Cysteine Desulfurization Research Articles

Anaerobic respiration of host-derived methionine sulfoxide protects intracellular Salmonella from the phagocyte NADPH oxidase

Intracellular Salmonella experiencing oxidative stress downregulates aerobic respiration. To maintain cellular energetics during periods of oxidative stress, intracellular Salmonella must utilize terminal electron acceptors of lower energetic value than molecular oxygen. We show here that intracellular Salmonella undergoes anaerobic respiration during adaptation to the respiratory burst of the phagocyte NADPH oxidase in macrophages and in mice. Reactive oxygen species generated by phagocytes oxidize methionine, generating methionine sulfoxide. Anaerobic Salmonella uses the molybdenum cofactor-containing DmsABC enzymatic complex to reduce methionine sulfoxide. The enzymatic activity of the methionine sulfoxide reductase DmsABC helps Salmonella maintain an alkaline cytoplasm that supports the synthesis of the antioxidant hydrogen sulfide via cysteine desulfuration while providing a source of methionine and fostering redox balancing by associated dehydrogenases. Our investigations demonstrate that nontyphoidal Salmonella responding to oxidative stress exploits the anaerobic metabolism associated with dmsABC gene products, a pathway that has accrued inactivating mutations in human-adapted typhoidal serovars.

An Iron-Catalyzed Protein Desulfurization Method Reminiscent of Aquatic Chemistry.

One pillar of protein chemical synthesis based on the application of ligation chemistries to cysteine is the group of reactions enabling the selective desulfurization of cysteine residues into alanines. Modern desulfurization reactions use a phosphine as a sink for sulfur under activation conditions involving the generation of sulfur-centered radicals. Here we show that cysteine desulfurization by a phosphine can be effected efficiently by micromolar concentrations of iron under aerobic conditions in hydrogen carbonate buffer, that is using conditions that are reminiscent of iron-catalyzed oxidation phenomena occurring in natural waters. Therefore, our work shows that chemical processes taking place in aquatic systems can be adapted to a chemical reactor for triggering a complex chemoselective transformation at the protein level, while minimizing the resort to harmful chemicals.

An Iron‐Catalyzed Protein Desulfurization Method Reminiscent of Aquatic Chemistry

AbstractOne pillar of protein chemical synthesis based on the application of ligation chemistries to cysteine is the group of reactions enabling the selective desulfurization of cysteine residues into alanines. Modern desulfurization reactions use a phosphine as a sink for sulfur under activation conditions involving the generation of sulfur‐centered radicals. Here we show that cysteine desulfurization by a phosphine can be effected efficiently by micromolar concentrations of iron under aerobic conditions in hydrogen carbonate buffer, that is using conditions that are reminiscent of iron‐catalyzed oxidation phenomena occurring in natural waters. Therefore, our work shows that chemical processes taking place in aquatic systems can be adapted to a chemical reactor for triggering a complex chemoselective transformation at the protein level, while minimizing the resort to harmful chemicals.

Threonine dehydratase enhances bacterial cadmium resistance via driving cysteine desulfuration and biomineralization of cadmium sulfide nanocrystals

Biomineralization is often used by microorganisms to sequester heavy metal ions and provides a potential means for remediating increasing levels of heavy metal pollution. Bacteria have been shown to utilize cysteine for the biomineralization of metal sulfide. Indeed, in the present study, the supplement of L-cysteine was found to significantly improve both cadmium resistance and removal abilities of a deep-sea bacterium Pseudomonas stutzeri 273 through cadmium sulfide (CdS) nanoparticle biomineralization. With a proteomic approach, threonine dehydratase of P. stutzeri 273 (psTD) was proposed to be a key factor enhancing bacterial cadmium resistance through catalyzing L-cysteine desulfuration, H2S generation and CdS nanoparticle biomineralization. Consistently, deletion of the gene encoding psTD in P. stutzeri 273 resulted in the decline of H2S generation, decrease of cadmium resistance, and reduction of cadmium removal ability, confirming the unique function of psTD directing the formation of CdS nanoparticles. Correspondingly, the single-enzyme biomineralization of CdS nanoparticle driven by psTD was further developed, and psTD was shown to act as a capping reagent for the mineralization reaction, which controlling the size and structure of nanocrystals. Our results provide important clues for the construction of engineered bacteria for cadmium bioremediation and widen the synthesis methods of nanomaterials.

