether it can really promote the catalytic reactivity of CDO, a series of computational models have been constructed, and quantum mechanical/molecular mechanical (QM/MM) calculations have been performed. Our calculation results reveal that WT-CDO and F2-CDO follow different mechanisms for the formation of the Cys-Tyr cofactor. In F2-CDO, the cofactor formation contains the H-abstraction, C-S bond formation, intramolecular F migration, and aromatization of the residue F2Y157, in which the Fe-coordinate dioxygen can be recovered after the formation cofactor; however, in the WT-CDO, the cofactor formation shows some differences. During the reaction, hydrogen peroxide is generated, and the final aromatization requires the assistance of one water molecule. Furthermore, the overall barriers of cofactor formation are always higher than l-cysteine oxidation for both WT-CDO and F2-CDO irrespective of the absence or presence of the cofactor. Thus, we can theoretically confirm that the Cys-Tyr cofactor is not essential for the oxidation activity of CDO, and cofactor formation is just an accompanying reaction but not a prerequisite for the oxidation reaction. These results may provide useful information for understanding the catalysis of CDO.

The clinical efficacy of DNA cytology test (CY) in gastric cancer (GC) has been retrospectively proposed using cancer-specific methylation of cysteine dioxygenase type 1 (CDO1). We confirmed the clinical utility of DNA CY in a prospective cohort. Four hundred GC samples were prospectively collected for washing cytology (UMIN000026191), and detection of the DNA methylation of CDO1 was assessed by quantitative methylation-specific PCR in the sediments. Endpoint was defined as the match rate between conventional CY1 and DNA CY1 (diagnostic sensitivity), and the DNA CY0 rate (diagnostic specificity) in pStage IA. DNA CY1 was detected in 45 cases (12.5%), while CY1 was seen in 31 cases (8.6%) of 361 chemotherapy-naïve samples, where the sensitivity and specificity of the DNA CY in the peritoneal solutions were 74.2% and 96.5%, respectively. The DNA CY was positive for 3.5/0/4.9/11.4/58.8% in pStage IA/IB/II/III/IV, respectively (P < .01). In the multivariate analysis, DNA CY1 was independently correlated with pathological tumor depth (pT) (P = .0012), female gender (P = .0099), CY1 (P = .0135), P1 (P = .019), and carcinoembryonic antigen (CEA) (P = .036). The combination of DNA CY1 and P factor nearly all covered the potential peritoneal dissemination (P1 and/or CY1 and/or DNA CY1) (58/61:95.1%). DNA CY1 had a significantly poorer prognosis than DNA CY0 in GC patients (P < .0001). DNA CY1 detected by CDO1 promoter DNA methylation has a great value to detect minimal residual disease of the peritoneum in GC clinics, representing poor prognosis as a novel single DNA marker.

ETHE1 is a member of a growing subclass of nonheme Fe enzymes that catalyzes transformations of sulfur-containing substrates without a cofactor. ETHE1 dioxygenates glutathione persulfide (GSSH) to glutathione (GSH) and sulfite in a reaction which is similar to that of cysteine dioxygenase (CDO), but with monodentate (vs bidentate) substrate coordination and a 2-His/1-Asp (vs 3-His) ligand set. In this study, we demonstrate that GSS- binds directly to the iron active site, causing coordination unsaturation to prime the site for O2 activation. Nitrosyl complexes without and with GSSH were generated and spectroscopically characterized as unreactive analogues for the invoked ferric superoxide intermediate. New spectral features from persulfide binding to the FeIII include the appearance of a low-energy FeIII ligand field transition, an energy shift of a NO- to FeIII CT transition, and two new GSS- to FeIII CT transitions. Time-dependent density functional theory calculations were used to simulate the experimental spectra to determine the persulfide orientation. Correlation of these spectral features with those of monodentate cysteine binding in isopenicillin N synthase (IPNS) shows that the persulfide is a poorer donor but still results in an equivalent frontier molecular orbital for reactivity. The ETHE1 persulfide dioxygenation reaction coordinate was calculated, and while the initial steps are similar to the reaction coordinate of CDO, an additional hydrolysis step is required in ETHE1 to break the S-S bond. Unlike ETHE1 and CDO, which both oxygenate sulfur, IPNS oxidizes sulfur through an initial H atom abstraction. Thus, factors that determine oxygenase vs oxidase reactivity were evaluated. In general, sulfur oxygenation is thermodynamically favored and has a lower barrier for reactivity. However, in IPNS, second-sphere residues in the active site pocket constrain the substrate, raising the barrier for sulfur oxygenation relative to oxidation via H atom abstraction.

