Thiol Dioxygenases Research Articles

A distinct co-expressed sulfurtransferase extends the physiological role of mercaptopropionate dioxygenase in Pseudomonas aeruginosa PAO1

Oxidation and assimilation of persulfides in bacteria is often catalyzed by a persulfide dioxygenase and sulfurtransferase in consecutive reactions. Enzymes responsible for the oxidation of persulfides have not been clearly defined in Pseudomonas aeruginosa PAO1. The characterized mercaptopropionate dioxygenase (MDO) in P. aeruginosa PAO1 has been proposed to catalyze the oxidation of 3-mercaptopropionate. However, the physiological role of MDO is uncertain given the expression of a sulfurtransferase (ST) enzyme on the same operon as the thiol dioxygenase. The st gene had a co-occurrence frequency with mdo of 0.94 demonstrating the co-expression and physiological link of the two genes. There are four tandem rhodanese domains in the ST enzyme with two of the domains containing potential catalytic Cys residues (Cys191 and Cys435) capable of forming a persulfide. Only Cys435 was accessible in thiol quantification assays, and results from H/D-X MS analyses further established the accessibility of the domain containing Cys435. Both thiosulfate and mercaptopyruvate served as sulfur donors to the ST enzyme, with Cys435 forming the persulfide intermediate. Kinetic investigations of MDO suggested the enzyme had a broader substrate specificity than previously identified, oxidizing both mercaptopropionate and mercaptopyruvate thiol and persulfide substrates. The results obtained from these investigations provide insight into the overall mechanism and physiological role of the mdo operon in sulfide oxidation and assimilation.

Cysteamine dioxygenase (ADO) governs cancer cell mitochondrial redox homeostasis through proline metabolism.

2-Aminoethanethiol dioxygenase (ADO) is a thiol dioxygenase that sulfinylates cysteamine and amino-terminal cysteines in polypeptides. The pathophysiological roles of ADO remain largely unknown. Here, we demonstrate that ADO expression represents a vulnerability in cancer cells, as ADO depletion led to loss of proliferative capacity and survival in cancer cells and reduced xenograft growth. In contrast, generation of the ADO knockout mouse revealed high tolerance for ADO depletion in adult tissues. To understand the mechanism underlying ADO's essentiality in cancer cells, we characterized the cell proteome and metabolome following depletion of ADO. This revealed that ADO depletion leads to toxic levels of polyamines which can be driven by ADO's substrate cysteamine. Polyamine accumulation in turn stimulated expression of proline dehydrogenase (PRODH) which resulted in mitochondrial hyperactivity and ROS production, culminating in cell toxicity. This work identifies ADO as a unique vulnerability in cancer cells, due to its essential role in maintenance of redox homeostasis through restraining polyamine levels and proline catabolism.

Cobalt(II)-Substituted Cysteamine Dioxygenase Oxygenation Proceeds through a Cobalt(III)-Superoxo Complex.

We investigated the metal-substituted catalytic activity of human cysteamine dioxygenase (ADO), an enzyme pivotal in regulating thiol metabolism and contributing to oxygen homeostasis. Our findings demonstrate the catalytic competence of cobalt(II)- and nickel(II)-substituted ADO in cysteamine oxygenation. Notably, Co(II)-ADO exhibited superiority over Ni(II)-ADO despite remaining significantly less active than the natural enzyme. Structural analyses through X-ray crystallography and cobalt K-edge excitation confirmed successful metal substitution with minimal structural perturbations. This provided a robust structural basis, supporting a conserved catalytic mechanism tailored to distinct metal centers. This finding challenges the proposed high-valent ferryl-based mechanism for thiol dioxygenases, suggesting a non-high-valent catalytic pathway in the native enzyme. Further investigation of the cysteamine-bound or a peptide mimic of N-terminus RGS5 bound Co(II)-ADO binary complex revealed the metal center's high-spin (S = 3/2) state. Upon reaction with O2, a kinetically and spectroscopically detectable intermediate emerged with a ground spin state of S = 1/2. This intermediate exhibits a characteristic 59Co hyperfine splitting (A = 67 MHz) structure in the EPR spectrum alongside UV-vis features, consistent with known low-spin Co(III)-superoxo complexes. This observation, unique for protein-bound thiolate-ligated cobalt centers in a protein, unveils the capacities for O2 activation in such metal environments. These findings provide valuable insights into the non-heme iron-dependent thiol dioxygenase mechanistic landscape, furthering our understanding of thiol metabolism regulation. The exploration of metal-substituted ADO sheds light on the intricate interplay between metal and catalytic activity in this essential enzyme.

