NifS-like Proteins Research Articles

Exploring the selenium-over-sulfur substrate specificity and kinetics of a bacterial selenocysteine lyase

Selenium is a vital micronutrient in many organisms. While traces are required for microbial utilization, excess amounts are toxic; thus, selenium can be regarded as a biological double-edged sword. Selenium is chemically similar to the essential element sulfur, but curiously, evolution has selected the former over the latter for a subset of oxidoreductases. Enzymes involved in sulfur metabolism are less discriminate in terms of preventing selenium incorporation; however, its specific incorporation into selenoproteins reveals a highly discriminate process that is not completely understood. We have identified SclA, a NifS-like protein in the nosocomial pathogen, Enterococcus faecalis, and characterized its enzymatic activity and specificity for l-selenocysteine over l-cysteine. It is known that Asp-146 is required for selenocysteine specificity in the human selenocysteine lyase. Thus, using computational biology, we compared the bacterial and mammalian enzymes and identified His-100, an Asp-146 ortholog in SclA, and generated site-directed mutants in order to study the residue’s potential role in the l-selenocysteine discrimination mechanism. The proteins were overexpressed, purified, and characterized for their biochemical properties. All mutants exhibited varying Michaelis-Menten behavior towards l-selenocysteine, but His-100 was not found to be essential for this activity. Additionally, l-cysteine acted as a competitive inhibitor of all enzymes with higher affinity than l-selenocysteine. Finally, we discovered that SclA exhibited low activity with l-cysteine as a poor substrate regardless of mutations. We conclude that His-100 is not required for l-selenocysteine specificity, underscoring the inherent differences in discriminatory mechanisms between bacterial NifS-like proteins and mammalian selenocysteine lyases.

The alr2505 (osiS) gene from Anabaena sp. strain PCC7120 encodes a cysteine desulfurase induced by oxidative stress

NifS-like cysteine desulfurases are widespread enzymes involved in the mobilization of sulfur from cysteine. The genome of the filamentous diazotrophic cyanobacterium Anabaena PCC 7120 contains four open reading frames potentially encoding NifS-like proteins. One of them, alr2505, belongs to the pkn22 operon, which enables Anabaena to cope with oxidative stress. The Alr2505 protein was purified and found to share all the features characteristic of cysteine desufurases. This is the first NifS-like enzyme to be functionally characterized in this bacterium. On the basis of the transcriptional profiling of all nifS-like genes in Anabaena, it is concluded that alr2505 is the only cysteine desulfurase-encoding gene induced by oxidative stress. The function of Alr2505, which was termed OsiS, is discussed.

SufS protein from Haloferax volcanii involved in Fe-S cluster assembly in haloarchaea

NifS-like proteins are pyridoxal 5'-phosphate (PLP)-dependent enzymes involved in sulphur transfer metabolism. These enzymes have been catalogued as cysteine desulphurases (CDs) which catalyse the conversion of L-cysteine into L-alanine and an enzyme-bound persulphide radical. This reaction, assisted by different scaffold protein machineries, seems to be the main source of sulphur for the synthesis of essential cofactors of the [Fe-S] cluster. CDs genes have been detected in the tree domains of life, but, up until now, there has been no biochemical characterisation or study into the physiological role of this enzyme in haloarchaea. In this study, we have cloned, expressed and characterised a cysteine desulphurase (SufS) from Haloferax volcanii and demonstrated that this protein is able to reconstitute the [Fe-S] cluster of halophilic ferredoxin.

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.

Chloroplast iron-sulfur cluster protein maturation requires the essential cysteine desulfurase CpNifS.

NifS-like proteins provide the sulfur (S) for the formation of iron-sulfur (Fe-S) clusters, an ancient and essential type of cofactor found in all three domains of life. Plants are known to contain two distinct NifS-like proteins, localized in the mitochondria (MtNifS) and the chloroplast (CpNifS). In the chloroplast, five different Fe-S cluster types are required in various proteins. These plastid Fe-S proteins are involved in a variety of biochemical pathways including photosynthetic electron transport and nitrogen and sulfur assimilation. In vitro, the chloroplastic cysteine desulfurase CpNifS can release elemental sulfur from cysteine for Fe-S cluster biogenesis in ferredoxin. However, because of the lack of a suitable mutant allele, the role of CpNifS has not been studied thus far in planta. To study the role of CpNifS in Fe-S cluster biogenesis in vivo, the gene was silenced by using an inducible RNAi (interference) approach. Plants with reduced CpNifS expression exhibited chlorosis, a disorganized chloroplast structure, and stunted growth and eventually became necrotic and died before seed set. Photosynthetic electron transport and carbon dioxide assimilation were severely impaired in the silenced plant lines. The silencing of CpNifS decreased the abundance of all chloroplastic Fe-S proteins tested, representing all five Fe-S cluster types. Mitochondrial Fe-S proteins and respiration were not affected, suggesting that mitochondrial and chloroplastic Fe-S assembly operate independently. These findings indicate that CpNifS is necessary for the maturation of all plastidic Fe-S proteins and, thus, essential for plant growth.

