Martha H. Stipanuk

Mammalian Cysteine Metabolism: New Insights into Regulation of Cysteine Metabolism

The mammalian liver tightly regulates its free cysteine pool, and intracellular cysteine in rat liver is maintained between 20 and 100 nmol/g even when sulfur amino acid intakes are deficient or excessive. By keeping cysteine levels within a narrow range and by regulating the synthesis of glutathione, which serves as a reservoir of cysteine, the liver addresses both the need to have adequate cysteine to support normal metabolism and the need to keep cysteine levels below the threshold of toxicity. Cysteine catabolism is tightly regulated via regulation of cysteine dioxygenase (CDO) levels in the liver, with the turnover of CDO protein being dramatically decreased when intracellular cysteine levels increase. This occurs in response to changes in the intracellular cysteine concentration via changes in the rate of CDO ubiquitination and degradation. Glutathione synthesis also increases when intracellular cysteine levels increase as a result of increased saturation of glutamate-cysteine ligase (GCL) with cysteine, and this contributes to removal of excess cysteine. When cysteine levels drop, GCL activity increases, and the increased capacity for glutathione synthesis facilitates conservation of cysteine in the form of glutathione (although the absolute rate of glutathione synthesis still decreases because of the lack of substrate). This increase in GCL activity is dependent on up-regulation of expression of both the catalytic and modifier subunits of GCL, resulting in an increase in total catalytic subunit plus an increase in the catalytic efficiency of the enzyme. An important role of cysteine utilization for coenzyme A synthesis in maintaining cellular cysteine levels in some tissues, and a possible connection between the necessity of controlling cellular cysteine levels to regulate the rate of hydrogen sulfide production, have been suggested by recent literature and are areas that deserve further study.

Dealing with methionine/homocysteine sulfur: cysteine metabolism to taurine and inorganic sulfur.

Synthesis of cysteine as a product of the transsulfuration pathway can be viewed as part of methionine or homocysteine degradation, with cysteine being the vehicle for sulfur conversion to end products (sulfate, taurine) that can be excreted in the urine. Transsulfuration is regulated by stimulation of cystathionine β-synthase and inhibition of methylene tetrahydrofolate reductase in response to changes in the level of S-adenosylmethionine, and this promotes homocysteine degradation when methionine availability is high. Cysteine is catabolized by several desulfuration reactions that release sulfur in a reduced oxidation state, generating sulfane sulfur or hydrogen sulfide (H2S), which can be further oxidized to sulfate. Cysteine desulfuration is accomplished by alternate reactions catalyzed by cystathionine β-synthase and cystathionine γ-lyase. Cysteine is also catabolized by pathways that require the initial oxidation of the cysteine thiol by cysteine dioxygenase to form cysteinesulfinate. The oxidative pathway leads to production of taurine and sulfate in a ratio of approximately 2:1. Relative metabolism of cysteine by desulfuration versus oxidative pathways is influenced by cysteine dioxygenase activity, which is low in animals fed low-protein diets and high in animals fed excess sulfur amino acids. Thus, desulfuration reactions dominate when cysteine is deficient, whereas oxidative catabolism dominates when cysteine is in excess. In rats consuming a diet with an adequate level of sulfur amino acids, about two thirds of cysteine catabolism occurs by oxidative pathways and one third by desulfuration pathways. Cysteine dioxygenase is robustly regulated in response to cysteine availability and may function to provide a pathway to siphon cysteine to less toxic metabolites than those produced by cysteine desulfuration reactions.

Cysteine dioxygenase: a robust system for regulation of cellular cysteine levels.

Cysteine catabolism in mammals is dependent upon cysteine dioxygenase (CDO), an enzyme that adds molecular oxygen to the sulfur of cysteine, converting the thiol to a sulfinic acid known as cysteinesulfinic acid (3-sulfinoalanine). CDO is one of the most highly regulated metabolic enzymes responding to diet that is known. It undergoes up to 45-fold changes in concentration and up to 10-fold changes in catalytic efficiency. This provides a remarkable responsiveness of the cell to changes in sulfur amino acid availability: the ability to decrease CDO activity and conserve cysteine when cysteine is scarce and to rapidly increase CDO activity and catabolize cysteine to prevent cytotoxicity when cysteine supply is abundant. CDO in both liver and adipose tissues responds to changes in dietary intakes of protein and/or sulfur amino acids over a range that encompasses the requirement level, suggesting that cysteine homeostasis is very important to the living organism.