A Nonheme Iron(III) Superoxide Complex Leads to Sulfur Oxygenation.

A new alkylthiolate-ligated nonheme iron complex, FeII(BNPAMe2S)Br (1), is reported. Reaction of 1 with O2 at -40 °C, or reaction of the ferric form with O2•- at -80 °C, gives a rare iron(III)-superoxide intermediate, [FeIII(O2)(BNPAMe2S)]+ (2), characterized by UV-vis, 57Fe Mössbauer, ATR-FTIR, EPR, and CSIMS. Metastable 2 then converts to an S-oxygenated FeII(sulfinate) product via a sequential O atom transfer mechanism involving an iron-sulfenate intermediate. These results provide evidence for the feasibility of proposed intermediates in thiol dioxygenases.

Improved resolution of 3-mercaptopropionate dioxygenase active site provided by ENDOR spectroscopy offers insight into catalytic mechanism

3-mercaptopropionate (3MPA) dioxygenase (MDO) is a mononuclear nonheme iron enzyme that catalyzes the O2-dependent oxidation of thiol-bearing substrates to yield the corresponding sulfinic acid. MDO is a member of the cysteine dioxygenase family of small molecule thiol dioxygenases and thus shares a conserved sequence of active site residues (Serine-155, Histidine-157, and Tyrosine-159), collectively referred to as the SHY-motif. It has been demonstrated that these amino acids directly interact with the mononuclear Fe-site, influencing steady-state catalysis, catalytic efficiency, O2-binding, and substrate coordination. However, the underlying mechanism by which this is accomplished is poorly understood. Here, pulsed electron paramagnetic resonance spectroscopy [1H Mims electron nuclear double resonance spectroscopy] is applied to validate density functional theory computational models for the MDO Fe-site simultaneously coordinated by substrate and nitric oxide (NO), (3MPA/NO)-MDO. The enhanced resolution provided by electron nuclear double resonance spectroscopy allows for direct observation of Fe-bound substrate conformations and H-bond donation from Tyr159 to the Fe-bound NO ligand. Further inclusion of SHY-motif residues within the validated model reveals a distinct channel restricting movement of the Fe-bound NO-ligand. It has been argued that the iron-nitrosyl emulates the structure of potential Fe(III)-superoxide intermediates within the MDO catalytic cycle. While the merit of this assumption remains unconfirmed, the model reported here offers a framework to evaluate oxygen binding at the substrate-bound Fe-site and possible reaction mechanisms. It also underscores the significance of hydrogen bonding interactions within the enzymatic active site.

Enzyme‐Catalyzed Oxidative Degradation of Ergothioneine

AbstractErgothioneine is a sulfur‐containing metabolite that is produced by bacteria and fungi, and is absorbed by plants and animals as a micronutrient. Ergothioneine reacts with harmful oxidants, including singlet oxygen and hydrogen peroxide, and may therefore protect cells against oxidative stress. Herein we describe two enzymes from actinobacteria that cooperate in the specific oxidative degradation of ergothioneine. The first enzyme is an iron‐dependent thiol dioxygenase that produces ergothioneine sulfinic acid. A crystal structure of ergothioneine dioxygenase from Thermocatellispora tengchongensis reveals many similarities with cysteine dioxygenases, suggesting that the two enzymes share a common mechanism. The second enzyme is a metal‐dependent ergothioneine sulfinic acid desulfinase that produces Nα‐trimethylhistidine and SO2. The discovery that certain actinobacteria contain the enzymatic machinery for O2‐dependent biosynthesis and O2‐dependent degradation of ergothioneine indicates that these organisms may actively manage their ergothioneine content.

