Cysteine Dioxygenase Activity Research Articles

Biochemical and Structural Characterization of OvoATh2: A Mononuclear Nonheme Iron Enzyme from Hydrogenimonas thermophila for Ovothiol Biosynthesis.

Ovothiol A and ergothioneine are thiol-histidine derivatives with sulfur substitutions at the δ-carbon or ε-carbon of the l-histidine imidazole ring, respectively. Both ovothiol A and ergothioneine have protective effects on many aging-related diseases, and the sulfur substitution plays a key role in determining their chemical and biological properties, while factors governing sulfur incorporation regioselectivities in ovothiol and ergothioneine biosynthesis in the corresponding enzymes (OvoA, Egt1, or EgtB) are not yet known. In this study, we have successfully obtained the first OvoA crystal structure, which provides critical information to explain their C-S bond formation regioselectivity. Furthermore, OvoATh2 exhibits several additional activities: (1) ergothioneine sulfoxide synthase activity akin to Egt1 in ergothioneine biosynthesis; (2) cysteine dioxygenase activity using l-cysteine and l-histidine analogues as substrates; (3) cysteine dioxygenase activity upon mutation of an active site tyrosine residue (Y406). The structural insights and diverse chemistries demonstrated by OvoATh2 pave the way for future comprehensive structure-function correlation studies.

The Effect of Dietary Cysteine Level on Cysteine Metabolism in Rats

Male, Sprague-Dawley rats were fed l-amino acid diets that contained 0.4% l methionine and either 0, 0.2% (control), or 2.6% l-cysteine (free base) for 5 or 20 days. Hepatic cysteine dioxygenase activity in rats fed 2.6% cysteine was 5 and 3-times as great as in pair-fed control rats at 5 and 20 days, respectively. Cysteine sulfinate decarboxylase activity in liver of rats fed 2.6% cysteine for 20 days was about 40% of the pair-fed control level. The activity of cysteine sulfinate aminotransferase and the rate of cysteine desulfhydration were not influenced by dietary cysteine content. Rats that had been fed these diets for 5 days were intubated with 5 g of diet that contained either 0.2% or 2.6% l-[35S]cysteine. The 24-hour urinary excretion of total 35S, 35SO4, and [35S]taurine by rats that had been fed 2.6% cysteine for 5 days and intubated with 0.2% l-[35S]cysteine was 1.4-, 1.8-, and 2.4-fold, respectively, that of pair-fed control rats. About 2.1, 1.8, and 9.1 times as much 35S, 35SO/4, and [35S]taurine, respectively, were excreted by rats fed 2.6% cysteine for 5 days and intubated with 2.6% l-[35S]cysteine as was excreted by pair-fed control rats. Urinary excretion of these metabolites was not different between rats fed 0 and 0.2% cysteine. These results indicate that the cysteine sulfinate pathway plays a major regulatory role in the metabolism of excess cysteine.cysteine cysteine desulfhydrase cysteine dioxygenase cysteine sulfinate decarboxylase taurine sulfate

Rats Fed a Low Protein Diet Supplemented with Sulfur Amino Acids Have Increased Cysteine Dioxygenase Activity and Increased Taurine Production in Hepatocytes

The metabolism of cysteine and cysteinesulfinate and the activities of key enzymes in cysteine catabolic pathways were investigated in hepatocytes isolated from rats fed a basal (100 g casein/kg) diet or the diet supplemented with L-methionine (3 or 10 g/kg diet) or the sulfur equivalent as L-cystine (2.4 or 8 g/kg diet). Cysteine dioxygenase activity was higher in hepatocytes from rats fed diets with the higher level of sulfur amino acid supplementation, and the higher enzyme activity was paralleled by a greater total catabolite production (taurine + sulfate) from cysteine. Taurine production as a percentage of total cysteine catabolism was significantly greater in hepatocytes from rats fed the diet with excess methionine or cystine (basal, 22%; excess methionine, 61%, excess cystine, 49%). Glutathione production was markedly lower in hepatocytes from rats fed excess sulfur amino acids such that total cysteine utilization was similar for all dietary treatments. Cysteinesulfinate decarboxylase activity and catabolism of cysteinesulfinate by hepatocytes were unaffected by the dietary supplementations. Results are in contrast to previous studies in which increased dietary protein resulted in decreased cysteinesulfinate decarboxylase activity and decreased partitioning of cysteinesulfinate to taurine vs. sulfate. Thus, sulfur amino acids may be less effective than protein in decreasing cysteinesulfinate decarboxylase activity and may result in a pattern of sulfur catabolite production from cysteine that favors taurine production.