Insights into multifaceted activities of CysK for therapeutic interventions.

CysK (O-acetylserine sulfhydrylase) is a pyridoxal-5' phosphate-dependent enzyme which catalyzes the second step of the de novo cysteine biosynthesis pathway by converting O-acetyl serine (OAS) into l-cysteine in the presence of sulfide. The first step of the cysteine biosynthesis involves formation of OAS from serine and acetyl CoA by CysE (serine acetyltransferase). Apart from role of CysK in cysteine biosynthesis, recent studies have revealed various additional roles of this enzyme in bacterial physiology. Other than the suggested regulatory role in cysteine production, other activities of CysK include involvement of CysK-in contact-dependent toxin activation in Gram-negative pathogens, as a transcriptional regulator of CymR by stabilizing the CymR-DNA interactions, in biofilm formation by providing cysteine and via another mechanism not yet understood, in ofloxacin and tellurite resistance as well as in cysteine desulfurization. Some of these activities involve binding of CysK to another cellular partner, where the complex is regulated by the availability of OAS and/or sulfide (H2S). The aim of this study is to present an overview of current knowledge of multiple functions performed by CysK and identifying structural features involved in alternate functions. Due to possible role in disease, promoting or inhibiting a "moonlighting" function of CysK could be a target for developing novel therapeutic interventions.

The role of sulfides in stress-induced changes of Eh in Escherichia coli cultures

Real-time monitoring of the state of bacterial cultures is important in both experiment and biotechnology. Using Eh and sulfide sensors, we demonstrated that the abrupt reversible reduction in Eh (Eh jump), occurring during transition of E. coli from exponential growth to starvation and antibiotic-induced stresses, is the result of sulfide excretion from the cells. Changes in the potential of sensors had a two-phase mode. The potential reduced within 10–15min and returned within 10–30min. In the parental strain, maximum amplitudes of Eh jumps (ΔEh) were 25±2mV, 57±6mV and 36±7mV under isoleucine starvation, glucose depletion and ciprofloxacin exposure that corresponded to 43±3nM, 96±5nM and 140±1nM of sulfide, respectively. In the glutathione-deficient mutant (ΔgshA), ΔEh values and sulfide concentration increased 1.5–4 times compared to the parent. Stress-induced sulfide excretion occurred in the background of inhibition of growth and respiration and a decrease in the membrane potential. The formation of sulfide caused by cysteine desulfurization may be related with maintaining of cysteine homeostasis under conditions of slow metabolism. There was a close relationship between transmembrane fluxes of sulfide, cysteine and glutathione.

P−B Desulfurization: An Enabling Method for Protein Chemical Synthesis and Site‐Specific Deuteration

AbstractCysteine‐mediated native chemical ligation is a powerful method for protein chemical synthesis. Herein, we report an unprecedentedly mild system (TCEP/NaBH4 or TCEP/LiBEt3H; TCEP=tris(2‐carboxyethyl)phosphine) for chemoselective peptide desulfurization to achieve effective protein synthesis via the native chemical ligation–desulfurization approach. This method, termed P−B desulfurization, features usage of common reagents, simplicity of operation, robustness, high yields, clean conversion, and versatile functionality compatibility with complex peptides/proteins. In addition, this method can be used for incorporating deuterium into the peptides after cysteine desulfurization by running the reaction in D2O buffer. Moreover, this method enables the clean desulfurization of peptides carrying post‐translational modifications, such as phosphorylation and crotonylation. The effectiveness of this method has been demonstrated by the synthesis of the cyclic peptides dichotomin C and E and synthetic proteins, including ubiquitin, γ‐synuclein, and histone H2A.

P-B Desulfurization: An Enabling Method for Protein Chemical Synthesis and Site-Specific Deuteration.