Crystal structure of mammalian cysteine dioxygenase: A novel mononuclear iron center for cysteine thiol oxidation

Cysteine dioxygenase is a mononuclear iron-dependent enzyme responsible for the oxidation of cysteine with molecular oxygen to form cysteine sulfinate. This reaction commits cysteine to either catabolism to sulfate and pyruvate or the taurine biosynthetic pathway. Cysteine dioxygenase is a member of the cupin superfamily of proteins. The crystal structure of recombinant rat cysteine dioxygenase has been determined to 1.5-Å resolution, and these results confirm the canonical cupin β-sandwich fold and the rare cysteinyltyrosine intramolecular cross-link (between Cys93 and Tyr157) seen in the recently reported murine cysteine dioxygenase structure. In contrast to the catalytically inactive mononuclear Ni(II) metallocenter present in the murine structure, crystallization of a catalytically competent preparation of rat cysteine dioxygenase revealed a novel tetrahedrally coordinated mononuclear iron center involving three histidines (His86, His88, and His140) and a water molecule. Attempts to acquire a structure with bound ligand using either cocrystallization or soaking crystals with cysteine revealed the formation of a mixed disulfide involving Cys164 near the active site, which may explain previously observed substrate inhibition. This work provides a framework for understanding the molecular mechanisms involved in thiol dioxygenation and sets the stage for exploration of the chemistry of both the novel mononuclear iron center and the catalytic role of the cysteinyl-tyrosine linkage.

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

There are only two known thiol dioxygenase activities in mammals, and they are ascribed to the enzymes cysteine dioxygenase (CDO) and cysteamine (2-aminoethanethiol) dioxygenase (ADO). Although many studies have been dedicated to CDO, resulting in the identification of its gene and even characterization of the tertiary structure of the protein, relatively little is known about cysteamine dioxygenase. The failure to identify the gene for this protein has significantly hampered our understanding of the metabolism of cysteamine, a product of the constitutive degradation of coenzyme A, and the synthesis of taurine, the final product of cysteamine oxidation and the second most abundant amino acid in mammalian tissues. In this study we identified a hypothetical murine protein homolog of CDO (hereafter called ADO) that is encoded by the gene Gm237 and belongs to the DUF1637 protein family. When expressed as a recombinant protein, ADO exhibited significant cysteamine dioxygenase activity in vitro. The reaction was highly specific for cysteamine; cysteine was not oxidized by the enzyme, and structurally related compounds were not competitive inhibitors of the reaction. When overexpressed in HepG2/C3A cells, ADO increased the production of hypotaurine from cysteamine. Similarly, when endogenous expression of the human ADO ortholog C10orf22 in HepG2/C3A cells was reduced by RNA-mediated interference, hypotaurine production decreased. Western blots of murine tissues with an antibody developed against ADO showed that the protein is ubiquitously expressed with the highest levels in brain, heart, and skeletal muscle. Overall, these data suggest that ADO is responsible for endogenous cysteamine dioxygenase activity.

Role of the Liver in Regulation of Body Cysteine and Taurine Levels: A Brief Review

The first-pass metabolism of dietary sulfur amino acids by the liver and the robust upregulation of hepatic cysteine dioxygenase activity in response to an increase in dietary protein or sulfur amino acid level gives the liver a primary role in the removal of excess cysteine and in the synthesis of taurine. Hepatic taurine synthesis is largely restricted by the low availability of cysteinesulfinate as substrate for cysteinesulfinate decarboxylase, and taurine production is increased when cysteinesulfinate increases in response to an increase in the hepatic cysteine concentration and the associated increase in cysteine dioxygenase activity. The upregulation of cysteine dioxygenase in the presence of cysteine is a consequence of diminished ubiquitination of cysteine dioxygenase and a slower rate of degradation by the 26S proteasome.

Evidence for a rate-limiting role of cysteinesulfinate decarboxylase activity in taurine biosynthesis in vivo

The relationship between activities of enzymes involved in cysteine oxidation and the apparent conversion of cysteine to taurine in vivo were investigated in the rat and cat. Both hepatic cysteinesulfinate decarboxylase activity and the oxidation in vivo of cysteine to taurine were lower in the kitten than in the adult female rat and lower in the latter than in the young male rat. Our data support the hypothesis that cysteinesulfinate decarboxylase plays a rate-limiting role in taurine biosynthesis.

Serum micronutrient concentrations and decline in physical function among older persons.

CONTEXT Maintaining independence of older persons is a public health priority, and identifying the factors that contribute to decline in physical function is needed to prevent or postpone the disablement process. The potential deleterious effect of poor nutrition on decline in physical function in older persons is unclear. OBJECTIVE To determine whether a low serum concentration of micronutrients is associated with subsequent decline in physical function among older men and women living in the community. DESIGN, SETTING, AND PARTICIPANTS Longitudinal study of 698 community-living persons 65 years or older who were randomly selected from a population registry in Tuscany, Italy. Participants completed the baseline examination from November 1, 1998, through May 28, 2000, and the 3-year follow-up assessments from November 1, 2001, through March 30, 2003. MAIN OUTCOME MEASURE Decline in physical function was defined as a loss of at least 1 point in the Short Physical Performance Battery during the 3-year follow-up. Odds ratios (ORs) were calculated for the lowest quartile of each nutrient using the other 3 quartiles combined as the reference group. Two additional and complementary analytical approaches were used to confirm the validity of the results. RESULTS The mean decline in the Short Physical Performance Battery score was 1.1 point. In a logistic regression analysis that was adjusted for potential confounders, only a low concentration of vitamin E (<1.1 microg/mL [<24.9 micromol/L]) was significantly associated with subsequent decline in physical function (OR, 1.62; 95% confidence interval, 1.11-2.36; P = .01 for association of lowest alpha-tocopherol quartile with at least a 1-point decline in physical function). In a general linear model, the concentration of vitamin E at baseline, when analyzed as a continuous measure, was significantly associated with the Short Physical Performance Battery score at follow-up after adjustment for potential confounders and Short Physical Performance Battery score at baseline (beta = .023; P = .01). In a classification and regression tree analysis, age older than 81 years and vitamin E (in participants aged 70-80 years) were identified as the strongest determinants of decline in physical function (physical decline in 84% and 60%, respectively; misclassification error rate, 0.33). CONCLUSIONS These results provide empirical evidence that a low serum concentration of vitamin E is associated with subsequent decline in physical function among community-living older adults. Clinical trials may be warranted to determine whether an optimal concentration of vitamin E reduces functional decline and the onset of disability in older persons.