The Activities of Rat Hepatic Cysteine Dioxygenase and Cysteinesulfinate Decarboxylase Are Regulated in a Reciprocal Manner in Response to Dietary Casein Level , ,

The catabolism of cysteine and cysteinesulfinate, the activities of key enzymes in cysteine catabolic pathways, and the effects of inhibitors of specific enzymes on cysteine catabolism were investigated in hepatocytes isolated from rats fed low (100 g casein/kg diet), moderate (300 g casein/kg diet) or high (600 g casein/kg diet) levels of dietary protein. Cysteine was catabolized predominantly by cysteinesulfinate-dependent pathways. Cysteine dioxygenase activity increased with increases in dietary casein level, and the higher enzyme activity was paralleled by a greater total catabolite production (taurine + hypotaurine + sulfate) from cysteine. However, taurine production did not closely follow cysteine dioxygenase activity. Taurine production doubled with an increase in dietary casein from 100 to 300 g/kg but did not increase with a further increase in dietary casein to 600 g/kg. Taurine production as a percentage of total catabolism decreased progressively with the increases in dietary casein and closely paralleled observed decreases in cysteinesulfinate decarboxylase activity. Thus, taurine production was limited at high protein levels by the decrease in cysteinesulfinate decarboxylase activity such that sulfate production from cysteinesulfinate was favored. D-Cysteinesulfinate inhibited cysteinesulfinate-dependent catabolism of cysteine, but inhibition of cysteinesulfinate decarboxylase was not specific.

Plant Cysteine Oxidase Oxygen-Sensing Function Is Conserved in Early Land Plants and Algae.

All aerobic organisms require O2 for survival. When their O2 is limited (hypoxia), a response is required to reduce demand and/or improve supply. A hypoxic response mechanism has been identified in flowering plants: the stability of certain proteins with N-terminal cysteine residues is regulated in an O2-dependent manner by the Cys/Arg branch of the N-degron pathway. These include the Group VII ethylene response factors (ERF-VIIs), which can initiate adaptive responses to hypoxia. Oxidation of their N-terminal cysteine residues is catalyzed by plant cysteine oxidases (PCOs), destabilizing these proteins in normoxia; PCO inactivity in hypoxia results in their stabilization. Biochemically, the PCOs are sensitive to O2 availability and can therefore act as plant O2 sensors. It is not known whether oxygen-sensing mechanisms exist in other phyla from the plant kingdom. Known PCO targets are only conserved in flowering plants, however PCO-like sequences appear to be conserved in all plant species. We sought to determine whether PCO-like enzymes from the liverwort, Marchantia polymorpha (MpPCO), and the freshwater algae, Klebsormidium nitens (KnPCO), have a similar function as PCO enzymes from Arabidopsis thaliana. We report that MpPCO and KnPCO show O2-sensitive N-terminal cysteine dioxygenase activity toward known AtPCO ERF-VII substrates as well as a putative endogenous substrate, MpERF-like, which was identified by homology to the Arabidopsis ERF-VIIs transcription factors. This work confirms functional and O2-dependent PCOs from Bryophyta and Charophyta, indicating the potential for PCO-mediated O2-sensing pathways in these organisms and suggesting PCO O2-sensing function could be important throughout the plant kingdom.

Formation Mechanism of Cofactor Cys-Tyr in the Cysteine Dioxygenases (CDO and F2-CDO) and Its Influence on Catalysis: A QM/MM Study.

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.