Cysteine-mediated native chemical ligation is a powerful method for protein chemical synthesis. Herein, we report an unprecedentedly mild system (TCEP/NaBH4 or TCEP/LiBEt3 H; TCEP=tris(2-carboxyethyl)phosphine) for chemoselective peptide desulfurization to achieve effective protein synthesis via the native chemical ligation-desulfurization approach. This method, termed P-B desulfurization, features usage of common reagents, simplicity of operation, robustness, high yields, clean conversion, and versatile functionality compatibility with complex peptides/proteins. In addition, this method can be used for incorporating deuterium into the peptides after cysteine desulfurization by running the reaction in D2 O buffer. Moreover, this method enables the clean desulfurization of peptides carrying post-translational modifications, such as phosphorylation and crotonylation. The effectiveness of this method has been demonstrated by the synthesis of the cyclic peptides dichotomin C and E and synthetic proteins, including ubiquitin, γ-synuclein, and histone H2A.

Biogenesis of Hydrogen Sulfide and Thioethers by Cystathionine Beta-Synthase.

The transsulfuration pathway enzymes cystathionine beta-synthase (CBS) and cystathionine gamma-lyase are thought to be the major source of hydrogen sulfide (H2S). In this study, we assessed the role of CBS in H2S biogenesis. We show that despite discouraging enzyme kinetics of alternative H2S-producing reactions utilizing cysteine compared with the canonical condensation of serine and homocysteine, our simulations of substrate competitions at biologically relevant conditions suggest that cysteine is able to partially compete with serine on CBS, thus leading to generation of appreciable amounts of H2S. The leading H2S-producing reaction is condensation of cysteine with homocysteine, while cysteine desulfuration plays a dominant role when cysteine is more abundant than serine and homocysteine is limited. We found that the serine-to-cysteine ratio is the main determinant of CBS H2S productivity. Abundance of cysteine over serine, for example, in plasma, allowed for up to 43% of CBS activity being responsible for H2S production, while excess of serine typical for intracellular levels effectively limited such activity to less than 1.5%. CBS also produced lanthionine from serine and cysteine and a third of lanthionine coming from condensation of two cysteines contributed to the H2S pool. Our study characterizes the H2S-producing potential of CBS under biologically relevant conditions and highlights the serine-to-cysteine ratio as the main determinant of H2S production by CBS in vivo. Our data clarify the function of CBS in H2S biogenesis and the role of thioethers as surrogate H2S markers. Antioxid. Redox Signal. 28, 311-323.

Estradiol of various saturation and its influence on hydrogen sulfide formation in the myocardium of female rats.

Hydrogen sulfide is signalling molecule that regulates vascular tone and myocardial contractility. Role of estradiol in hydrogen sulfide production in cardiovascular system of rats remains unclear and this becames research objective. Effect of estradiol on hydrogen sulfide level, cystathionine-γ -lyase activity and its kinetic parameters - Michaelis constant (Km) and maximum rate (Vmax) in rats’ heart was estimated. Experiments were conducted on 45 white nonlinear female rats. Deficit of sex hormones level in females was modulated by castration - ovariectomy and hormone replacement therapy with estradiol. It is shown that the production of hydrogen sulfide in the myocardium is determined by the level of estradiol: castration of females causes a significant decrease in myocardial hydrogen sulfide content, cystathionine-γ-lyase activity, maximum rate of hydrogen sulfide formation from cysteine in participation of cystathionine-γ-lyase, as well as increase in Michaelis constant of this enzyme in comparison with the controls. Hormone replacement therapy with estradiol brings nearer hydrogen sulfide level, cystathionine-γ-lyase activity and kinetic parameters of cysteine desulfuration reaction in the rats’ myocardium to the levels of the control group. Thus, estradiol is involved in regulation of hydrogen sulfide production in cardiovascular system of rats, namely it increases cystathionine-γ-lyase activity and its affinity to cysteine.

