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Martha H Stipanuk - One of the best experts on this subject based on the ideXlab platform.
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effects of a block in Cysteine catabolism on energy balance and fat metabolism in mice
Annals of the New York Academy of Sciences, 2016Co-Authors: Julie Niewiadomski, Lawrence L Hirschberger, Heather B Roman, James Q Zhou, Jason W Locasale, Martha H StipanukAbstract:: To gain further insights into the effects of elevated Cysteine levels on energy metabolism and the possible mechanisms underlying these effects, we conducted studies in Cysteine Dioxygenase (Cdo1)-null mice. Cysteine Dioxygenase (CDO) catalyzes the first step of the major pathway for Cysteine catabolism. When CDO is absent, tissue and plasma Cysteine levels are elevated, resulting in enhanced flux of Cysteine through desulfhydration reactions. When Cdo1-null mice were fed a high-fat diet, they gained more weight than their wild-type controls, regardless of whether the diet was supplemented with taurine. Cdo1-null mice had markedly lower leptin levels, higher feed intakes, and markedly higher abundance of hepatic stearoyl-CoA desaturase 1 (SCD1) compared to wild-type control mice, and these differences were not affected by the fat or taurine content of the diet. Thus, reported associations of elevated Cysteine levels with greater weight gain and with elevated hepatic Scd1 expression are also seen in the Cdo1-null mouse model. Hepatic accumulation of acylcarnitines suggests impaired mitochondrial β-oxidation of fatty acids in Cdo1-null mice. The strong associations of elevated Cysteine levels with excess H2 S production and impairments in energy metabolism suggest that H2 S signaling could be involved.
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From the ‡Division of Nutritional Sciences, §Macromolecular Diffraction Facility at Cornell High Energy Synchrotron Source
2015Co-Authors: Martha H StipanukAbstract: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 path-way. Cysteine Dioxygenase is a member of the cupin superfamily of proteins. The crystal structure of recombinant rat Cysteine dioxyge-nase has been determined to 1.5-A ̊ resolution, and these results confirm the canonical cupin-sandwich fold and the rare cysteinyl-tyrosine 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) metallo-center present in the murine structure, crystallization of a catalyti-cally competent preparation of rat Cysteine Dioxygenase revealed a novel tetrahedrally coordinatedmononuclear iron center involving three histidines (His86, His88, and His140) and a water molecule
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primary hepatocytes from mice lacking Cysteine Dioxygenase show increased Cysteine concentrations and higher rates of metabolism of Cysteine to hydrogen sulfide and thiosulfate
Amino Acids, 2014Co-Authors: Jakub Krijt, Lawrence L Hirschberger, Heather B Roman, Halina Jurkowska, Kiyoshi Sasakura, Tetsuo Nagano, Kenjiro Hanaoka, Martha H StipanukAbstract:The oxidation of Cysteine in mammalian cells occurs by two routes: a highly regulated direct oxidation pathway in which the first step is catalyzed by Cysteine Dioxygenase (CDO) and by desulfhydration-oxidation pathways in which the sulfur is released in a reduced oxidation state. To assess the effect of a lack of CDO on production of hydrogen sulfide (H2S) and thiosulfate (an intermediate in the oxidation of H2S to sulfate) and to explore the roles of both cystathionine γ-lyase (CTH) and cystathionine β-synthase (CBS) in Cysteine desulfhydration by liver, we investigated the metabolism of Cysteine in hepatocytes isolated from Cdo1-null and wild-type mice. Hepatocytes from Cdo1-null mice produced more H2S and thiosulfate than did hepatocytes from wild-type mice. The greater flux of Cysteine through the Cysteine desulfhydration reactions catalyzed by CTH and CBS in hepatocytes from Cdo1-null mice appeared to be the consequence of their higher Cysteine levels, which were due to the lack of CDO and hence lack of catabolism of Cysteine by the Cysteinesulfinate-dependent pathways. Both CBS and CTH appeared to contribute substantially to Cysteine desulfhydration, with estimates of 56 % by CBS and 44 % by CTH in hepatocytes from wild-type mice, and 63 % by CBS and 37 % by CTH in hepatocytes from Cdo1-null mice.