Особливості метаболічних перетворень гомоцистеїну та цистеїну в гепатоцитах щурів за умов нутрітивного дисбалансу

The work is devoted to the study of the homocysteine concentration in blood plasma and the activity of the enzymes of cysteine catabolic transformations γ-glutamate-cysteine ligase and cysteine dioxygenase in rats’ hepatocytes under the conditions of consumption of excessive sucrose content on the background of alimentary protein deprivation. It was found, that the consumption of a high-sucrose diet in animals is accompanied by the increase of homocysteine concentration in plasma by 57% compared to control, whereas the excess of sucrose under the conditions of protein deficiency leads to hypohomocysteinemia (the content of homocysteine decreased by 43% compared to control). The increased plasma homocysteine levels in excess-consuming sucrose can be considered as a prognostic marker of functional disorders of the transsulfuration pathway in the liver and used in the diagnosis of hepatopathologies. Since, the cysteine content in liver cells increased under the conditions of high-sucrose diet consumption, the increasing γ-GCS, a key enzyme of glutathione synthesis, is probably associated with the maintenance of intercellular glutathione stores. Our data of the occurrence hypohomocysteinemia under the conditions of the consumption of excessive amounts of sucrose on the background of dietary protein deficiency highlight gaps in the understanding of the correlation between the metabolic processes of methionine, homocysteine and cysteine in the liver. Under the excessive consumption of sucrose on the background of protein deficiency, the exogenous protein deficiency can be considered as a key factor in reducing the γ-GCS activity (36%) and the cysteine content, because not only the amount of this amino acid is reduced but also its synthesis is disrupted. At the same time, the maximum increase in cysteine dioxygenase activity in rats’ hepatocytes under the conditions of high-sucrose/low-protein diet consumption on the background of decreased γ-GCS activity indicates the utilization of excess cysteine with the formation of taurine and sulfates.

Dietary taurine intake affects growth and taurine synthesis regulation in golden pompano, Trachinotus ovatus (Linnaeus 1758)

Taurine serves an important role in many species, including fish. The purpose of this study was to investigate the effects of varying dietary taurine levels on the growth, antioxidant capacity, and tissue deposition of taurine in golden pompano. In addition, the mechanism of taurine synthesis was studied via enzyme activity and gene expression of the following enzymes: cystathionine β-synthase (CBS), cystathionine γ-lyase (CSE), cysteine dioxygenase (CDO), cysteine sulfinic acid decarboxylase (CSAD), and 2-aminoethanethiol dioxygenase (ADO). Taurine concentrations of 1.3, 4.4, 7.4, 10.5 and 12.7 g kg−1 were added to the basic feed, hereafter labelled the T0, T1, T2, T3 and T4 diets, respectively. A control diet was supplemented with 1.3 g kg−1 taurine. Following 8 weeks of treatment, the results showed that the specific growth rate increased with the increase in dietary taurine content; the growth rate peaked at the 10.5 g kg−1 dietary taurine level (P < 0.05). The hepatosomatic index was significantly reduced as dietary taurine levels increased (P < 0.05). Crude protein and crude lipid in the body increased significantly with the intake of exogenous taurine (P < 0.05). The increase in dietary taurine significantly increased the total antioxidant capacity, as well as the superoxide dismutase, catalase, and glutathione peroxidase activities; however, the malondialdehyde concentration correspondingly decreased (P < 0.05). The taurine content in whole fish, liver, heart, eye, kidney, brain, muscle and gill significantly increased with the increase in dietary taurine (P < 0.05). The activity of CSE, CDO, CSAD and ADO decreased with the increase in dietary taurine (P < 0.05). Taurine intake had no significant impact on CBS activity (P > 0.05). The gene expression of CBS, CSE, CDO, CSAD and ADO exhibited a downward trend with an increase of taurine (P < 0.05). These results indicate that taurine plays an important role in the growth of golden pompano. Polynomial regression analysis revealed that the taurine requirements of golden pompano could be satisfied if the taurine content reached at least 10.0 g kg−1. In addition, taurine has a regulating effect on taurine synthesis in golden pompano.

Taurine requirement and metabolism response of tiger puffer Takifugu rubripes to graded taurine supplementation