BACKGROUND: Cysteine biology is important for the chemosensitivity of cancer cells. Our research has focused on the epigenetic silencing of cysteine dioxygenase type 1 (CDO1) in colorectal cancer (CRC). In this study, we describe detection of CDO1 methylation in the plasma of CRC patients using methylation specific PCR (Q-MSP) and extensive analysis of the PCR reaction.
METHODS: DNA was extracted from plasma, and analysed for methylation of the CDO1 gene using Q-MSP. The detection rate of CDO1 gene methylation was calculated and compared with that of diluted DNA extracted from \"positive control\" DLD1 cells. CDO1 gene methylation in the plasma of 40 CRC patients that were clinicopathologically analysed was then determined.
RESULTS: (1) The cloned sequence analysis detected 93.3% methylation of the promoter CpG islands of the CDO1 gene of positive control DLD1 cells and 4.7% methylation of the negative control HepG2 CDO1 gene. (2) DLD1 CDO1 DNA could not be detected in this assay if the extracted DNA was diluted ∼1000 fold. The more DNA that was used for the PCR reaction, the more effectively it was amplified in Q-MSP. (3) By increasing the amount of DNA used, methylated CDO1 could be clearly detected in the plasma of 8 (20%) of the CRC patients. However, the percentage of CRC patients detected by methylated CDO1 in plasma was lower than that detected by CEA (35.9%) or CA19-9 (23.1%) in preoperative serum. Combination of CEA/CA19-9 plus plasma methylated CDO1 could increase the rate of detection of curable CRC patients (39.3%) as compared to CEA/CA19-9 (25%).
CONCLUSIONS: We have described detection of CDO1 methylation in the plasma of CRC patients. Although CDO1 methylation was not detected as frequently as conventional tumor markers, analysis of plasma CDO1 methylation in combination with CEA/CA19-9 levels increases the detection rate of curable CRC patients.

Cysteine dioxygenase (CDO) is a vital enzyme for human health involved in the biodegradation of toxic cysteine and thereby regulation of the cysteine concentration in the body. The enzyme belongs to the group of nonheme iron dioxygenases and utilizes molecular oxygen to transfer two oxygen atoms to cysteinate to form cysteine sulfinic acid products. The mechanism for this reaction is currently disputed, with crystallographic studies implicating a persulfenate intermediate in the catalytic cycle. To resolve the dispute we have performed quantum mechanics/molecular mechanics (QM/MM) calculations on substrate activation by CDO enzymes using an enzyme monomer and a large QM active region. We find a stepwise mechanism, whereby the distal oxygen atom of the iron(II)-superoxo complex attacks the sulfur atom of cysteinate to form a ring structure, followed by dioxygen bond breaking and the formation of a sulfoxide bound to an iron(IV)-oxo complex. A sulfoxide rotation precedes the second oxygen atom transfer to the substrate to give cysteine sulfinic acid products. The reaction takes place on several low-lying spin-state surfaces via multistate reactivity patterns. It starts in the singlet ground state of the iron(II)-superoxo reactant and then proceeds mainly on the quintet and triplet surfaces. The initial and rate-determining attack of the superoxo group on the cysteinate sulfur atom involves a spin-state crossing from singlet to quintet. We have also investigated an alternative mechanism via a persulfenate intermediate, with a realignment of hydrogen bonding interactions in the substrate binding pocket. However, this alternative mechanism of proximal oxygen atom attack on the sulfur atom of cysteinate is computed to be a high-energy pathway, and therefore, the persulfenate intermediate is unlikely to participate in the catalytic cycle of CDO enzymes.

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Cells exposed to hyperosmotic conditions maintain their volume by accumulating organic osmolytes. Taurine is considered as an osmolyte in brain cells. Accumulation of other osmolytes (sorbitol, myo-inositol and betaine), was shown in renal cells to result from an upregulation of the expression of the genes regulating osmolyte cell content. We have investigated the gene expression of the taurine transporter (TauT) and of the taurine biosynthetic enzymes, cysteine dioxygenase (CDO) and cysteine sulfinate decarboxylase (CSD) by measuring their mRNA levels in brain of salt-loaded rats. mRNA levels of genes previously identified as osmosensitive, namely aldose reductase (AR), myo-inositol transporter (SMIT) and betaine transporter (BGT1) were also determined. In whole brain, TauT-, SMIT- and BGT1-mRNA levels were significantly increased following acute salt-loading but SMIT-mRNA levels only remained elevated following chronic salt-loading while CDO-, CSD- and AR-mRNA levels remained unchanged in both conditions. Following acute salt-loading, mRNA levels of TauT, CDO, CSD, SMIT, BGT1 and AR were increased in cerebral cortex while SMIT- and BGT1-mRNA levels only were increased in striatum and habenula.TauT, CDO and CSD genes may be upregulated in brain of salt-loaded rats but the upregulation of the TauT gene appears more widespread. TauT, CDO and CSD are thus putative osmosensitive genes. However the actual pattern (amplitude, time course and regional occurrence) of the upregulation of each of the putative (TauT, CDO and CSD) and established (AR, SMIT and BGT1) osmosensitive genes differs markedly. This indicates that there exist other factors in brain cells which can selectively prevent the upregulation of these genes by hyperosmolarity.

Effects of some cysteine analogs and other compounds on in vitro cysteine oxidase were studied in rat brain microsomes. Among the tested compounds, maximum inhibition of microsomal cysteine oxidase was by alpha,alpha\'-dipyridyl and the least inhibition by dithiothreitol. Kinetic and dialysis studies found L-homocysteine to be a competitive and reversible inhibitor of cysteine oxidase. Epinephrine was shown to inhibit cysteine oxidase, whereas pyridoxal HCl activated cysteine oxidase at the same concentration. Except for the Mg2+ ion, other metallic ions inhibited cysteine oxidase activity in the following order: Zn2+ greater than Cu2+ greater than Li+ greater than Ca2+ greater than Co2+ greater than K+ greater than Mn2+. A 12 mM concentration of Mg2+ ion was required to obtain maximum cysteine oxidase activity.