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the Cysteine dioxgenase knockout mouse altered Cysteine metabolism in nonhepatic tissues leads to excess h2s hs production and evidence of pancreatic and lung toxicity
Antioxidants & Redox Signaling, 2013Co-Authors: Heather B Roman, Jakub Krijt, Viktor Kožich, Alessandro Valli, Lawrence L Hirschberger, Martha H StipanukAbstract:Abstract Aims: To define the consequences of loss of Cysteine Dioxygenase (CDO) on Cysteine metabolism at the tissue level, we determined levels of relevant metabolites and enzymes and evidence of H2S/HS− (gaseous hydrogen sulfide and its conjugate base) toxicity in liver, pancreas, kidney, and lung of CDO−/− mice that were fed either a taurine-free or taurine-supplemented diet. Results: CDO−/− mice had low tissue and serum taurine and hypotaurine levels and high tissue levels of Cysteine, consistent with the loss of CDO. CDO−/− mice had elevated urinary excretion of thiosulfate, high tissue and serum cystathionine and lanthionine levels, and evidence of inhibition and destabilization of cytochrome c oxidase, which is consistent with excess production of H2S/HS−. Accumulation of cystathionine and lanthionine appeared to result from cystathionine β-synthase (CBS)-mediated Cysteine desulfhydration. Very high levels of hypotaurine in pancreas of wild-type mice and very high levels of cystathionine and lanthi...
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Nutrient Metabolism Variations in Dietary Protein but Not in Dietary Fat Plus Cellulose or Carbohydrate Levels Affect Cysteine Metabolism in Rat Isolated Hepatocytes1'2
2013Co-Authors: Deborah L Bella, Young Hye, Martha H StipanukAbstract:ABSTRACT To determine if previously observed ef fects of dietary protein on hepatic Cysteine metabolism were due specifically to increases in dietary protein or to the accompanying decreases in dietary carbohydrate, two experiments were conducted. In one experiment, rats were fed diets that contained different levels of protein vs. an isocaloric mixture of fat + cellulose and a constant amount of carbohydrate. In the other, rats were fed diets that contained a constant amount of pro tein but different levels of carbohydrate vs. an isocaloric mixture of fat + cellulose. Diets were fed for 2-3 wk and hepatocytes were then isolated. Hepatic Cysteine Dioxygenase activity increased and Cysteinesulfinate decarboxylase and y-glutamylCysteine synthetase ac tivities decreased in a stepwise manner when protein was added to the diet at the expense of fat + cellulose. Changes in Cysteine Dioxygenase, Cysteinesulfinate de carboxylase and y-glutamylCysteine synthetase activi ties were consistent with changes in rates of Cysteine catabolism, taurine production and glutathione synthe sis, respectively, by intact hepatocytes incubated with 0.2 mmol/L Cysteine. When the carbohydrate to fat + cellulose ratio was varied, but the protein level was held constant, little or no change in enzyme activities or lev els of metabolite production was observed. Regulation of the activities of enzymes involved in Cysteine metabo lism is predominantly due to changes in dietary protein intake and not to the associated changes in intake of other dietary macronutrients. J. Nutr. 126: 2179
Brad S Pierce - One of the best experts on this subject based on the ideXlab platform.
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Oxidative Uncoupling in Cysteine Dioxygenase Is Gated by a Proton-Sensitive Intermediate
2014Co-Authors: Joshua K. Crowell, Brad S PierceAbstract:Cysteine Dioxygenase (CDO) is a non-heme mononuclear iron enzyme that catalyzes the O2-dependent oxidation of l-Cysteine (Cys) to produce Cysteine sulfinic acid (CSA). This enzyme catalyzes the first committed step in Cys catabolism; thus, it is central to mammalian sulfur metabolism and redox homeostasis. Ironically, despite nearly 45 years of continued research on CDO, essentially no information has been reported with respect to its kinetic mechanism. In this work, the timing of chemical steps in the CDO kinetic mechanism is investigated by pH/pD-dependent steady-state kinetics and solvent isotope effects on kcat, kcat/KM, and (O2/CSA) coupling. Normal solvent kinetic isotope effects of 1.45 ± 0.05 and 2.0 ± 0.1 are observed in kcat–pL and kcat/KM–pL profiles, respectively. Proton inventory experiments within the pL-independent region (pL 8.5) suggest multiple solvent-exchangeable protons in flight for both kcat and kcat/KM data. The influence of solvent viscosity was also investigated to probe non-chemical steps and to verify that the apparent isotope effects were not attributable to increased solvent viscosity of D2O reactions relative to H2O. Although solvent viscosity did have a modest influence on kcat and kcat/KM, the response is not sufficient to account for the observed solvent isotope effects. This suggests that product release is only partially rate-limiting for CDO catalysis. Most crucially, proton inventory of (O2/CSA) coupling indicates that a proton-sensitive transition state directly follows O2 activation. Thus, protonation of a transient species preceding Cys oxidation is gated by protons in flight. This behavior provides valuable insight into the kinetically masked transients generated during catalysis
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second sphere interactions between the c93 y157 cross link and the substrate bound fe site influence the o2 coupling efficiency in mouse Cysteine Dioxygenase
Biochemistry, 2013Co-Authors: Elizabeth J Blaesi, Michael D Pecore, Joshua K Crowell, Brad S PierceAbstract:Cysteine Dioxygenase (CDO) is a non-heme iron enzyme that catalyzes the O2-dependent oxidation of l-Cysteine (l-Cys) to produce Cysteinesulfinic acid (CSA). Adjacent to the Fe site of CDO is a cova...