An eight week feeding trial was conducted to investigate the effect of taurine supplementation on growth performance, sulfur amino acid concentrations, the activity and gene expression of enzymes related to taurine synthesis, and gene expression of taurine transporter (TAUT) in tiger puffer (Takifugu rubripes). Test diets were respectively supplemented with 0, 3, 6, 9, 12 and 20 g kg−1 taurine (the T0, T3, T6, T9, T12 and T20 diets) and a control diet was supplemented with 12 g kg−1 cysteine (the Cys diet). All experimental diets were fed to triplicate groups of 25 fish (initial weight 20.05 ± 0.06 g). Dose responses were observed in terms of specific growth rate and feed efficiency ratio. Dietary taurine requirement was estimated at 5.25 and 8.23 g kg−1 of dry matter of the diet based on regression analysis for specific growth rate and feed efficiency ratio, respectively. Free taurine concentrations of muscle and liver tissues increased with the increase of dietary taurine concentration. However, there was no significant difference in free taurine concentrations in muscle or liver between the Cys and T0 groups (p > .05). Free methionine concentration of liver tissue gradually increased with the increase of dietary taurine, which implied that exogenous taurine appeared to spare hepatic methionine. Cysteine sulfinic acid decarboxylase (CSD) activity and mRNA level in liver were down-regulated in the T9, T12 and T20 groups compared with the T0 group (p < .05), while no statistical difference was observed in cysteine dioxygenase (CDO) activity and expression between the T0 group and the taurine supplementation groups (the T3, T6, T9, T12 and T20 groups (p > .05). TAUT mRNA levels in muscle had a decrease trend in fish fed diets containing 1.7 to 8.2 g kg−1 taurine (the T0, T3 and T6 groups), while there was no significant difference when dietary taurine concentrations increased from 8.2 to 21.9 g kg−1 (the T6, T9, T12 and T20 groups). In conclusion, tiger puffer cannot synthesize taurine in the liver at the level of cysteine used in this study, whereas a sparing effect on the methionine demand may be observed in liver by dietary taurine supplementation. Our data indicates that taurine is a conditionally essential amino acid for tiger puffer, and the taurine requirement was estimated ranged from 5.25 to 8.23 g kg−1 of dry matter of the diet.

Cysteine Dioxygenase Enzyme Activity and Gene Expression in the Dimorphic Pathogenic Fungus Histoplasma capsulatum Is in both the Mold and Yeast Morphotypes and Exhibits Substantial Strain Variation.

In the dimorphism (mold/yeast) Histoplasma capsulatum (Hc) literature are reports that yeast (the so-called pathogenic form) uniquely expresses a cysteine dioxygenase (CDO, approx. 10,500 dal) activity which the mold morphotype (the so-called saprophytic soil form) does not express (C.F., Kumar et al., Biochem 22, 762, 1983). This yeast-specific CDO activity is postulated to play a critical role in the mold-to-yeast shift. A number of years ago, our lab isolated the gene encoding the Hc cysteine dioxygenase (CDO1, Genbank accession AY804144) and noted significant expression in the mold morphotype of several Histoplasma strains and also determined that the predicted protein would be over double the 10,500 dal reported by Kumar et al. Our report demonstrates (in the class 1 Downs strain, the class 2 G271B strain and two Panamanian strains, 184AS and 186AS) that the CDO1 gene is expressed in both the mold and yeast morphotypes and both morphotypes show significant CDO activity. Furthermore, we show via a FLAG-tag analysis that the expressed protein is approximately 24.7 ± 2.4 kd, in agreement with the putative protein sequence (determined from cDNA sequence) which yields 23.8 kd and is consistent with most other eukaryotic CDO enzymes. Additionally, we demonstrate that intracellular cysteine levels are actually significantly higher in the mold form of the two Panamanian strains, 184AS and 186AS, equal in both mold and yeast in the class 1 Downs strain and significantly higher in yeast of the more pathogenic class 2 G217B strain.

Comparison of the sulfur-oxygenation of cysteine and S-carboxymethyl-l-cysteine in human hepatic cytosol and the rôle of cysteine dioxygenase.

To determine the Km , Vmax , cofactor, activator and inhibitor requirements of human cysteine dioxygenase and S-carboxymethyl-l-cysteine S-oxygenase with respect to both l-Cysteine and S-carboxymethyl-l-cysteine as substrates. In vitro human hepatic cytosolic fraction enzyme assays were optimised for cysteine dioxygenase activity using l-Cysteine as substrate and the effect of various cofactors, activators and inhibitors on the S-oxidations of both l-Cysteine and S-carboxymethyl-l-cysteine were investigated. The results of the invitro reaction phenotyping investigation found that although both cysteine dioxygenase and S-carboxymethyl-l-cysteine S-oxygenase required Fe2+ for catalytic activity both enzymes showed considerable divergence in cofactor, activator and inhibitor specificities. Cysteine dioxygenase has no cofactor but uses NAD+ and NADH(H+ ) as pharmacological chaperones and is not inhibited by S-carboxymethyl-l-cysteine. S-carboxymethyl-l-cysteine S-oxygenase requires tetrahydrobiopterin as a cofactor, is not activated by NAD+ and NADH(H+ ) but is activated by l-Cysteine. Additionally, the sulfydryl alkylating agent, N-ethylmaleimide, activated carboxymethyl-l-cysteine S-oxygenase but inhibited cysteine dioxygenase. Human hepatic cytosolic fraction cysteine dioxygenase activity is not responsible for the S-oxidation of the substituted cysteine, S-carboxymethyl-l-cysteine.