S9-1 Sulfurtransferases and their role in hydrogen sulfide generation

The rhodanese superfamily (EC 2.8.1.x) includes tandem-domain thiosulfate:cyanide sulfurtransferase (TST, EC 2.8.1.1) and the strictly related 3-mercaptopyruvate:cyanide sulfurtransferase (MPST, EC 2.8.1.2). TST is the key enzyme, in the active center of which the persulfide group is created. The highly reactive sulfane sulfur atom of this group can be transferred to a substrate, e.g., cyanide detoxification, or can be passed to another protein acceptor participating in different cellular processes, e.g., biosynthesis of cofactors or thionucleosides. MPST transfers the sulfur atom from mercaptopyruvate, a product of cysteine transamination, to acceptors, such as cyanide (forming thiocyanate) or sulfite (forming thiosulfate – a sulfane sulfur-containing compound). Several metabolic reactions are known to generate sulfane sulfur in vitro , but their relative importance in vivo is not yet known. Current knowledge about sulfur donors assumes that the primary source of sulfane sulfur in eukaryotic cells relies on l -cysteine desulfuration pathway. Gamma-cystathionase (CTH, EC 4.4.1.1), an enzyme on the pathway, participates in endogenous production of H 2 S from l -cysteine. A product of CTH reaction, thiocystine, has been suggested to serve as the sulfane sulfur donor substrate for rhodanese. The potential role of the sulfane sulfur pool as an H 2 S reservoir and factors influencing the activity of sulfane sulfur-generating enzymes are under debate. The current study demonstrates that the MPST-dependent mitochondrial H 2 S-producing pathway is affected by oxidative stress, what may attenuate the production of H 2 S, and leads to various pathophysiological conditions. The intriguing problem is to identify possible sulfane sulfur donor(s) and/or acceptor(s), and to determine the regulatory role of sulfur in cellular metabolism.

Enzymology of H2S Biogenesis, Decay and Signaling

Hydrogen sulfide (H2S), produced by the desulfuration of cysteine or homocysteine, functions as a signaling molecule in an array of physiological processes including regulation of vascular tone, the cellular stress response, apoptosis, and inflammation. The low steady-state levels of H2S in mammalian cells have been recently shown to reflect a balance between its synthesis and its clearance. The subversion of enzymes in the cytoplasmic trans-sulfuration pathway for producing H2S from cysteine and/or homocysteine versus producing cysteine from homocysteine, presents an interesting regulatory problem. It is not known under what conditions the enzymes operate in the canonical trans-sulfuration pathway and how their specificity is switched to catalyze the alternative H2S-producing reactions. Similarly, it is not known if and whether the mitochondrial enzymes, which oxidize sulfide and persulfide (or sulfane sulfur), are regulated to increase or decrease H2S or sulfane-sulfur pools. In this review, we focus on the enzymology of H2S homeostasis and discuss H2S-based signaling via persulfidation and thionitrous acid.

Mammalian Frataxin Controls Sulfur Production and Iron Entry during de Novo Fe4S4 Cluster Assembly

Iron-sulfur (Fe-S) cluster-containing proteins are essential components of cells. In eukaryotes, Fe-S clusters are synthesized by the mitochondrial iron-sulfur cluster (ISC) machinery and the cytosolic iron-sulfur assembly (CIA) system. In the mammalian ISC machinery, preassembly of the Fe-S cluster on the scaffold protein (ISCU) involves a cysteine desulfurase complex (NFS1/ISD11) and frataxin (FXN), the protein deficient in Friedreich's ataxia. Here, by comparing the biochemical and spectroscopic properties of quaternary (ISCU/NFS1/ISD11/FXN) and ternary (ISCU/NFS1/ISD11) complexes, we show that FXN stabilizes the quaternary complex and controls iron entry to the complex through activation of cysteine desulfurization. Furthermore, we show for the first time that in the presence of iron and L-cysteine, an [Fe(4)S(4)] cluster is formed within the quaternary complex that can be transferred to mammalian aconitase (mACO2) to generate an active enzyme. In the absence of FXN, although the ternary complex can assemble an Fe-S cluster, the cluster is inefficiently transferred to ACO2. Taken together, these data help to unravel further the Fe-S cluster assembly process and the molecular basis of Friedreich's ataxia.