Iron sulfur cluster biosynthesis. Human NFU mediates sulfide delivery to ISU in the final step of [2Fe–2S] cluster assembly

Human NFU forms a complex with NifS-like proteins and is a functionally competent reducing agent for cysteinyl persulfide bond cleavage, releasing inorganic sulfide for incorporation into the ISU-bound [2Fe-2S] cluster.

Selenocysteine Lyase.

Selenocysteine is a naturally occurring analog of cysteine in which the sulfur atom of the latter is replaced with selenium. This seleno-amino acid occurs as a specific component of various selenoproteins and selenium-dependent enzymes. Incorporation of selenocysteine into these proteins occurs cotranslationally as directed by the UGA codon. For this process, a special tRNA having an anticodon complimentary to UGA, tRNASec, is utilized. In Escherichia coli and related bacteria, this tRNA first is amino acylated with serine, and the seryl-tRNASec is converted to selenocysteyl-tRNASec. The specific incorporation of selenocysteine into proteins directed by the UGA codon depends on the synthesis of selenocysteyl-tRNASec. Included in the selenium delivery protein category are rhodaneses that mobilize selenium from inorganic sources and NIFS-like proteins that liberate elemental selenium from selenocysteine. The NIFS protein from Azotobacter vinelandii was found to serve as an efficient catalyst in vitro for delivery of selenium from free selenocysteine to Escherichia coli selenophosphate synthetase for selenophosphate formation. The widespread distribution of selenocysteine lyase in numerous bacterial species was reported and the bacterial enzymes, like the pig liver enzyme, required pyridoxal phosphate as cofactor. Three NIFS-like genes were isolated from E. coli by Esaki and coworkers and the expressed gene products were isolated and characterized. One of these NIFS-like proteins also exhibited a high preference for selenocysteine over cysteine. M. vannielii, an anaerobic methane-producing organism, that grows in a mineral medium containing formate as sole organic carbon source, synthesizes several specific selenoenzymes required for growth and energy production under these conditions.

Slr0077 of Synechocystis has cysteine desulfurase as well as cystine lyase activity

NifS-like proteins activate sulfur for a variety of biosynthetic purposes. The genome of the cyanobacterium Synechocystis contains 4 nifS-related sequences of which only the slr0077 gene seems to be essential. In this report the heterologous production of the Slr0077 protein, its purification, and catalytic properties are described. Slr0077 produces alanine as well as pyruvate from cyst(e)ine as substrate; the product ratio depends on the redox conditions. Alanine is the typical product of orthodox NifS proteins, pyruvate formation is typical of the cystine lyase of Synechocystis which is the most peculiar member of the NifS protein family. The specific activities of Slr0077 for both reaction types are low as compared to the prototypic enzymes. Upon reaction with thiol-alkylating agents Slr0077 is not readily inactivated unlike NifS. The unique properties of Slr0077 add to the emerging picture that the NifS family of proteins comprises enzymes with a variety of distinct reactivities.

Snapshots of the Cystine Lyase C-DES during Catalysis: STUDIES IN SOLUTION AND IN THE CRYSTALLINE STATE

The cystine lyase (C-DES) of Synechocystis is a pyridoxal-5'-phosphate-dependent enzyme distantly related to the family of NifS-like proteins. The crystal structure of an N-terminal modified variant has recently been determined. Herein, the reactivity of this enzyme variant was investigated spectroscopically in solution and in the crystalline state to follow the course of the reaction and to determine the catalytic mechanism on a molecular level. Using the stopped-flow technique, the reaction with the preferred substrate cystine was found to follow biphasic kinetics leading to the formation of absorbing species at 338 and 470 nm, attributed to the external aldimine and the alpha-aminoacrylate; the reaction with cysteine also exhibited biphasic behavior but only the external aldimine accumulated. The same reaction intermediates were formed in crystals as seen by polarized absorption microspectrophotometry, thus indicating that C-DES is catalytically competent in the crystalline state. The three-dimensional structure of the catalytically inactive mutant C-DES(K223A) in the presence of cystine showed the formation of an external aldimine species, in which two alternate conformations of the substrate were observed. The combined results allow a catalytic mechanism to be proposed involving interactions between cystine and the active site residues Arg-360, Arg-369, and Trp-251*; these residues reorient during the beta-elimination reaction, leading to the formation of a hydrophobic pocket that stabilizes the enolimine tautomer of the aminoacrylate and the cysteine persulfide product.