Use of a Tyrosine Analogue To Modulate the Two Activities of a Nonheme Iron Enzyme OvoA in Ovothiol Biosynthesis, Cysteine Oxidation versus Oxidative C-S Bond Formation.

Ovothiol is a histidine thiol derivative. The biosynthesis of ovothiol involves an extremely efficient trans-sulfuration strategy. The nonheme iron enzyme OvoA catalyzed oxidative coupling between cysteine and histidine is one of the key steps. Besides catalyzing the oxidative coupling between cysteine and histidine, OvoA also catalyzes the oxidation of cysteine to cysteine sulfinic acid (cysteine dioxygenase activity). Thus far, very little mechanistic information is available for OvoA-catalysis. In this report, we measured the kinetic isotope effect (KIE) in OvoA-catalysis using the isotopically sensitive branching method. In addition, by replacing an active site tyrosine (Tyr417) with 2-amino-3-(4-hydroxy-3-(methylthio)phenyl)propanoic acid (MtTyr) through the amber suppressor mediated unnatural amino acid incorporation method, the two OvoA activities (oxidative coupling between cysteine and histidine, and cysteine dioxygenase activity) can be modulated. These results suggest that the two OvoA activities branch out from a common intermediate and that the active site tyrosine residue plays some key roles in controlling the partitioning between these two pathways.

The Cysteine Dioxygenase Homologue from Pseudomonas aeruginosa Is a 3-Mercaptopropionate Dioxygenase

Thiol dioxygenation is the initial oxidation step that commits a thiol to important catabolic or biosynthetic pathways. The reaction is catalyzed by a family of specific non-heme mononuclear iron proteins each of which is reported to react efficiently with only one substrate. This family of enzymes includes cysteine dioxygenase, cysteamine dioxygenase, mercaptosuccinate dioxygenase, and 3-mercaptopropionate dioxygenase. Using sequence alignment to infer cysteine dioxygenase activity, a cysteine dioxygenase homologue from Pseudomonas aeruginosa (p3MDO) has been identified. Mass spectrometry of P. aeruginosa under standard growth conditions showed that p3MDO is expressed in low levels, suggesting that this metabolic pathway is available to the organism. Purified recombinant p3MDO is able to oxidize both cysteine and 3-mercaptopropionic acid in vitro, with a marked preference for 3-mercaptopropionic acid. We therefore describe this enzyme as a 3-mercaptopropionate dioxygenase. Mössbauer spectroscopy suggests that substrate binding to the ferrous iron is through the thiol but indicates that each substrate could adopt different coordination geometries. Crystallographic comparison with mammalian cysteine dioxygenase shows that the overall active site geometry is conserved but suggests that the different substrate specificity can be related to replacement of an arginine by a glutamine in the active site.

Structures of Arg- and Gln-type bacterial cysteine dioxygenase homologs.

In some bacteria, cysteine is converted to cysteine sulfinic acid by cysteine dioxygenases (CDO) that are only ∼15-30% identical in sequence to mammalian CDOs. Among bacterial proteins having this range of sequence similarity to mammalian CDO are some that conserve an active site Arg residue ("Arg-type" enzymes) and some having a Gln substituted for this Arg ("Gln-type" enzymes). Here, we describe a structure from each of these enzyme types by analyzing structures originally solved by structural genomics groups but not published: a Bacillus subtilis "Arg-type" enzyme that has cysteine dioxygenase activity (BsCDO), and a Ralstonia eutropha "Gln-type" CDO homolog of uncharacterized activity (ReCDOhom). The BsCDO active site is well conserved with mammalian CDO, and a cysteine complex captured in the active site confirms that the cysteine binding mode is also similar. The ReCDOhom structure reveals a new active site Arg residue that is hydrogen bonding to an iron-bound diatomic molecule we have interpreted as dioxygen. Notably, the Arg position is not compatible with the mode of Cys binding seen in both rat CDO and BsCDO. As sequence alignments show that this newly discovered active site Arg is well conserved among "Gln-type" CDO enzymes, we conclude that the "Gln-type" CDO homologs are not authentic CDOs but will have substrate specificity more similar to 3-mercaptopropionate dioxygenases.

