Assembly of iron-sulfur clusters mediated by cysteine desulfurases,IscS, CsdB and CSD,from Escherichia coli

Cysteine desulfurase plays a principal role in the assembly of iron-sulfur clusters by mobilizing the sulfur atom of L-cysteine. The active site cysteine residue of the enzyme attacks the sulfur atom of L-cysteine to form a cysteine persulfide residue, and the substrate-derived sulfur atom of this residue is incorporated into iron-sulfur clusters. Escherichia coli has three cysteine desulfurases named IscS, CsdB and CSD. We found that each of them facilitates the formation of the iron-sulfur cluster of ferredoxin in vitro. Since IscU, an iron-sulfur protein of E. coli, is believed to function as a scaffold for the cluster assembly in vivo, we examined whether IscS, CsdB and CSD interact with IscU to deliver the sulfur atom to IscU. By surface plasmon resonance analysis, we found that only IscS interacts with IscU. We isolated the IscS/IscU complex, determined the residues involved in the formation of the complex, and obtained data suggesting that the sulfur transfer from IscS to IscU is initiated by the attack of Cys63 of IscU on the S gamma atom of the cysteine persulfide residue transiently produced on IscS.     

Transfer of sulfur from IscS to IscU during Fe/S cluster assembly

The cysteine desulfurase enzymes NifS and IscS provide sulfur for the biosynthesis of Fe/S proteins. NifU and IscU have been proposed to serve as template or scaffold proteins in the initial Fe/S cluster assembly events, but the mechanism of sulfur transfer from NifS or IscS to NifU or IscU has not been elucidated. We have employed [(35)S]cysteine radiotracer studies to monitor sulfur transfer between IscS and IscU from Escherichia coli and have used direct binding measurements to investigate interactions between the proteins. IscS catalyzed transfer of (35)S from [(35)S]cysteine to IscU in the absence of additional thiol reagents, suggesting that transfer can occur directly and without involvement of an intermediate carrier. Surface plasmon resonance studies and isothermal titration calorimetry measurements further revealed that IscU binds to IscS with high affinity (K(d) approximately 2 microm) in support of a direct transfer mechanism. Transfer was inhibited by treatment of IscU with iodoacetamide, and (35)S was released by reducing reagents, suggesting that transfer of persulfide sulfur occurs to cysteinyl groups of IscU. A deletion mutant of IscS lacking C-terminal residues 376-413 (IscSDelta376-413) displayed cysteine desulfurase activity similar to the full-length protein but exhibited lower binding affinity for IscU, decreased ability to transfer (35)S to IscU, and reduced activity in assays of Fe/S cluster assembly on IscU. The findings with IscSDelta376-413 provide additional support for a mechanism of sulfur transfer involving a direct interaction between IscS and IscU and suggest that the C-terminal region of IscS may be important for binding IscU.     

The SufE sulfur-acceptor protein contains a conserved core structure that mediates interdomain interactions in a variety of redox protein complexes

The isc and suf operons in Escherichia coli represent alternative genetic systems optimized to mediate the essential metabolic process of iron-sulfur cluster (Fe-S) assembly under basal or oxidative-stress conditions, respectively. Some of the proteins in these two operons share strong sequence homology, e.g. the cysteine desulfurases IscS and SufS, and presumably play the same role in the oxygen-sensitive assembly process. However, other proteins in these operons share no significant homology and occur in a mutually exclusive manner in Fe-S assembly operons in other organisms (e.g. IscU and SufE). These latter proteins presumably play distinct roles adapted to the different assembly mechanisms used by the two systems. IscU has three invariant cysteine residues that function as a template for Fe-S assembly while accepting a sulfur atom from IscS. SufE, in contrast, does not function as an Fe-S assembly template but has been suggested to function as a shuttle protein that uses a persulfide linkage to a single invariant cysteine residue to transfer a sulfur atom from SufS to an alternative Fe-S assembly template. Here, we present and analyze the 2.0A crystal structure of E.coli SufE. The structure shows that the persulfide-forming cysteine occurs at the tip of a loop with elevated B-factors, where its side-chain is buried from solvent exposure in a hydrophobic cavity located beneath a highly conserved surface. Despite the lack of sequence homology, the core of SufE shows strong structural similarity to IscU, and the sulfur-acceptor site in SufE coincides with the location of the cysteine residues mediating Fe-S cluster assembly in IscU. Thus, a conserved core structure is implicated in mediating the interactions of both SufE and IscU with the mutually homologous cysteine desulfurase enzymes present in their respective operons. A similar core structure is observed in a domain found in a variety of Fe-S cluster containing flavoenzymes including xanthine dehydrogenase, where it also mediates interdomain interactions. Therefore, the core fold of SufE/IscU has been adapted to mediate interdomain interactions in diverse redox protein systems in the course of evolution.     