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second sphere interactions between the c93 y157 cross link and the substrate bound fe site influence the o2 coupling efficiency in mouse Cysteine Dioxygenase
Biochemistry, 2013Co-Authors: Elizabeth J Blaesi, Michael D Pecore, Joshua K Crowell, Brad S PierceAbstract:Cysteine Dioxygenase (CDO) is a non-heme iron enzyme that catalyzes the O₂-dependent oxidation of l-Cysteine (l-Cys) to produce Cysteinesulfinic acid (CSA). Adjacent to the Fe site of CDO is a covalently cross-linked Cysteine-tyrosine pair (C93-Y157). While several theories have been proposed for the function of the C93-Y157 pair, the role of this post-translational modification remains unclear. In this work, the steady-state kinetics and O₂/CSA coupling efficiency were measured for wild-type CDO and selected active site variants (Y157F, C93A, and H155A) to probe the influence of second-sphere enzyme-substrate interactions on catalysis. In these experiments, it was observed that both kcat and the O₂/CSA coupling efficiency were highly sensitive to the presence of the C93-Y157 cross-link and its proximity to the substrate carboxylate group. Complementary electron paramagnetic resonance (EPR) experiments were performed to obtain a more detailed understanding of the second-sphere interactions identified in O₂/CSA coupling experiments. Samples of the catalytically inactive substrate-bound Fe(III)-CDO species were treated with cyanide, resulting in a low-spin (S = ¹/₂) ternary complex. Remarkably, both the presence of the C93-Y157 pair and interactions with the Cys carboxylate group could be readily identified by perturbations to the rhombic EPR signal. Spectroscopically validated active site quantum mechanics/molecular mechanics and density functional theory computational models are provided to suggest a potential role for Y157 in the positioning of the substrate Cys in the active site and to verify the orientation of the g-tensor relative to the CDO Fe site molecular axis.
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Second-Sphere Interactions between the C93–Y157 Cross-Link and the Substrate-Bound Fe Site Influence the O2 Coupling Efficiency in Mouse Cysteine Dioxygenase
2013Co-Authors: Elizabeth J Blaesi, Michael D Pecore, Joshua K Crowell, Brad S PierceAbstract:Cysteine Dioxygenase (CDO) is a non-heme iron enzyme that catalyzes the O2-dependent oxidation of l-Cysteine (l-Cys) to produce Cysteinesulfinic acid (CSA). Adjacent to the Fe site of CDO is a covalently cross-linked Cysteine–tyrosine pair (C93–Y157). While several theories have been proposed for the function of the C93–Y157 pair, the role of this post-translational modification remains unclear. In this work, the steady-state kinetics and O2/CSA coupling efficiency were measured for wild-type CDO and selected active site variants (Y157F, C93A, and H155A) to probe the influence of second-sphere enzyme–substrate interactions on catalysis. In these experiments, it was observed that both kcat and the O2/CSA coupling efficiency were highly sensitive to the presence of the C93–Y157 cross-link and its proximity to the substrate carboxylate group. Complementary electron paramagnetic resonance (EPR) experiments were performed to obtain a more detailed understanding of the second-sphere interactions identified in O2/CSA coupling experiments. Samples of the catalytically inactive substrate-bound FeIII–CDO species were treated with cyanide, resulting in a low-spin (S = 1/2) ternary complex. Remarkably, both the presence of the C93–Y157 pair and interactions with the Cys carboxylate group could be readily identified by perturbations to the rhombic EPR signal. Spectroscopically validated active site quantum mechanics/molecular mechanics and density functional theory computational models are provided to suggest a potential role for Y157 in the positioning of the substrate Cys in the active site and to verify the orientation of the g-tensor relative to the CDO Fe site molecular axis
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single turnover of substrate bound ferric Cysteine Dioxygenase with superoxide anion enzymatic reactivation product formation and a transient intermediate