Characterization of a NifS-like chloroplast protein from Arabidopsis. Implications for its role in sulfur and selenium metabolism.

NifS-like proteins catalyze the formation of elemental sulfur (S) and alanine from cysteine (Cys) or of elemental selenium (Se) and alanine from seleno-Cys. Cys desulfurase activity is required to produce the S of iron (Fe)-S clusters, whereas seleno-Cys lyase activity is needed for the incorporation of Se in selenoproteins. In plants, the chloroplast is the location of (seleno) Cys formation and a location of Fe-S cluster formation. The goal of these studies was to identify and characterize chloroplast NifS-like proteins. Using seleno-Cys as a substrate, it was found that 25% to 30% of the NifS activity in green tissue in Arabidopsis is present in chloroplasts. A cDNA encoding a putative chloroplast NifS-like protein, AtCpNifS, was cloned, and its chloroplast localization was confirmed using immunoblot analysis and in vitro import. AtCpNIFS is expressed in all major tissue types. The protein was expressed in Escherichia coli and purified. The enzyme contains a pyridoxal 5' phosphate cofactor and is a dimer. It is a type II NifS-like protein, more similar to bacterial seleno-Cys lyases than to Cys desulfurases. The enzyme is active on both seleno-Cys and Cys but has a much higher activity toward the Se substrate. The possible role of AtCpNifS in plastidic Fe-S cluster formation or in Se metabolism is discussed.

The AtNFS2 gene from Arabidopsis thaliana encodes a NifS-like plastidial cysteine desulphurase

NifS-like proteins are cysteine desulphurases required for the mobilization of sulphur from cysteine. They are present in all organisms, where they are involved in iron-sulphur (Fe-S) cluster biosynthesis. In eukaryotes, these enzymes are present in mitochondria, which are the major site for Fe-S cluster assembly. The genome of the model plant Arabidopsis thaliana contains two putative NifS-like proteins. A cDNA corresponding to one of them was cloned by reverse-transcription PCR, and named AtNFS2. The corresponding transcript is expressed in many plant tissues. It encodes a protein highly related (75% similarity) to the slr0077-gene product from Synechocystis PCC 6803, and is predicted to be targeted to plastids. Indeed, a chimaeric AtNFS2-GFP fusion protein, containing one-third of AtNFS2 from its N-terminal end, was addressed to chloroplasts. Overproduction in Escherichia coli and purification of recombinant AtNFS2 protein enabled one to demonstrate that it bears a pyridoxal 5'-phosphate-dependent cysteine desulphurase activity in vitro, thus being the first NifS homologue characterized to date in plants. The putative physiological functions of this gene are discussed, including the attractive hypothesis of a possible role in Fe-S cluster assembly in plastids.

The iscS gene is essential for the biosynthesis of 2-selenouridine in tRNA and the selenocysteine-containing formate dehydrogenase H.

Three NifS-like proteins, IscS, CSD, and CsdB, from Escherichia coli catalyze the removal of sulfur and selenium from L-cysteine and L-selenocysteine, respectively, to form L-alanine. These enzymes are proposed to function as sulfur-delivery proteins for iron-sulfur cluster, thiamin, 4-thiouridine, biotin, and molybdopterin. Recently, it was reported that selenium mobilized from free selenocysteine is incorporated specifically into a selenoprotein and tRNA in vivo, supporting the involvement of the NifS-like proteins in selenium metabolism. We here report evidence that a strain lacking IscS is incapable of synthesizing 5-methylaminomethyl-2-selenouridine and its precursor 5-methylaminomethyl-2-thiouridine (mnm(5)s(2)U) in tRNA, suggesting that the sulfur atom released from L-cysteine by the action of IscS is incorporated into mnm(5)s(2)U. In contrast, neither CSD nor CsdB was essential for production of mnm(5)s(2)U and 5-methylaminomethyl-2-selenouridine. The lack of IscS also caused a significant loss of the selenium-containing polypeptide of formate dehydrogenase H. Together, these results suggest a dual function of IscS in sulfur and selenium metabolism.

Selenium is mobilized in vivo from free selenocysteine and is incorporated specifically into formate dehydrogenase H and tRNA nucleosides.