Enzyme-Catalyzed Oxidative Degradation of Ergothioneine.

Ergothioneine is a sulfur-containing metabolite that is produced by bacteria and fungi, and is absorbed by plants and animals as a micronutrient. Ergothioneine reacts with harmful oxidants, including singlet oxygen and hydrogen peroxide, and may therefore protect cells against oxidative stress. Herein we describe two enzymes from actinobacteria that cooperate in the specific oxidative degradation of ergothioneine. The first enzyme is an iron-dependent thiol dioxygenase that produces ergothioneine sulfinic acid. A crystal structure of ergothioneine dioxygenase from Thermocatellispora tengchongensis reveals many similarities with cysteine dioxygenases, suggesting that the two enzymes share a common mechanism. The second enzyme is a metal-dependent ergothioneine sulfinic acid desulfinase that produces Nα-trimethylhistidine and SO2 . The discovery that certain actinobacteria contain the enzymatic machinery for O2 -dependent biosynthesis and O2 -dependent degradation of ergothioneine indicates that these organisms may actively manage their ergothioneine content.

Non-standard amino acid incorporation into thiol dioxygenases.

Thiol dioxygenases (TDOs) are non‑heme Fe(II)‑dependent enzymes that catalyze the O2-dependent oxidation of thiol substrates to their corresponding sulfinic acids. Six classes of TDOs have thus far been identified and two, cysteine dioxygenase (CDO) and cysteamine dioxygenase (ADO), are found in eukaryotes. All TDOs belong to the cupin superfamily of enzymes, which share a common β‑barrel fold and two cupin motifs: G(X)5HXH(X)3-6E(X)6G and G(X)5-7PXG(X)2H(X)3N. Crystal structures of TDOs revealed that these enzymes contain a relatively rare, neutral 3‑His iron‑binding facial triad. Despite this shared metal-binding site, TDOs vary greatly in their secondary coordination spheres. Site‑directed mutagenesis has been used extensively to explore the impact of changes in secondary sphere residues on substrate specificity and enzymatic efficiency. This chapter summarizes site-directed mutagenesis studies of eukaryotic TDOs, focusing on the tools and practicality of non‑standard amino acid incorporation.

Unveiling the mechanism of cysteamine dioxygenase: A combined HPLC-MS assay and metal-substitution approach.

Mammalian cysteamine dioxygenase (ADO), a mononuclear non-heme Fe(II) enzyme with three histidine ligands, plays a key role in cysteamine catabolism and regulation of the N-degron signaling pathway. Despite its importance, the catalytic mechanism of ADO remains elusive. Here, we describe an HPLC-MS assay for characterizing thiol dioxygenase catalytic activities and a metal-substitution approach for mechanistic investigation using human ADO as a model. Two proposed mechanisms for ADO differ in oxygen activation: one involving a high-valent ferryl-oxo intermediate. We hypothesized that substituting iron with a metal that has a disfavored tendency to form high-valent states would discriminate between mechanisms. This chapter details the expression, purification, preparation, and characterization of cobalt-substituted ADO. The new HPLC-MS assay precisely measures enzymatic activity, revealing retained reactivity in the cobalt-substituted enzyme. The results obtained favor the concurrent dioxygen transfer mechanism in ADO. This combined approach provides a powerful tool for studying other non-heme iron thiol oxidizing enzymes.

Oxygen-Sensing Protein Cysteamine Dioxygenase from Mandarin Fish Involved in the Arg/N-Degron Pathway and Siniperca chuatsi Rhabdovirus Infection.