Biochemistry, 2011Co-Authors: Joshua Crawford, Brad S PierceAbstract:Cysteine Dioxygenase (CDO) is a non-heme mononuclear iron enzyme that catalyzes the O2-dependent oxidation of l-Cysteine (Cys) to produce Cysteine sulfinic acid (CSA). In this study we demonstrate that the catalytic cycle of CDO can be “primed” by one electron through chemical oxidation to produce CDO with ferric iron in the active site (FeIII-CDO, termed 2). While catalytically inactive, the substrate-bound form of FeIII-CDO (2a) is more amenable to interrogation by UV–vis and EPR spectroscopy than the ‘as-isolated’ FeII-CDO enzyme (1). Chemical-rescue experiments were performed in which superoxide (O2•-) anions were introduced to 2a to explore the possibility that a FeIII-superoxide species represents the first intermediate within the catalytic pathway of CDO. In principle, O2•– can serve as a suitable acceptor for the remaining 3-electrons necessary for CSA formation and regeneration of the active FeII-CDO enzyme (1). Indeed, addition of O2•– to 2a resulted in the rapid formation of a transient species...
Sam P. De Visser - One of the best experts on this subject based on the ideXlab platform.
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sulfoxide synthase versus Cysteine Dioxygenase reactivity in a nonheme iron enzyme
Journal of the American Chemical Society, 2017Co-Authors: Abayomi S Faponle, Florian P Seebeck, Sam P. De VisserAbstract:The sulfoxide synthase EgtB represents a unique family of nonheme iron enzymes that catalyze the formation of a C-S bond between N-α-trimethyl histidine and γ-glutamyl Cysteine, which is the key step in the biosynthesis of ergothioneine, an important amino acid related to aging. A controversy has arisen regarding its catalytic mechanism related to the function of the active-site Tyr377 residue. The biosynthesis of ergothioneine in EgtB shows structural similarities to Cysteine Dioxygenase which transfers two oxygen atoms to the thiolate group of Cysteine. The question, therefore, is how do EgtB enzymes catalyze the C-S bond-formation reaction, while also preventing a dioxygenation of its cysteinate substrate? In this work we present a quantum mechanics/molecular mechanics study into the mechanism of sulfoxide synthase enzymes as compared to Cysteine Dioxygenase enzymes and present pathways for both reaction channels in EgtB. We show that EgtB contains a conserved tyrosine residue that reacts via proton-coupled electron transfer with the iron(III)-superoxo species and creates an iron(III)-hydroperoxo intermediate, thereby preventing the possible thiolate dioxygenation side reaction. The nucleophilic C-S bond-formation step happens subsequently concomitant to relay of the proton of the iron(II)-hydroperoxo back to Tyr377. This is the rate-determining step in the reaction cycle and is followed by hydrogen-atom transfer from the CE1-H group of trimethyl histidine substrate to iron(II)-superoxo. In the final step, a quick and almost barrierless sulfoxidation leads to the sulfoxide product complexes. The work highlights a unique machinery and active-site setup of the enzyme that drives the sulfoxide synthase reaction.
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structure and mechanism leading to formation of the Cysteine sulfinate product complex of a biomimetic Cysteine Dioxygenase model
Chemistry: A European Journal, 2015Co-Authors: Madleen Sallmann, Devesh Kumar, Suresh Kumar, Petko Chernev, Joscha Nehrkorn, Alexander Schnegg, Holger Dau, Christian Limberg, Sam P. De VisserAbstract:Cysteine Dioxygenase is a unique nonheme iron enzyme that is involved in the metabolism of Cysteine in the body. It contains an iron active site with an unusual 3-His ligation to the protein, which contrasts with the structural features of common nonheme iron Dioxygenases. Recently, some of us reported a truly biomimetic model for this enzyme, namely a trispyrazolylborato iron(II) cysteinato complex, which not only has a structure very similar to the enzyme-substrate complex but also represents a functional model: Treatment of the model with dioxygen leads to Cysteine dioxygenation, as shown by isolating the Cysteine part of the product in the course of the work-up. However, little is known on the conversion mechanism and, so far, not even the structure of the actual product complex had been characterised, which is also unknown in case of the enzyme. In a multidisciplinary approach including density functional theory calculations and X-ray absorption spectroscopy, we have now determined the structure of the actual sulfinato complex for the first time. The Cys-SO2 (-) functional group was found to be bound in an η(2) -O,O-coordination mode, which, based on the excellent resemblance between model and enzyme, also provides the first support for a corresponding binding mode within the enzymatic product complex. Indeed, this is again confirmed by theory, which had predicted a η(2) -O,O-binding mode for synthetic as well as the natural enzyme.