Selenophosphate synthetase (SPS), the selD gene product from Escherichia coli, catalyzes the biosynthesis of monoselenophosphate, AMP, and orthophosphate in a 1:1:1 ratio from selenide and ATP. It was recently demonstrated that selenium delivered from selenocysteine by an E. coli NifS-like protein could replace free selenide in the in vitro SPS assay for selenophosphate formation (G. M. Lacourciere, H. Mihara, T. Kurihara, N. Esaki, and T. C. Stadtman, J. Biol. Chem. 275:23769-23773, 2000). During growth of E. coli in the presence of 0.1 microM (75)SeO(3)(2-) and increasing amounts of L-selenocysteine, a concomitant decrease in (75)Se incorporation into formate dehydrogenase H and nucleosides of bulk tRNA was observed. This is consistent with the mobilization of selenium from L-selenocysteine in vivo and its use in selenophosphate formation. The ability of E. coli to utilize selenocysteine as a selenium source for selenophosphate biosynthesis in vivo supports the participation of the NifS-like proteins in selenium metabolism.

Utilization of selenocysteine as a source of selenium for selenophosphate biosynthesis.

Selenophosphate synthetase (SPS), the selD gene product from Escherichia coli, catalyzes the biosynthesis of monoselenophosphate from selenide and ATP. Characterization of selenophosphate synthetase revealed the determined K(m) value for selenide is far above the optimal concentration needed for growth and approached levels which are toxic. Selenocysteine lyase enzymes, which decompose selenocysteine to elemental selenium (Se(0)) and alanine, were considered as candidates for the control of free selenium levels in vivo. The ability of a lyase protein to generate Se(0) in the proximity of SPS maybe an attractive solution to selenium toxicity as well as the high K(m) value for selenide. Recently, three E. coli NifS-like proteins, CsdB, CSD, and IscS, were characterized. All three proteins exhibit lyase activity on L-cysteine and L-selenocysteine and produce sulfane sulfur, S(0), or Se(0) respectively. Each lyase can effectively mobilize Se(0) from L-selenocysteine for selenophosphate biosynthesis.

Escherichia coli NifS-like Proteins Provide Selenium in the Pathway for the Biosynthesis of Selenophosphate

Selenophosphate synthetase (SPS), the selD gene product from Escherichia coli, catalyzes the biosynthesis of monoselenophosphate, AMP, and orthophosphate in a 1:1:1 ratio from selenide and ATP. Kinetic characterization revealed the K(m) value for selenide approached levels that are toxic to the cell. Our previous demonstration that a Se(0)-generating system consisting of l-selenocysteine and the Azotobacter vinelandii NifS protein can replace selenide for selenophosphate biosynthesis in vitro suggested a mechanism whereby cells can overcome selenide toxicity. Recently, three E. coli NifS-like proteins, CsdB, CSD, and IscS, have been overexpressed and characterized. All three enzymes act on selenocysteine and cysteine to produce Se(0) and S(0), respectively. In the present study, we demonstrate the ability of each E. coli NifS-like protein to function as a selenium delivery protein for the in vitro biosynthesis of selenophosphate by E. coli wild-type SPS. Significantly, the SPS (C17S) mutant, which is inactive in the standard in vitro assay with selenide as substrate, was found to exhibit detectable activity in the presence of CsdB, CSD, or IscS and l-selenocysteine. Taken together the ability of the NifS-like proteins to generate a selenium substrate for SPS and the activation of the SPS (C17S) mutant suggest a selenium delivery function for the proteins in vivo.

Crystal structure of the cystine C-S lyase from Synechocystis: stabilization of cysteine persulfide for FeS cluster biosynthesis.

FeS clusters are versatile cofactors of a variety of proteins, but the mechanisms of their biosynthesis are still unknown. The cystine C-S lyase from Synechocystis has been identified as a participant in ferredoxin FeS cluster formation. Herein, we report on the crystal structure of the lyase and of a complex with the reaction products of cystine cleavage at 1.8- and 1.55-A resolution, respectively. The sulfur-containing product was unequivocally identified as cysteine persulfide. The reactive persulfide group is fixed by a hydrogen bond to His-114 in the center of a hydrophobic pocket and is thereby shielded from the solvent. Binding and stabilization of the cysteine persulfide represent an alternative to the generation of a protein-bound persulfide by NifS-like proteins and point to the general importance of persulfidic compounds for FeS cluster assembly.

Role of a NifS-like protein from the cyanobacterium Synechocystis PCC 6803 in the maturation of FeS proteins.