Mechanisms involved in the regulation of key enzymes of cysteine metabolism in rat liver in vivo

Little is known about mechanisms of regulation of cysteine dioxygenase (CDO), gamma-glutamylcysteine synthetase (GCS), and cysteine-sulfinate decarboxylase (CSDC) in response to diet. Enzyme activity and Western and Northern or dot blot analyses were conducted on liver samples from rats fed a basal low-protein diet or diets with graded levels of protein or methionine for 2 wk. Higher levels of CDO activity and CDO protein but not of CDO mRNA were observed in liver of rats fed methionine or protein-supplemented diets, indicating that CDO activity is regulated by changes in enzyme concentration. Lower concentrations of the heavy or catalytic subunit of GCS (GCS-HS) mRNA and protein, as well as a lower activity state of GCS-HS in rats fed methionine- or protein-supplemented diets, indicated that dietary regulation of GCS occurs by both pretranslational and posttranslational mechanisms. Lower CSDC activity, CSDC protein concentration, and CSDC mRNA concentration were found in rats fed the highest level of protein, and regulation appeared to involve changes in mRNA concentration. Regulation of key enzymes of cysteine metabolism in response to diet determines the use of cysteine for synthesis of its essential metabolites.Little is known about mechanisms of regulation of cysteine dioxygenase (CDO), γ-glutamylcysteine synthetase (GCS), and cysteine-sulfinate decarboxylase (CSDC) in response to diet. Enzyme activity and Western and Northern or dot blot analyses were conducted on liver samples from rats fed a basal low-protein diet or diets with graded levels of protein or methionine for 2 wk. Higher levels of CDO activity and CDO protein but not of CDO mRNA were observed in liver of rats fed methionine or protein-supplemented diets, indicating that CDO activity is regulated by changes in enzyme concentration. Lower concentrations of the heavy or catalytic subunit of GCS (GCS-HS) mRNA and protein, as well as a lower activity state of GCS-HS in rats fed methionine- or protein-supplemented diets, indicated that dietary regulation of GCS occurs by both pretranslational and posttranslational mechanisms. Lower CSDC activity, CSDC protein concentration, and CSDC mRNA concentration were found in rats fed the highest level of protein, and regulation appeared to involve changes in mRNA concentration. Regulation of key enzymes of cysteine metabolism in response to diet determines the use of cysteine for synthesis of its essential metabolites.

Synthesis of Amino Acid Cofactor in Cysteine Dioxygenase Is Regulated by Substrate and Represents a Novel Post-translational Regulation of Activity

Cysteine dioxygenase (CDO) catalyzes the conversion of cysteine to cysteinesulfinic acid and is important in the regulation of intracellular cysteine levels in mammals and in the provision of oxidized cysteine metabolites such as sulfate and taurine. Several crystal structure studies of mammalian CDO have shown that there is a cross-linked cofactor present in the active site of the enzyme. The cofactor consists of a thioether bond between the γ-sulfur of residue cysteine 93 and the aromatic side chain of residue tyrosine 157. The exact requirements for cofactor synthesis and the contribution of the cofactor to the catalytic activity of the enzyme have yet to be fully described. In this study, therefore, we explored the factors necessary for cofactor biogenesis in vitro and in vivo and examined what effect cofactor formation had on activity in vitro. Like other cross-linked cofactor-containing enzymes, formation of the Cys-Tyr cofactor in CDO required a transition metal cofactor (Fe2+) and O2. Unlike other enzymes, however, biogenesis was also strictly dependent upon the presence of substrate. Cofactor formation was also appreciably slower than the rates reported for other enzymes and, indeed, took hundreds of catalytic turnover cycles to occur. In the absence of the Cys-Tyr cofactor, CDO possessed appreciable catalytic activity, suggesting that the cofactor was not essential for catalysis. Nevertheless, at physiologically relevant cysteine concentrations, cofactor formation increased CDO catalytic efficiency by ∼10-fold. Overall, the regulation of Cys-Tyr cofactor formation in CDO by ambient cysteine levels represents an unusual form of substrate-mediated feed-forward activation of enzyme activity with important physiological consequences.