Mammalia cysteamine (2-aminoethanethiol) dioxygenase (ADO) controls the stability of the regulator of G protein signaling 4 (RGS4) through the Cys branch of the Arg/N-degron pathway, thereby affecting the response of the body to hypoxia. However, the oxygen-sensing function of ADO remains unknown in teleost fish. Mandarin fish (Siniperca chuatsi) is one of the most important freshwater economic fishes in China. As the scale of the rearing density continues to increase, hypoxia has become an important factor threatening the growth of mandarin fish. Herein, the molecular characterization, the oxygen-sensing enzyme function, and the role in virus infection of ADO from mandarin fish (scADO) were explored. Bioinformation analysis results showed that scADO had all the molecular foundations for achieving thiol dioxygenase function: three histidine residues coordinated with Fe(II), PCO/ADO domain, and a "jelly roll" β-barrel structure. The expression pattern analysis showed that scAdo was highly expressed in the immune-related tissues, liver, and kidneys and responded to hypoxia on the expression level. Protein degradation experiment results revealed that scADO could lead to the degradation of RGS4 protein through the Cys branch of the Arg/N-degron pathway. Furthermore, the expression levels of scADO responded to fish virus infection. scADO could significantly promote the replication of Siniperca chuatsi rhabdovirus, and this was associated with its thiol dioxygenase activity. These findings not only demonstrate scADO as an oxygen-sensing protein in teleost fish, but are also of considerable importance for clarifying the contribution of the mechanism of hypoxia to the outbreaks of fish viruses.

Abstract 4783: ADO is essential for redox homeostasis in liver cancer

Abstract 2-Aminoethanethiol dioxygenase (ADO) is a thiol dioxygenase that plays a direct role in both metabolism and protein stability. Oxidation of the metabolite cysteamine produces hypotaurine, while oxidation of N-terminal cysteines targets protein substrates for N-degron pathway-mediated degradation. Despite these known functions, the (patho)physiological roles of ADO remain obscure. By analyzing TCGA datasets we discovered that high ADO expression is associated with poor outcome for patients with hepatocellular carcinoma (HCC) (HR 1.2, p<0.001). HCC is linked to viral hepatitis, alcohol, metabolic syndrome, non-alcoholic fatty liver disease and chronic inflammation. It is the third leading cause of cancer-related deaths worldwide and is significantly more common in males than females. With poor outcomes, there is a need to better understand HCC biology and vulnerabilities. To assess the functional roles of ADO, we created ADO knockout mice as well as two liver cancer cell line models where ADO was depleted by siRNA, doxycycline-inducible shRNA, or CRISPR/Cas9. While male ADO knockout mice were viable, fertile and healthy, depletion of ADO in liver cancer models significantly reduced cancer cell proliferation and colony formation. ADO was also essential for supporting xenograft growth when implanted subcutaneously in immune-compromised mice. Taken together, this suggested that ADO depletion represents a cancer-specific vulnerability. Comprehensive metabolomic, transcriptomic and proteomic characterization of HCC cells isogenic for ADO demonstrated that loss of ADO resulted in dysregulation of glutathione, ascorbate, polyamine and proline metabolism. Consistent with this, ADO depleted cells had high levels of reactive oxygen species (ROS) measured by CellROX flowcytometry, and an increased ratio of oxidized to reduced glutathione, indicative of oxidative stress. Since mitochondria represent a substantial source of ROS, we assessed mitochondrial mass and function using MitotrackerTM flow cytometry and Seahorse stress tests, respectively. ADO depleted cancer cells had higher mitochondrial mass and higher maximal respiration rates compared to control cells. Finally, exogenously supplied antioxidants could rescue the survival of HCC cells with ADO depletion, demonstrating that the observed loss of viability is due to oxidative stress. This work shows that ADO is essential for redox homeostasis in liver cancer models and suggests that interfering with ADO function may represent a novel targeting strategy for HCC. Citation Format: Sandy C-E Lee, Andrea H. Pyo, Helia Mohammadi, Ji Zhang, Anna Dvorkin-Gheva, Lucie Malbeteau, Stephen Chung, Shahbaz Khan, Thomas Kislinger, Julie A. Reisz, Courtney Jones, Marianne Koritzinsky. ADO is essential for redox homeostasis in liver cancer. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 4783.