In Azotobacter vinelandii and Escherichia coli NifS or NifS-like proteins are involved in FeS protein assembly by mobilizing sulfur from free cysteine. This sulfur together with Fe(2+) is then incorporated into apo-FeS proteins to form an FeS center. A different activity termed C-DES [for cyst(e)ine desulfurylase] was recently isolated from the cyanobacterium Synechocystis PCC 6714 which also mobilized sulfur and which was able to incorporate the FeS center into apoferredoxin. In the genome of the cyanobacterium Synechocystis PCC 6803, there are three open reading frames (orfs) that are similar to NifS and one that is similar to C-DES, indicating that this bacterium might contain both activities, NifS and C-DES. One orf from Synechocystis PCC 6803 encoding a NifS-like protein, slr0387, was overexpressed in E. coli and purified. The molecular mass of the recombinant protein was determined to be about 82 kDa, indicating that it is a homodimer. The absorption spectrum was typical for PLP-containing proteins with an absorption maximum at 390 nm at pH 9.0 and at 425 nm at pH 6.5. The pH dependence of the absorption spectrum correlated with enzyme activity. Maximal activity measured as sulfide production was observed between pH 8.5 and 10. The activity decreased at lower pH values and was undetectable at pH 5.5. pH-dependent changes in the absorption spectrum and activity were attributed to protonation of the Schiff base formed by a lysine side chain and the PLP cofactor. Studies on substrate specificity demonstrated that cysteine derivatives other than cysteine methyl ester and cysteine-sulfinic acid could not serve as substrates for this enzyme. In particular, cystine was not a substrate for the Synechocystis NifS-like protein, whereas it is the best substrate for C-DES. In the presence of Fe(2+), cysteine, and a reductant, the NifS-like protein was able to produce holoferredoxin from apoferredoxin. The implications of two different activities for FeS center biosynthesis in Synechocystis are discussed.

Crystal structure of a NifS-like protein from Thermotoga maritima: implications for iron sulphur cluster assembly

NifS-like proteins are ubiquitous, homodimeric, proteins which belong to the α-family of pyridoxal-5′-phoshate dependent enzymes. They are proposed to donate elementary sulphur, generated from cysteine, via a cysteinepersulphide intermediate during iron sulphur cluster biosynthesis, an important albeit not well understood process. Here, we report on the crystal structure of a NifS-like protein from the hyperthermophilic bacterium Thermotoga maritima (tmNifS) at 2.0 Å resolution. The tmNifS is structured into two domains, the larger bearing the pyridoxal-5′-phosphate-binding active site, the smaller hosting the active site cysteine in the middle of a highly flexible loop, 12 amino acid residues in length. Once charged with sulphur the loop could possibly deliver S 0 directly to regions far remote from the protein. Based on the three-dimensional structures of the native as well as the substrate complexed form and on spectrophotometric results, a mechanism of sulphur activation is proposed. The His99, which stacks on top of the pyridoxal-5′-phosphate co-factor, is assigned a crucial role during the catalytic cycle by acting as an acid-base catalyst and is believed to have a p K a value depending on the co-factor redox state.

SufS is a NifS-like protein, and SufD is necessary for stability of the [2Fe-2S] FhuF protein in Escherichia coli.

Escherichia coli fhuF mutants, a sufS::MudI mutant, and a sufD::MudI mutant were found to have the same phenotype: the inability to use ferrioxamine B as an iron source in a plate assay. In addition, the sufS and sufD genes were shown to be regulated by the iron-dependent Fur repressor. Sequence analysis revealed that the sufS open reading frame corresponds to orf f406. The protein SufS belongs to the family of NifS-like proteins, which supply sulfur for [Fe-S] centers. The protein FhuF contains a [2Fe-2S] center. A mutation in the upstream sufD gene (orf f423) caused the same phenotype. The T7 expression system and a His tag allow the isolation in good yield of the FhuF protein from a wild-type strain. In contrast, overproduction of the protein in a DeltasufD strain failed. Radioactive labeling of N-His-FhuF with [35S]methionine showed that the protein was unstable in the DeltasufD mutant.

CDNA cloning and characterization of mouse nifS-like protein, m-Nfs1: mitochondrial localization of eukaryotic NifS-like proteins

We have isolated a mouse cDNA which shows significant sequence similarity to the yeast nifS-like gene (y- NFS1), and termed it m- Nfs1. The deduced protein sequence (459 amino acids long) has several characteristic features common to those of bacterial NifS proteins, but distinct from them by its amino-terminal extension which contains a typical mitochondrial targeting presequence. m-Nfs1 was found to be a soluble 47-kDa protein in the matrix fraction of mouse liver mitochondria. The m- Nfs1 gene was ubiquitously expressed in most tissues, suggesting its housekeeping function in vivo. We also found that the y-NFS1 protein was localized in the mitochondrial matrix in yeast cells. These results suggest that both eukaryotic NifS-like proteins may play some roles in mitochondrial functions.