CysK from Lactobacillus casei encodes a protein with O-acetylserine sulfhydrylase and cysteine desulfurization activity

A gene encoding an O-acetyl-L-serine sulfhydrylase (cysK) was cloned from Lactobacillus casei FAM18110 and expressed in Escherichia coli. The purified recombinant enzyme synthesized cysteine from sulfide and O-acetyl-L-serine at pH 5.5 and pH 7.4. At pH 7.4, the apparent K(M) for O-acetyl-L-serine (OAS) and sulfide were 0.6 and 6.7 mM, respectively. Furthermore, the enzyme showed cysteine desulfurization activity in the presence of dithiothreitol at pH 7.5, but not at pH 5.5. The apparent K(M) for L-cysteine was 0.7 mM. The synthesis of cystathionine from homocysteine and serine or OAS was not observed. When expressed in a cysMK mutant of Escherichia coli, the cloned gene complemented the cysteine auxotrophy of the mutant. These findings suggested that the gene product is mainly involved in cysteine biosynthesis in L. casei. Quantitative real-time PCR and a mass spectrometric assay based on selected reaction monitoring demonstrated that L. casei FAM18110 is constitutively overexpressing cysK.

Isd11p Protein Activates the Mitochondrial Cysteine Desulfurase Nfs1p Protein

Cysteine desulfurases perform pyridoxal phosphate (PLP)-dependent desulfuration of cysteine. The key steps of the enzymatic cycle include substrate binding to PLP, formation of a covalent persulfide intermediate at the active site cysteine, and transfer of sulfur to recipients for use in various metabolic pathways. In Saccharomyces cerevisiae, the cysteine desulfurase Nfs1p and an accessory protein, Isd11p, are found primarily in mitochondria, and both are essential for cell viability. Although cysteine desulfurases are conserved from bacteria to humans, Isd11p is found only in eukaryotes and not in prokaryotes. Here we show that Isd11p activates Nfs1p. The enzyme without Isd11p was inactive and did not form the [(35)S]persulfide intermediate from the substrate [(35)S]cysteine. Addition of Isd11p to inactive Nfs1p induced formation of the persulfide. Remarkably, in a two-step assay, [(35)S]cysteine could be bound to the inactive Nfs1p in a PLP-dependent manner, and the enzyme could be subsequently induced to form the persulfide by addition of Isd11p. A mutant form of Isd11p with the (15)LYK(17) motif changed to (15)AAA(17) was able to bind but failed to activate Nfs1p, thus separating these two functions of Isd11p. Finally, compared with Nfs1p with or without the bound Isd11p mutant, the Nfs1p·Isd11p complex was more resistant to inactivation by an alkylating agent. On the basis of these novel findings, we propose that interaction of Isd11p with Nfs1p activates the enzyme by inducing a conformational change, thereby promoting formation of the persulfide intermediate at the active site cysteine. Such a conformational change may protect the active site cysteine from alkylating agents.

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

Regulation of body cysteine levels is dependent on the regulation of cysteine dioxygenase (CDO), the first enzyme in the pathway for cysteine oxidation to taurine or pyruvate + sulfate. To gain insight into the physiological function of CDO, we generated CDO+/− mice and crossed them to generate CDO−/−, CDO+/−, and CDO+/+ mice. CDO null mice exhibited increased postnatal mortality, a marked growth deficit, and connective tissue pathology. CDO−/− mice had extremely low plasma and tissue taurine levels and somewhat elevated cysteine levels, consistent with the lack of flux through CDO‐dependent catabolic pathways. However, plasma sulfate levels were slightly higher in CDO−/− mice than in CDO+/− or CDO+/+ mice and tissue levels of acid‐labile sulfide were elevated, indicating an increase in cysteine catabolism by cysteine desulfuration pathways in the null mice. Supplementation of mice with taurine improved survival of male pups but otherwise had little effect on the phenotype of the CDO−/− mice. Because H2S has been identified as an important gaseous signaling molecule, pathology may be due to dysregulation of H2S production. Control of cysteine levels by regulation of CDO may be necessary to maintain low H2S levels and facilitate the use of H2S as a signaling molecule. Supported by NIH grant DK056649..

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.