Cyanide replaces substrate in obligate-ordered addition of nitric oxide to the non-heme mononuclear iron AvMDO active site.

Thiol dioxygenases are a subset of non-heme mononuclear iron oxygenases that catalyze the O2-dependent oxidation of thiol-bearing substrates to yield sulfinic acid products. Cysteine dioxygenase (CDO) and 3-mercaptopropionic acid (3MPA) dioxygenase (MDO) are the most extensively characterized members of this enzyme family. As with many non-heme mononuclear iron oxidase/oxygenases, CDO and MDO exhibit an obligate-ordered addition of organic substrate before dioxygen. As this substrate-gated O2-reactivity extends to the oxygen-surrogate, nitric oxide (NO), EPR spectroscopy has long been used to interrogate the [substrate:NO:enzyme] ternary complex. In principle, these studies can be extrapolated to provide information about transient iron-oxo intermediates produced during catalytic turnover with dioxygen. In this work, we demonstrate that cyanide mimics the native thiol-substrate in ordered-addition experiments with MDO cloned from Azotobacter vinelandii (AvMDO). Following treatment of the catalytically active Fe(II)-AvMDO with excess cyanide, addition of NO yields a low-spin (S = 1/2) (CN/NO)-Fe-complex. Continuous wave and pulsed X-band EPR characterization of this complex produced in wild-type and H157N variant AvMDO reveal multiple nuclear hyperfine features diagnostic of interactions within the first- and outer-coordination sphere of the enzymatic Fe-site. Spectroscopically validated computational models indicate simultaneous coordination of two cyanide ligands replaces the bidentate (thiol and carboxylate) coordination of 3MPA allowing for NO-binding at the catalytically relevant O2-binding site. This promiscuous substrate-gated reactivity of AvMDO with NO provides an instructive counterpoint to the high substrate-specificity exhibited by mammalian CDO for L-cysteine.

Kinetic Measurements to Investigate the Oxygen-Sensing Properties of Plant Cysteine Oxidases.

Enzymatic O2 sensors transduce the availability of O2 within the cell into a physiological, typically adaptive response. One such O2-sensing enzymatic family is the N-terminal cysteine dioxygenases in plants (plant cysteine oxidases [PCOs]). In vitro kineticstudies have determined the O2-sensing capacity of PCOs. Here we describe the rationale and experimental protocol for an assay with which the O2 sensitivity of Arabidopsis thaliana PCOs (AtPCOs) can be measured. We explain each step from the recombinant protein synthesis of AtPCOs to the steady-state kineticassays of AtPCOs for primary substrate and O2 from which kinetic parameters can be derived. The same techniques can be applied to other N-terminal cysteine thiol dioxygenases, e.g. 2-aminoethanethiol dioxygenase (ADO), and similar principles can be applied to determine kinetic characteristics of other oxygenase enzymes towards O2.

Differences in the Second Coordination Sphere Tailor the Substrate Specificity and Reactivity of Thiol Dioxygenases.