A chromogenic assay of substrate depletion by thiol dioxygenases

A fast and easy method for enzyme activity assays using the chromogenic Ellman reagent, 5,5′-dithiobis(2-nitrobenzoic acid), was developed. The method was used to measure the activity of the nonheme mono-iron enzyme cysteine dioxygenase. Quantifying the depletion of the substrate, cysteine, allowed standard kinetic parameters to be determined for the enzyme from Rattus norvegicus. The assay was also used to quickly test the effects of ionic strength, pH, enzyme storage conditions, and potential inhibitors and activators. This assay facilitates a higher throughput than available HPLC-based assays, as it enjoys the advantages of fewer sample handling steps, implementation in a 96-well format, and speed. In addition, the relative specificity of Ellman’s reagent, coupled with its reaction with a wide range of thiols, means that this assay is applicable to many enzymes. Finally, the use of readily available reagents and instrumentation means that this assay can be used by practically any research group to compare results with those of other groups.

Branched-chain amino acids inhibit the TGF-beta-induced down-regulation of taurine biosynthetic enzyme cysteine dioxygenase in HepG2 cells

Taurine deficiency has been suggested to contribute to the pathogenesis and complications of advanced hepatic diseases. The molecular basis for a low level of taurine associated with hepatic failure is largely unknown. Using carbon tetrachloride (CCl4)-induced cirrhotic rat model, we found that the activity and expression of cysteine dioxygenase (CDO), a rate-limiting enzyme in taurine synthesis, were significantly decreased in the liver of these rats. To investigate the underlying mechanisms for the suppression, we examined the effects of pathological cytokines on CDO expression in human hepatoma HepG2 cells. Among the several cytokines, transforming growth factor-β (TGF-β), one of the key mediators of fibrogenesis, suppressed Cdo1 gene transcription through the MEK/ERK pathway. Finally, we further examined potential effects of branched-chain amino acids (BCAA) on CDO expression, as it has been reported that oral BCAA supplementation increased plasma taurine level in the patients with liver cirrhosis. BCAA, especially leucine, promoted Cdo1 gene transcription, and attenuated TGF-β-mediated suppression of Cdo1 gene expression. These results indicate that the low plasma level of taurine in advanced hepatic disease is due to decreased hepatic CDO expression, which can be partly attributed to suppressive effect of TGF-β on Cdo1 gene transcription. Furthermore, our observation that BCAA promotes Cdo1 expression suggests that BCAA may be therapeutically useful to improve hepatic taurine metabolism and further suppress dysfunctions associated with low level of taurine in hepatic diseases.Electronic supplementary materialThe online version of this article (doi:10.1007/s00726-014-1693-3) contains supplementary material, which is available to authorized users.

Addition of an external electron donor to in vitro assays of cysteine dioxygenase precludes the need for exogenous iron

Cysteine dioxygenase (CDO) utilizes a 3-His facial triad for coordination of its metal center. Recombinant CDO present in cellular lysate exists primarily in the ferrous form and exhibits significant catalytic activity. Removal of CDO from the reducing cellular environment during purification results in the loss of bound iron and oxidation of greater than 99% of the remaining metal centers. The as-isolated recombinant enzyme has comparable activity as the background level of l-cysteine oxidation confirming that CDO is inactive under the aerobic conditions required for catalysis. Including exogenous ferrous iron in assays resulted in non-enzymatic product formation; however, addition of an external reductant in assays of the purified protein resulted in the recovery of CDO activity. EPR spectroscopy of CDO in the presence of a reductant confirms that the recovered activity is consistent with reduction of iron to the ferrous form. The as-isolated enzyme in the presence of l-cysteine was nearly unreactive with the dioxygen analog, but had increased affinity when pre-incubated with an external reductant. These studies shed light on the discrepancies among reported kinetic parameters for CDO and also juxtapose the stability of the 3-His and 2-His/1-carboxylate ferrous enzymes in the presence of dioxygen.