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Mechanism of S-Oxygenation by a Cysteine Dioxygenase Model Complex
2012Co-Authors: Devesh Kumar, Narahari G. Sastry, David P. Goldberg, Sam P. De VisserAbstract:In this work, we present the first computational study on a biomimetic Cysteine Dioxygenase model complex, [FeII(LN3S)]+, in which LN3S is a tetradentate ligand with a bis(imino)pyridyl scaffold and a pendant arylthiolate group. The reaction mechanism of sulfur dioxygenation with O2 was examined by density functional theory (DFT) methods and compared with results obtained for Cysteine Dioxygenase. The reaction proceeds via multistate reactivity patterns on competing singlet, triplet, and quintet spin state surfaces. The reaction mechanism is analogous to that found for Cysteine Dioxygenase enzymes (Kumar, D.; Thiel, W.; de Visser, S. P. J. Am. Chem. Soc. 2011, 133, 3869–3882); hence, the computations indicate that this complex can closely mimic the enzymatic process. The catalytic mechanism starts from an iron(III)–superoxo complex and the attack of the terminal oxygen atom of the superoxo group on the sulfur atom of the ligand. Subsequently, the dioxygen bond breaks to form an iron(IV)–oxo complex with a bound sulfenato group. After reorganization, the second oxygen atom is transferred to the substrate to give a sulfinic acid product. An alternative mechanism involving the direct attack of dioxygen on the sulfur, without involving any iron–oxygen intermediates, was also examined. Importantly, a significant energetic preference for dioxygen coordinating to the iron center prior to attack at sulfur was discovered and serves to elucidate the function of the metal ion in the reaction process. The computational results are in good agreement with experimental observations, and the differences and similarities of the biomimetic complex and the enzymatic Cysteine Dioxygenase center are highlighted
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theoretical study on the mechanism of the oxygen activation process in Cysteine Dioxygenase enzymes
Journal of the American Chemical Society, 2011Co-Authors: Devesh Kumar, Walter Thiel, Sam P. De VisserAbstract: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 t...
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iron containing enzymes versatile catalysts of hydroxylation reactions in nature
2011Co-Authors: Sam P. De Visser, Devesh KumarAbstract:Nonheme iron(IV)-oxo oxidants in enzymes: Spectroscopic properties and reactivity patterns Heme iron(IV)-oxo oxidants in enzymes: Spectroscopic properties and reactivity patterns Mechanism and function of taurine/ -ketoglutarate Dioxygenase enzymes, an update Mechanism and function of Cysteine Dioxygenase enzymes Mechanism and function of heme peroxidase enzymes Mechanism and function of cytochrome P450 enzymes Biomimetic studies of mononuclear nonheme iron containing oxidants Biomimetic studies of mononuclear porphyrin containing oxidants Density functional calibration studies on iron-containing systems Density functional theory studies on isomerisation reactions catalyzed by cytochrome P450 enzymes Quantum mechanics/molecular mechanics studies of peroxidase enzymes Theoretical modelling of nonheme iron containing oxidants
Thomas C Brunold - One of the best experts on this subject based on the ideXlab platform.