In recent years, considerable progress has been made toward elucidating the geometric and electronic structures of thiol dioxygenases (TDOs). TDOs catalyze the conversion of substrates with a sulfhydryl group to their sulfinic acid derivatives via the addition of both oxygen atoms from molecular oxygen. All TDOs discovered to date belong to the family of cupin-type mononuclear nonheme Fe(II)-dependent metalloenzymes. While most members of this enzyme family bind the Fe cofactor by two histidines and one carboxylate side chain (2-His-1-carboxylate) to provide a monoanionic binding motif, TDOs feature a neutral three histidine (3-His) facial triad. In this Account, we present a bioinformatics analysis and multiple sequence alignment that highlight the significance of the secondary coordination sphere in tailoring the substrate specificity and reactivity among the different TDOs. These insights provide the framework within which important structural and functional features of the distinct TDOs are discussed.The best studied TDO is cysteine dioxygenase (CDO), which catalyzes the conversion of cysteine to cysteine sulfinic acid in both eukaryotes and prokaryotes. Crystal structures of resting and substrate-bound mammalian CDOs revealed two surprising structural motifs in the first- and second coordination spheres of the Fe center. The first is the presence of the abovementioned neutral 3-His facial triad that coordinates the Fe ion. The second is the existence of a covalent cross-link between the sulfur of Cys93 and an ortho carbon of Tyr157 (mouse CDO numbering scheme). While the exact role of this cross-link remains incompletely understood, various studies established that it is needed for proper substrate Cys positioning and gating solvent access to the active site. Intriguingly, bacterial CDOs lack the Cys-Tyr cross-link; yet, they are as active as cross-linked eukaryotic CDOs.The other known mammalian TDO is cysteamine dioxygenase (ADO). Initially, it was believed that ADO solely catalyzes the oxidation of cysteamine to hypotaurine. However, it has recently been shown that ADO additionally oxidizes N-terminal cysteine (Nt-Cys) peptides, which indicates that ADO may play a much more significant role in mammalian physiology than was originally anticipated. Though predicted on the basis of sequence alignment, site-directed mutagenesis, and spectroscopic studies, it was not until last year that two crystal structures, one of wild-type mouse ADO (solved by us) and the other of a variant of nickel-substituted human ADO, finally provided direct evidence that this enzyme also features a 3-His facial triad. These structures additionally revealed several features that are unique to ADO, including a putative cosubstrate O2 access tunnel that is lined by two Cys residues. Disulfide formation under conditions of high O2 levels may serve as a gating mechanism to prevent ADO from depleting organisms of Nt-Cys-containing molecules.The combination of kinetic and spectroscopic studies in conjunction with structural characterizations of TDOs has furthered our understanding of enzymatic sulfhydryl substrate regulation. In this article, we take advantage of the fact that the ADO X-ray crystal structures provided the final piece needed to compare and contrast key features of TDOs, an essential family of metalloenzymes found across all kingdoms of life.

Abstract 1431: ADO contributes to tumour initiating phenotypes

Abstract 2-aminoethanethiol dioxygenase (ADO) is a thiol dioxygenase that plays a role in both metabolism and protein stability. ADO directly metabolizes cysteamine to produce hypotaurine and taurine in mammals. ADO has also been recently identified to promote oxygen dependent stability of a subset of substrates involved in the N-degron pathway in mammals (IL32, RGS4 and RGS5). The ability of ADO to target protein stability of signaling molecules suggests that it may have the potential to transduce rapid responses to hypoxia and affect tumour initiation and progression phenotypes. Here, we have successfully knocked down and knocked out ADO using two independent siRNAs and clustered regularly interspaced short palindromic repeats associated protein 9 (CRISPR-Cas9) system, respectively. We have assessed proliferation and migration through the Incucyte® ZOOM system by imaging cell confluency over time. Survival was assessed through a clonogenic assay. siRNA mediated knockdown of ADO in cervical (HeLa and SiHa), pancreatic (Panc1 and Capan2) and liver (SNU499 and Huh6) cancer cell lines drastically reduced proliferation, survival, and migration in normoxia. These results were also replicated in hypoxia (0.2% O2) across all 6 cell lines. Out of the 6 cell lines, the liver cancer cell lines were most drastically affected by the knockdown of ADO. This phenotype was replicated in the ADO KO cell lines. Taken together, these data suggest that expression of ADO may contribute to phenotypes that induce aggressive tumour phenotypes by targeting the stability of specific proteins and altering cellular metabolism in mammals. Citation Format: Sandy Che-Eun Serena Lee, Andrea Hye An Pyo, Marianne Koritzinsky. ADO contributes to tumour initiating phenotypes [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1431.

Charge Maintenance during Catalysis in Nonheme Iron Oxygenases.