嗜酸氧化亚铁硫杆菌ISC铁硫簇系统的合成

【目的】铁硫簇是最古老的一种氧化还原中心,它普遍存在于所有生命体内,在光合作用、呼吸作用和固氮作用这三个地球生命最基本的代谢途径中扮演着重要的角色。【方法】以嗜酸氧化亚铁硫杆菌(A.ferrooxidans ATCC 23270)基因组为模板,克隆表达其ISC铁硫簇组装的3个核心蛋白,IscS(半胱氨酸脱硫酶蛋白)、IscU(支架蛋白)和IscA(铁供体蛋白)。【结果】研究发现IscS能催化半胱氨酸脱硫,为铁硫簇的组装提供硫,支架蛋白IscU不具备结合铁的能力,IscA具有较强的铁结合能力。【结论】铁硫簇体外组装证明Fe-IscA在体外能将结合的铁传递给IscS,并在IscU上进行铁硫簇的组装。     

Structural Changes during Cysteine Desulfurase CsdA and Sulfur Acceptor CsdE Interactions Provide Insight into the trans-Persulfuration

In Escherichia coli, three cysteine desulfurases (IscS, SufS, and CsdA) initiate the delivery of sulfur for various biological processes such as the biogenesis of Fe-S clusters. The sulfur generated as persulfide on a cysteine residue of cysteine desulfurases is further transferred to Fe-S scaffolds (e.g. IscU) or to intermediate cysteine-containing sulfur acceptors (e.g. TusA, SufE, and CsdE) prior to its utilization. Here, we report the structures of CsdA and the CsdA-CsdE complex, which provide insight into the sulfur transfer mediated by the trans-persulfuration reaction. Analysis of the structures indicates that the conformational flexibility of the active cysteine loop in CsdE is essential for accepting the persulfide from the cysteine of CsdA. Additionally, CsdA and CsdE invoke a different binding mode than those of previously reported cysteine desulfurase (IscS) and sulfur acceptors (TusA and IscU). Moreover, the conservation of interaction-mediating residues between CsdA/SufS and CsdE/SufE further suggests that the SufS-SufE interface likely resembles that of CsdA and CsdE.     

The cysteine-desulfurase IscS promotes the production of the rhodanese RhdA in the persulfurated form

After heterologous expression in Escherichia coli, the Azotobacter vinelandii rhodanese RhdA is purified in a persulfurated form (RhdA-SSH). We identified l-cysteine as the most effective sulfur source in producing RhdA-SSH. An E. coli soluble extract was required for in vitro persulfuration of RhdA, and the addition of pyridoxal-5'-phosphate increased RhdA-SSH production, indicating a likely involvement of a cysteine desulfurase. We were able to show the formation of a covalent complex between IscS and RhdA. By combining a time-course fluorescence assay and mass spectrometry analysis, we demonstrated the transfer of sulfur from E. coli IscS to RhdA.     

Expression,purification and characterization of a cysteine desulfurase,IscS, from <Emphasis Type="Italic">Acidithiobacillus ferrooxidans</Emphasis>

Iron–sulfur clusters are one of the most common types of redox center in nature. Three proteins of IscS (a cysteine desulfurase), IscU (a scaffold protein) and IscA (an iron chaperon) encoded by the operon iscSUA are involved in the iron–sulfur cluster assembly in Acidithiobacillus ferrooxidans. In this study the gene of IscS from A. ferrooxidans ATCC 23270 was cloned and expressed in Escherichia coli, the protein was purified by one-step affinity chromatography to homogeneity. The molecular mass of recombinant IscS was 46 kDa by SDS-PAGE. The IscS was a pyridoxal phosphate-containing protein, that catalyzed the elimination of S from l-cysteine to yield l-alanine and elemental sulfur or H₂S, depending on whether or not a reducing agent was added to the reaction mixture. Jia Zeng and Yanfei Zhang contributed equally to this work.     