Desulfurization of cysteine‐containing peptides resulting from sample preparation for protein characterization by mass spectrometry

AbstractIn this study, we have examined two cysteine modifications resulting from sample preparation for protein characterization by mass spectrometry (MS): (1) a previously observed conversion of cysteine into dehydroalanine, now found in the case of disulfide mapping and (2) a novel modification corresponding to conversion of cysteine into alanine. Using model peptides, the conversion of cysteine into dehydroalanine via β‐elimination of a disulfide bond was seen to result from the conditions of typical tryptic digestion (37°C, pH 7.0–9.0) without disulfide reduction and alkylation. Furthermore, the surprising conversion of cysteine into alanine was shown to occur by heating cysteine‐containing peptides in the presence of a phosphine (tris(2‐carboxyethyl)phosphine hydrochloride (TCEP)). The formation of alanine from cysteine, investigated by performing experiments in H2O or D2O, suggested a radical‐based desulfurization mechanism unrelated to β‐elimination. Importantly, an understanding of the mechanism and conditions favorable for cysteine desulfurization provides insight for the establishment of improved sample preparation procedures of protein analysis. Copyright © 2010 John Wiley & Sons, Ltd.

Development of Assay Methods for Endogenous Inorganic Sulfur Compounds and Sulfurtransferases and Evaluation of the Physiological Functions of Bound Sulfur

Inorganic sulfur compounds, such as S(2-), SO(3)(2-) and S(2)O(3)(2-), are produced from sulfur- containing amino acids as intermediary metabolites in mammalian tissues through complex pathways and are ultimately incorporated into sulfate. Reduced sulfur is also produced via the desulfuration of cysteine by several sulfurtransferases present in mammalian tissues; these enzymes include gamma-cystathionase (gamma-CST), and 3-mercaptopyruvate sulfurtransferase (3-MST). This reduced sulfur is then incorporated into pools of active reduced sulfur (sulfane sulfur; polysulfides, polythionates, thiosulfate, thiosulfonates and elemental sulfur) that are involved in the detoxication of cyanide and in the biosynthesis of iron-sulfur cluster. Sulfane sulfur is labile and is reduced to H(2)S by reducing agents. The physiological function of these sulfur species is less clear. We have found that a reduced sulfur species is commonly present in mammalian sera and tissues as a high molecular weight material and as both a high and a low molecular weight material, respectively; we designated this sulfur species as "bound sulfur." Bound sulfur can be easily liberated as sulfide by reduction with DTT. This review describes sensitive and specific assay method for determining the presence of inorganic sulfur compounds as well as bound sulfur and related sulfurtransferases in biological samples. The physiological functions of bound sulfur in rat tissues were also evaluated using these assay methods. Bound sulfur was found to be located primarily in the rat liver cytosolic fraction in the form of high molecular weight components. The capacity of bound sulfur production was enriched in the cytosol fraction and depended on gamma-CST. Bound sulfur also affected redox regulation by modifying active thiol residues in some liver cytosol enzymes and effectively inhibited cytochrome P-450-dependent lipid peroxidation induced by CCl(4) and t-BuOOH.

Characterization of Arabidopsis thaliana SufE2 and SufE3: FUNCTIONS IN CHLOROPLAST IRON-SULFUR CLUSTER ASSEMBLY AND NAD SYNTHESIS

In this study we characterize two novel chloroplast SufE-like proteins from Arabidopsis thaliana. Other SufE-like proteins, including the previously described A. thaliana CpSufE, participate in sulfur mobilization for Fe-S biosynthesis through activation of cysteine desulfurization by NifS-like proteins. In addition to CpSufE, the Arabidopsis genome encodes two other proteins with SufE domains, SufE2 and SufE3. SufE2 has plastid targeting information. Purified recombinant SufE2 could activate the cysteine desulfurase activity of CpNifS 40-fold. SufE2 expression was flower-specific and high in pollen; we therefore hypothesize that SufE2 has a specific function in pollen Fe-S cluster biosynthesis. SufE3, also a plastid targeted protein, was expressed at low levels in all major plant organs. The mature SufE3 contains two domains, one SufE-like and one with similarity to the bacterial quinolinate synthase, NadA. Indeed SufE3 displayed both SufE activity (stimulating CpNifS cysteine desulfurase activity 70-fold) and quinolinate synthase activity. The full-length protein was shown to carry a highly oxygen-sensitive (4Fe-4S) cluster at its NadA domain, which could be reconstituted by its own SufE domain in the presence of CpNifS, cysteine and ferrous iron. Knock-out of SufE3 in Arabidopsis is embryolethal. We conclude that SufE3 is the NadA enzyme of A. thaliana, involved in a critical step during NAD biosynthesis.