Correlating crosslink formation with enzymatic activity in cysteine dioxygenase

Cysteine dioxygenase (CDO) from rat and other mammals exhibits a covalent post-translational modification between the residues C93 and Y157 that is in close proximity to the active site, and whose presence enhances the enzyme's activity. Protein with and without C93-Y157 crosslink migrates as distinct bands in SDS-PAGE, allowing quantification of the relative ratios between the two forms by densitometry of the respective bands. Expression of recombinant rat wild type CDO in Escherichia coli typically produces 40–50% with the C93-Y157 crosslink. A strategy was developed to increase the ratio of the non-crosslinked form in an enzyme preparation of reasonable quantity and purity, allowing direct assessment of the activity of non-crosslinked CDO and mechanism of formation of the crosslink. The presence of ferrous iron and oxygen is a prerequisite for C93-Y157 crosslink formation. Absence of oxygen during protein expression increased the fraction of non-crosslinked CDO, while presence of the metal chelator EDTA had little effect. Metal affinity chromatography was used to enrich non-crosslinked content. Both the enzymatic rate of cysteine oxidation and the amount of cross-linking between C93 and Y157 increased significantly upon exposure of CDO to air/oxygen and substrate cysteine in the presence of iron in a hitherto unreported two-phase process. The instantaneous activity was proportional to the amount of crosslinked enzyme present, demonstrating that the non-crosslinked form has negligible enzymatic activity. The biphasic kinetics suggest the existence of an as yet uncharacterised intermediate in crosslink formation and enzyme activation.

Knockout of the murine cysteine dioxygenase gene results in severe impairment in ability to synthesize taurine and an increased catabolism of cysteine to hydrogen sulfide

Cysteine homeostasis is dependent on the regulation of cysteine dioxygenase (CDO) in response to changes in sulfur amino acid intake. CDO oxidizes cysteine to cysteinesulfinate, which is further metabolized to either taurine or to pyruvate plus sulfate. To gain insight into the physiological function of CDO and the consequence of a loss of CDO activity, mice carrying a null CDO allele (CDO(+/-) mice) were crossed to generate CDO(-/-), CDO(+/-), and CDO(+/+) mice. CDO(-/-) mice exhibited postnatal mortality, growth deficit, and connective tissue pathology. CDO(-/-) mice had extremely low taurine levels and somewhat elevated cysteine levels, consistent with the lack of flux through CDO-dependent catabolic pathways. However, plasma sulfate levels were slightly higher in CDO(-/-) mice than in CDO(+/-) or CDO(+/+) mice, and tissue levels of acid-labile sulfide were elevated, indicating an increase in cysteine catabolism by cysteine desulfhydration pathways. Null mice had lower hepatic cytochrome c oxidase levels, suggesting impaired electron transport capacity. Supplementation of mice with taurine improved survival of male pups but otherwise had little effect on the phenotype of the CDO(-/-) mice. H(2)S has been identified as an important gaseous signaling molecule as well as a toxicant, and pathology may be due to dysregulation of H(2)S production. Control of cysteine levels by regulation of CDO may be necessary to maintain low H(2)S/sulfane sulfur levels and facilitate the use of H(2)S as a signaling molecule.

Simplified cysteine dioxygenase activity assay allows simultaneous quantitation of both substrate and product

A high-performance liquid chromatography (HPLC) method for enzyme activity assays using a hydrophilic interaction liquid chromatography (HILIC) column in combination with an evaporative light scattering detector was developed. The method was used to measure the activity of the non-heme mono-iron enzyme cysteine dioxygenase. The substrate cysteine and the product cysteine sulfinic acid are very weak chromophores, making direct ultraviolet (UV) detection without derivatization rather insensitive; moreover, derivatization of cysteine is often not efficient. Using the system described, underivatized substrate and product in samples from cysteine dioxygenase activity assays could be separated and analyzed. Furthermore, it was possible to quantify cysteic acid, the noncatalytic oxidation product of cysteine sulfinic acid. Acetone was used both to stop the enzymatic reaction by protein precipitation and as an organic mobile phase, making sample preparation very easy and the assay highly reproducible.