Here, the choice of the first coordination shell of the metal center is analyzed from the perspective of charge maintenance in a binary enzyme-substrate complex and an O2-bound ternary complex in the nonheme iron oxygenases. Comparing homogentisate 1,2-dioxygenase and gentisate dioxygenase highlights the significance of charge maintenance after substrate binding as an important factor that drives the reaction coordinate. We then extend the charge analysis to several common types of nonheme iron oxygenases containing either a 2-His-1-carboxylate facial triad or a 3-His or 4-His ligand motif, including extradiol and intradiol ring-cleavage dioxygenases, thiol dioxygenases, α-ketoglutarate-dependent oxygenases, and carotenoid cleavage oxygenases. After forming the productive enzyme-substrate complex, the overall charge of the iron complex at the 0, +1, or +2 state is maintained in the remaining catalytic steps. Hence, maintaining a constant charge is crucial to promote the reaction of the iron center beginning from the formation of the Michaelis or ternary complex. The charge compensation to the iron ion is tuned not only by protein-derived carboxylate ligands but also by substrates. Overall, these analyses indicate that charge maintenance at the iron center is significant when all the necessary components form a productive complex. This charge maintenance concept may apply to most oxygen-activating metalloenzymes systems that do not draw electrons and protons step-by-step from a separate reactant, such as NADH, via a reductase. The charge maintenance perception may also be useful in proposing catalytic pathways or designing prototypical reactions using artificial or engineered enzymes for biotechnological applications.

Spectroscopic investigation of iron(III) cysteamine dioxygenase in the presence of substrate (analogs): implications for the nature of substrate-bound reaction intermediates.

Thiol dioxygenases (TDOs) are a class of metalloenzymes that oxidize various thiol-containing substrates to their corresponding sulfinic acids. Originally established by X-ray crystallography for cysteine dioxygenase (CDO), all TDOs are believed to contain a 3-histidine facial triad that coordinates the necessary Fe(II) cofactor. However, very little additional information is available for cysteamine dioxygenase (ADO), the only other mammalian TDO besides CDO. Previous spectroscopic characterizations revealed that ADO likely binds substrate cysteamine in a monodentate fashion, while a mass spectrometry study provided evidence that a thioether crosslink can form between Cys206 and Tyr208 (mouse ADO numbering). In the present study, we have used electronic absorption and electron paramagnetic resonance (EPR) spectroscopies to investigate the species formed upon incubation of Fe(III)ADO with sulfhydryl-containing substrates and the superoxide surrogates azide and cyanide. Our data reveal that azide is unable to coordinate to cysteamine-bound Fe(III)ADO, suggesting that the Fe(III) center lacks an open coordination site or azide competes with cysteamine for the same binding site. Alternatively, cyanide binds to either cysteamine- or Cys-bound Fe(III)ADO to yield a low-spin (S = 1/2) EPR signal that is distinct from that observed for cyanide/Cys-bound Fe(III)CDO, revealing differences in the active-site pockets between ADO and CDO. Finally, EPR spectra obtained for cyanide/cysteamine adducts of wild-type Fe(III)ADO and its Tyr208Phe variant are superimposable, implying that either an insignificant fraction of as-isolated wild-type enzyme is crosslinked or that formation of the thioether bond has minimal effects on the electronic structure of the iron cofactor.

Crystal structure of human cysteamine dioxygenase provides a structural rationale for its function as an oxygen sensor

Cysteamine dioxygenase (ADO) plays a vital role in regulating thiol metabolism and preserving oxygen homeostasis in humans by oxidizing the sulfur of cysteamine and N-terminal cysteine-containing proteins to their corresponding sulfinic acids using O2 as a cosubstrate. However, as the only thiol dioxygenase that processes both small-molecule and protein substrates, how ADO handles diverse substrates of disparate sizes to achieve various reactions is not understood. The knowledge gap is mainly due to the three-dimensional structure not being solved, as ADO cannot be directly compared with other known thiol dioxygenases. Herein, we report the first crystal structure of human ADO at a resolution of 1.78 Å with a nickel-bound metal center. Crystallization was achieved through both metal substitution and C18S/C239S double mutations. The metal center resides in a tunnel close to an entry site flanked by loops. While ADO appears to use extensive flexibility to handle substrates of different sizes, it also employs proline and proline pairs to maintain the core protein structure and to retain the residues critical for catalysis in place. This feature distinguishes ADO from thiol dioxygenases that only oxidize small-molecule substrates, possibly explaining its divergent substrate specificity. Our findings also elucidate the structural basis for ADO functioning as an oxygen sensor by modifying N-degron substrates to transduce responses to hypoxia. Thus, this work fills a gap in structure–function relationships of the thiol dioxygenase family and provides a platform for further mechanistic investigation and therapeutic intervention targeting impaired oxygen sensing.