A sulfurtransferase is essential for activity of formate dehydrogenases in Escherichia coli

l-Cysteine desulfurases provide sulfur to several metabolic pathways in the form of persulfides on specific cysteine residues of an acceptor protein for the eventual incorporation of sulfur into an end product. IscS is one of the three Escherichia coli l-cysteine desulfurases. It interacts with FdhD, a protein essential for the activity of formate dehydrogenases (FDHs), which are iron/molybdenum/selenium-containing enzymes. Here, we address the role played by this interaction in the activity of FDH-H (FdhF) in E. coli. The interaction of IscS with FdhD results in a sulfur transfer between IscS and FdhD in the form of persulfides. Substitution of the strictly conserved residue Cys-121 of FdhD impairs both sulfur transfer from IscS to FdhD and FdhF activity. Furthermore, inactive FdhF produced in the absence of FdhD contains both metal centers, albeit the molybdenum cofactor is at a reduced level. Finally, FdhF activity is sulfur-dependent, as it shows reversible sensitivity to cyanide treatment. Conclusively, FdhD is a sulfurtransferase between IscS and FdhF and is thereby essential to yield FDH activity.     

Substitutions in an active site loop of Escherichia coli IscS result in specific defects in Fe-S cluster and thionucleoside biosynthesis in vivo

IscS catalyzes the fragmentation of l-cysteine to l-alanine and sulfane sulfur in the form of a cysteine persulfide in the active site of the enzyme. In Escherichia coli IscS, the active site cysteine Cys(328) resides in a flexible loop that potentially influences both the formation and stability of the cysteine persulfide as well as the specificity of sulfur transfer to protein substrates. Alanine-scanning substitution of this 14 amino acid region surrounding Cys(328) identified additional residues important for IscS function in vivo. Two mutations, S326A and L333A, resulted in strains that were severely impaired in Fe-S cluster synthesis in vivo. The mutant strains were deficient in Fe-S cluster-dependent tRNA thionucleosides (s(2)C and ms(2)i(6)A) yet showed wild type levels of Fe-S-independent thionucleosides (s(4)U and mnm(5)s(2)U) that require persulfide formation and transfer. In vitro, the mutant proteins were similar to wild type in both cysteine desulfurase activity and sulfur transfer to IscU. These results indicate that residues in the active site loop can selectively affect Fe-S cluster biosynthesis in vivo without detectably affecting persulfide delivery and suggest that additional assays may be necessary to fully represent the functions of IscS in Fe-S cluster formation.     

Biochemical characterization of iron-sulfur cluster assembly in the scaffold IscU of Escherichia coli

Iron-sulfur cluster is one of the most common prosthetic groups, and it functions in numerous biological processes. However, little is currently known about the mechanisms of iron-sulfur cluster biosynthesis. In this study, we cloned and purified iron-sulfur cluster assembly proteins from Escherichia coli and assembled the cluster in vitro. The results showed that the assembly of iron-sulfur cluster is completed in about 20 min. Although iron or sulfur binds with IscU equivalently, 2-fold amount of iron or cysteine compared with that of IscU is better for the cluster formation, while high concentrations of IscS (IscS/IscU > 1: 10) do not facilitate the cluster formation. Environmental pH plays an important role in iron-sulfur cluster assembly; the cluster was well assembled at pH 7.6–8.0, but was inhibited at pH less than 7.4. On supply of a catalytic amount of IscS (1/50 of IscU) and excess of other substrates, with increasing each of IscU, iron, or cysteine concentration, the iron-sulfur cluster assembly process developed from first order reaction, mixed order reaction to zero order reaction, and up to 64% of apo-IscU was converted to the [2Fe-2S] cluster-bound IscU under the optimal laboratory conditions.     