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Spectroscopic and Computational Investigation of Iron(III) Cysteine Dioxygenase: Implications for the Nature of the Putative Superoxo-Fe(III) Intermediate
2015Co-Authors: Elizabeth J. Blaesi, Brian G Fox, Thomas C BrunoldAbstract:Cysteine Dioxygenase (CDO) is a mononuclear, non-heme iron-dependent enzyme that converts exogenous Cysteine (Cys) to Cysteine sulfinic acid using molecular oxygen. Although the complete catalytic mechanism is not yet known, several recent reports presented evidence for an Fe(III)-superoxo reaction intermediate. In this work, we have utilized spectroscopic and computational methods to investigate the as-isolated forms of CDO, as well as Cys-bound Fe(III)CDO, both in the absence and presence of azide (a mimic of superoxide). An analysis of our electronic absorption, magnetic circular dichroism, and electron paramagnetic resonance data of the azide-treated as-isolated forms of CDO within the framework of density functional theory (DFT) computations reveals that azide coordinates directly to the Fe(III), but not the Fe(II) center. An analogous analysis carried out for Cys-Fe(III)CDO provides compelling evidence that at physiological pH, the iron center is six coordinate, with hydroxide occupying the sixth coordination site. Upon incubation of this species with azide, the majority of the active sites retain hydroxide at the iron center. Nonetheless, a modest perturbation of the electronic structure of the Fe(III) center is observed, indicating that azide ions bind near the active site. Additionally, for a small fraction of active sites, azide displaces hydroxide and coordinates directly to the Cys-bound Fe(III) center to generate a low-spin (S = 1/2) Fe(III) complex. In the DFT-optimized structure of this complex, the central nitrogen atom of the azide moiety lies within 3.12 Å of the Cysteine sulfur. A similar orientation of the superoxide ligand in the putative Fe(III)-superoxo reaction intermediate would promote the attack of the distal oxygen atom on the sulfur of substrate Cys
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Spectroscopic and Computational Investigation of the H155A Variant of Cysteine Dioxygenase: Geometric and Electronic Consequences of a Third-Sphere Amino Acid Substitution
2015Co-Authors: Elizabeth J. Blaesi, Brian G Fox, Thomas C BrunoldAbstract:Cysteine Dioxygenase (CDO) is a mononuclear, non-heme iron(II)-dependent enzyme that utilizes molecular oxygen to catalyze the oxidation of l-Cysteine (Cys) to Cysteinesulfinic acid. Although the kinetic consequences of various outer-sphere amino acid substitutions have previously been assessed, the effects of these substitutions on the geometric and electronic structures of the active site remained largely unexplored. In this work, we have performed a spectroscopic and computational characterization of the H155A CDO variant, which was previously shown to display a rate of Cys oxidation ∼100-fold decreased relative to that of wild-type (WT) CDO. Magnetic circular dichroism and electron paramagnetic resonance spectroscopic data indicate that the His155 → Ala substitution has a significant effect on the electronic structure of the Cys-bound Fe(II)CDO active site. An analysis of these data within the framework of density functional theory calculations reveals that Cys-bound H155A Fe(II)CDO possesses a six-coordinate Fe(II) center, differing from the analogous WT CDO species in the presence of an additional water ligand. The enhanced affinity of the Cys-bound Fe(II) center for a sixth ligand in the H155A CDO variant likely stems from the increased level of conformational freedom of the Cysteine–tyrosine cross-link in the absence of the H155 imidazole ring. Notably, the nitrosyl adduct of Cys-bound Fe(II)CDO [which mimics the (O2/Cys)–CDO intermediate] is essentially unaffected by the H155A substitution, suggesting that the primary role played by the H155 side chain in CDO catalysis is to discourage the binding of a water molecule to the Cys-bound Fe(II)CDO active site
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spectroscopic and computational characterization of the no adduct of substrate bound fe ii Cysteine Dioxygenase insights into the mechanism of o2 activation
Biochemistry, 2013Co-Authors: Elizabeth J Blaesi, Jessica D Gardner, Brian G Fox, Thomas C BrunoldAbstract:Cysteine Dioxygenase (CDO) is a mononuclear nonheme iron(II)-dependent enzyme critical for maintaining appropriate Cysteine (Cys) and taurine levels in eukaryotic systems. Because CDO possesses both an unusual 3-His facial ligation sphere to the iron center and a rare Cys–Tyr cross-link near the active site, the mechanism by which it converts Cys and molecular oxygen to Cysteine sulfinic acid is of broad interest. However, as of yet, direct experimental support for any of the proposed mechanisms is still lacking. In this study, we have used NO as a substrate analogue for O2 to prepare a species that mimics the geometric and electronic structures of an early reaction intermediate. The resultant unusual S = 1/2 {FeNO}7 species was characterized by magnetic circular dichroism, electron paramagnetic resonance, and electronic absorption spectroscopies as well as computational methods including density functional theory and semiempirical calculations. The NO adducts of Cys- and selenoCysteine (Sec)-bound Fe(II)...