Emerging roles for thiol dioxygenases as oxygen sensors.

Cysteine dioxygenases, 3-mercaptopropionate dioxygenases and mercaptosuccinate dioxygenases are all thiol dioxygenases (TDOs) that catalyse oxidation of thiol molecules to sulphinates. They are Fe(II)-dependent dioxygenases with a cupin fold that supports a 3xHis metal-coordinating triad at the active site. They also have other, broadly common features including arginine residues involved in substrate carboxylate binding and a conserved trio of residues at the active site featuring a tyrosine important in substrate binding catalysis. Recently, N-terminal cysteinyl dioxygenase enzymes (NCOs) have been identified in plants (plant cysteine oxidases, PCOs), while human 2-aminoethanethiol dioxygenase (ADO) has been shown to act as both an NCO and a small molecule TDO. Although the cupin fold and 3xHis Fe(II)-binding triad seen in the small molecule TDOs are conserved in NCOs, other active site features and aspects of the overall protein architecture are quite different. Furthermore, the PCOs and ADO appear to act as biological O2 sensors, as shown by kinetic analyses and hypoxic regulation of the stability of their biological targets (N-terminal cysteine oxidation triggers protein degradation via the N-degron pathway). Here, we discuss the emergence of these two subclasses of TDO including structural features that could dictate their ability to bind small molecule or polypeptide substrates. These structural features may also underpin the O2 -sensing capability of the NCOs. Understanding how these enzymes interact with their substrates, including O2 , could reveal strategies to manipulate their activity, relevant to hypoxic disease states and plant adaptive responses to flooding.

Structure of 3-mercaptopropionic acid dioxygenase with a substrate analog reveals bidentate substrate binding at the iron center

Thiol dioxygenases are a subset of nonheme iron oxygenases that catalyze the formation of sulfinic acids from sulfhydryl-containing substrates and dioxygen. Among this class, cysteine dioxygenases (CDOs) and 3-mercaptopropionic acid dioxygenases (3MDOs) are the best characterized, and the mode of substrate binding for CDOs is well understood. However, the manner in which 3-mercaptopropionic acid (3MPA) coordinates to the nonheme iron site in 3MDO remains a matter of debate. A model for bidentate 3MPA coordination at the 3MDO Fe-site has been proposed on the basis of computational docking, whereas steady-state kinetics and EPR spectroscopic measurements suggest a thiolate-only coordination of the substrate. To address this gap in knowledge, we determined the structure of Azobacter vinelandii 3MDO (Av3MDO) in complex with the substrate analog and competitive inhibitor, 3-hydroxypropionic acid (3HPA). The structure together with DFT computational modeling demonstrates that 3HPA and 3MPA associate with iron as chelate complexes with the substrate-carboxylate group forming an additional interaction with Arg168 and the thiol bound at the same position as in CDO. A chloride ligand was bound to iron in the coordination site assigned as the O2-binding site. Supporting HYSCORE spectroscopic experiments were performed on the (3MPA/NO)-bound Av3MDO iron nitrosyl (S = 3/2) site. In combination with spectroscopic simulations and optimized DFT models, this work provides an experimentally verified model of the Av3MDO enzyme–substrate complex, effectively resolving a debate in the literature regarding the preferred substrate-binding denticity. These results elegantly explain the observed 3MDO substrate specificity, but leave unanswered questions regarding the mechanism of substrate-gated reactivity with dioxygen.