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Spectroscopic and Computational Characterization of the NO Adduct of Substrate-Bound Fe(II) Cysteine Dioxygenase: Insights into the Mechanism of O2 Activation
2013Co-Authors: Elizabeth J. Blaesi, Jessica D Gardner, Brian G Fox, Thomas C BrunoldAbstract:Cysteine Dioxygenase (CDO) is a mononuclear nonheme iron(II)-dependent enzyme critical for maintaining appropriate Cysteine (Cys) and taurine levels in eukaryotic systems. Because CDO possesses both an unusual 3-His facial ligation sphere to the iron center and a rare Cys–Tyr cross-link near the active site, the mechanism by which it converts Cys and molecular oxygen to Cysteine sulfinic acid is of broad interest. However, as of yet, direct experimental support for any of the proposed mechanisms is still lacking. In this study, we have used NO as a substrate analogue for O2 to prepare a species that mimics the geometric and electronic structures of an early reaction intermediate. The resultant unusual S = 1/2 {FeNO}7 species was characterized by magnetic circular dichroism, electron paramagnetic resonance, and electronic absorption spectroscopies as well as computational methods including density functional theory and semiempirical calculations. The NO adducts of Cys- and selenoCysteine (Sec)-bound Fe(II)CDO exhibit virtually identical electronic properties; yet, CDO is unable to oxidize Sec. To explore the differences in reactivity between Cys- and Sec-bound CDO, the geometries and energies of viable O2-bound intermediates were evaluated computationally, and it was found that a low-energy quintet-spin intermediate on the Cys reaction pathway adopts a different geometry for the Sec-bound adduct. The absence of a low-energy O2 adduct for Sec-bound CDO is consistent with our experimental data and may explain why Sec is not oxidized by CDO
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spectroscopic and computational characterization of substrate bound mouse Cysteine Dioxygenase nature of the ferrous and ferric Cysteine adducts and mechanistic implications
Biochemistry, 2010Co-Authors: Jessica D Gardner, Brad S Pierce, Brian G Fox, Thomas C BrunoldAbstract:Cysteine Dioxygenase (CDO) is a mononuclear non-heme Fe-dependent Dioxygenase that catalyzes the initial step of oxidative Cysteine catabolism. Its active site consists of an Fe(II) ion ligated by three histidine residues from the protein, an interesting variation on the more common 2-His-1-carboxylate motif found in many other non-heme Fe(II)-dependent enzymes. Multiple structural and kinetic studies of CDO have been carried out recently, resulting in a variety of proposed catalytic mechanisms; however, many open questions remain regarding the structure/function relationships of this vital enzyme. In this study, resting and substrate-bound forms of CDO in the Fe(II) and Fe(III) states, both of which are proposed to have important roles in this enzyme's catalytic mechanism, were characterized by utilizing various spectroscopic methods. The nature of the substrate/active site interactions was also explored using the Cysteine analogue selenoCysteine (Sec). Our electronic absorption, magnetic circular dichroism, and resonance Raman data exhibit features characteristic of direct S (or Se) ligation to both the high-spin Fe(II) and Fe(III) active site ions. The resulting Cys- (or Sec-) bound species were modeled and further characterized using density functional theory computations to generate experimentally validated geometric and electronic structure descriptions. Collectively, our results yield a more complete description of several catalytically relevant species and provide support for a reaction mechanism similar to that established for many structurally related 2-His-1-carboxylate Fe(II)-dependent Dioxygenases.
Lawrence L Hirschberger - One of the best experts on this subject based on the ideXlab platform.
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effects of a block in Cysteine catabolism on energy balance and fat metabolism in mice
Annals of the New York Academy of Sciences, 2016Co-Authors: Julie Niewiadomski, Lawrence L Hirschberger, Heather B Roman, James Q Zhou, Jason W Locasale, Martha H StipanukAbstract:: To gain further insights into the effects of elevated Cysteine levels on energy metabolism and the possible mechanisms underlying these effects, we conducted studies in Cysteine Dioxygenase (Cdo1)-null mice. Cysteine Dioxygenase (CDO) catalyzes the first step of the major pathway for Cysteine catabolism. When CDO is absent, tissue and plasma Cysteine levels are elevated, resulting in enhanced flux of Cysteine through desulfhydration reactions. When Cdo1-null mice were fed a high-fat diet, they gained more weight than their wild-type controls, regardless of whether the diet was supplemented with taurine. Cdo1-null mice had markedly lower leptin levels, higher feed intakes, and markedly higher abundance of hepatic stearoyl-CoA desaturase 1 (SCD1) compared to wild-type control mice, and these differences were not affected by the fat or taurine content of the diet. Thus, reported associations of elevated Cysteine levels with greater weight gain and with elevated hepatic Scd1 expression are also seen in the Cdo1-null mouse model. Hepatic accumulation of acylcarnitines suggests impaired mitochondrial β-oxidation of fatty acids in Cdo1-null mice. The strong associations of elevated Cysteine levels with excess H2 S production and impairments in energy metabolism suggest that H2 S signaling could be involved.
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primary hepatocytes from mice lacking Cysteine Dioxygenase show increased Cysteine concentrations and higher rates of metabolism of Cysteine to hydrogen sulfide and thiosulfate
Amino Acids, 2014Co-Authors: Jakub Krijt, Lawrence L Hirschberger, Heather B Roman, Halina Jurkowska, Kiyoshi Sasakura, Tetsuo Nagano, Kenjiro Hanaoka, Martha H StipanukAbstract:The oxidation of Cysteine in mammalian cells occurs by two routes: a highly regulated direct oxidation pathway in which the first step is catalyzed by Cysteine Dioxygenase (CDO) and by desulfhydration-oxidation pathways in which the sulfur is released in a reduced oxidation state. To assess the effect of a lack of CDO on production of hydrogen sulfide (H2S) and thiosulfate (an intermediate in the oxidation of H2S to sulfate) and to explore the roles of both cystathionine γ-lyase (CTH) and cystathionine β-synthase (CBS) in Cysteine desulfhydration by liver, we investigated the metabolism of Cysteine in hepatocytes isolated from Cdo1-null and wild-type mice. Hepatocytes from Cdo1-null mice produced more H2S and thiosulfate than did hepatocytes from wild-type mice. The greater flux of Cysteine through the Cysteine desulfhydration reactions catalyzed by CTH and CBS in hepatocytes from Cdo1-null mice appeared to be the consequence of their higher Cysteine levels, which were due to the lack of CDO and hence lack of catabolism of Cysteine by the Cysteinesulfinate-dependent pathways. Both CBS and CTH appeared to contribute substantially to Cysteine desulfhydration, with estimates of 56 % by CBS and 44 % by CTH in hepatocytes from wild-type mice, and 63 % by CBS and 37 % by CTH in hepatocytes from Cdo1-null mice.
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the Cysteine dioxgenase knockout mouse altered Cysteine metabolism in nonhepatic tissues leads to excess h2s hs production and evidence of pancreatic and lung toxicity
Antioxidants & Redox Signaling, 2013Co-Authors: Heather B Roman, Jakub Krijt, Viktor Kožich, Alessandro Valli, Lawrence L Hirschberger, Martha H StipanukAbstract:Abstract Aims: To define the consequences of loss of Cysteine Dioxygenase (CDO) on Cysteine metabolism at the tissue level, we determined levels of relevant metabolites and enzymes and evidence of H2S/HS− (gaseous hydrogen sulfide and its conjugate base) toxicity in liver, pancreas, kidney, and lung of CDO−/− mice that were fed either a taurine-free or taurine-supplemented diet. Results: CDO−/− mice had low tissue and serum taurine and hypotaurine levels and high tissue levels of Cysteine, consistent with the loss of CDO. CDO−/− mice had elevated urinary excretion of thiosulfate, high tissue and serum cystathionine and lanthionine levels, and evidence of inhibition and destabilization of cytochrome c oxidase, which is consistent with excess production of H2S/HS−. Accumulation of cystathionine and lanthionine appeared to result from cystathionine β-synthase (CBS)-mediated Cysteine desulfhydration. Very high levels of hypotaurine in pancreas of wild-type mice and very high levels of cystathionine and lanthi...
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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
American Journal of Physiology-endocrinology and Metabolism, 2011Co-Authors: Iori Ueki, Alessandro Valli, Heather B Roman, Krista Fieselmann, Rachel M Peters, Lawrence L HirschbergerAbstract: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 me...
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synthesis of amino acid cofactor in Cysteine Dioxygenase is regulated by substrate and represents a novel post translational regulation of activity
Journal of Biological Chemistry, 2008Co-Authors: John E Dominy, Lawrence L Hirschberger, Jesse Hwang, Sheng Zhang, Martha H StipanukAbstract:Abstract 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.