Facilitated transfer of IscU-[2Fe2S] clusters by chaperone-mediated ligand exchange

The scaffold protein IscU and molecular chaperones HscA and HscB play central roles in biological assembly of iron-sulfur clusters and maturation of iron-sulfur proteins. However, the structure of IscU-FeS complexes and the molecular mechanism whereby the chaperones facilitate cluster transfer to acceptor proteins are not well understood. We have prepared amino acid substitution mutants of Escherichia coli IscU in which potential ligands to the FeS cluster (Cys-37, Cys-63, His-105, and Cys-106) were individually replaced with alanine. The properties of the IscU-FeS complexes formed were investigated by measuring both their ability to transfer preformed FeS clusters to apo-ferredoxin and the activity of the IscU proteins in catalyzing cluster assembly on apo-ferredoxin using inorganic iron with inorganic sulfide or with IscS and cysteine as a sulfur source. The ability of the HscA/HscB chaperone system to accelerate ATP-dependent cluster transfer from each IscU substitution mutant to apo-ferredoxin was also determined. All of the mutants formed FeS complexes with a stoichiometry similar to the wild-type holo-protein, i.e., IscU(2)[2Fe2S], raising the possibility that different cluster ligation states may occur during iron-sulfur protein maturation. Spectroscopic properties of the mutants and the kinetics of transfer of performed IscU-FeS clusters to apo-ferredoxin indicate that the most stable form of holo-IscU involves iron coordination by Cys-63 and Cys-106. Results of studies on the ability of mutants to catalyze formation of holo-ferredoxin using iron and different sulfur sources were consistent with proposed roles for Cys-63 and Cys-106 in FeS cluster binding and also indicated an essential role for Cys-106 in sulfide transfer to IscU from IscS. Measurements of the ability of the chaperones HscA and HscB to facilitate cluster transfer from holo-IscU to apo-ferredoxin showed that only IscU(H105A) behaved similarly to wild-type IscU in exhibiting ATP-dependent stimulation of cluster transfer. IscU(C63A) and IscU(C106A) displayed elevated rates of cluster transfer in the ±ATP whereas IscU(C37A) exhibited low rates of cluster transfer ±ATP. In interpreting these findings, we propose that IscU(2)[2Fe2S] is able undergo structural isomerization to yield conformers having different cysteine residues bound to the cluster. On the basis of the crystal structure of HscA complexed with an IscU-derived peptide, we propose that the chaperone binds and stabilizes an isomer of IscU(2)[2Fe2S] in which the cluster is bound by cysteine residues 37 and 63 and that the [2Fe2S] cluster, being held less tightly than that coordinated by Cys-63 and Cys-106 in free IscU(2)[2Fe2S], is more readily transferred to acceptor proteins such as apo-ferredoxin.     

Interplay of IscA and IscU in biogenesis of iron-sulfur clusters

Increasing evidence suggests that sulfur in ubiquitous iron-sulfur clusters is derived from L-cysteine via cysteine desulfurases. In Escherichia coli, the major cysteine desulfurase activity for biogenesis of iron-sulfur clusters has been attributed to IscS. The gene that encodes IscS is a member of an operon iscSUA, which also encodes two highly conserved proteins: IscU and IscA. Previous studies suggested that both IscU and IscA may act as the iron-sulfur cluster assembly scaffold proteins. However, recent evidence indicated that IscA is an iron-binding protein that can provide iron for the iron-sulfur cluster assembly in IscU (Ding, H., Harrison, K., and Lu, J. (2005) J. Biol. Chem. 280, 30432-30437). To further elucidate the function of IscA in biogenesis of iron-sulfur clusters, we evaluate the iron-sulfur cluster binding activity of IscA and IscU under physiologically relevant conditions. When equal amounts of IscA and IscU are incubated with an equivalent amount of ferrous iron in the presence of IscS, L-cysteine and dithiothreitol, iron-sulfur clusters are assembled in IscU, but not in IscA, suggesting that IscU is a preferred iron-sulfur cluster assembly scaffold protein. In contrast, when equal amounts of IscA and IscU are incubated with an equivalent amount of ferrous iron in the presence of IscS and dithiothreitol but without L-cysteine, nearly all iron is bound to IscA. The iron binding in IscA appears to prevent the formation of the biologically inaccessible ferric hydroxide under aerobic conditions. Subsequent addition of L-cysteine efficiently mobilizes the iron center in IscA and transfers the iron for the iron-sulfur cluster assembly in IscU. The results suggest an intriguing interplay between IscA and IscU in which IscA acts as an iron chaperon that recruits "free" iron and delivers the iron for biogenesis of iron-sulfur clusters in IscU under aerobic conditions.     

Distinct iron-sulfur cluster assembly complexes exist in the cytosol and mitochondria of human cells

Iron-sulfur (Fe-S) clusters are cofactors found in many proteins that have important redox, catalytic or regulatory functions. In mammalian cells, almost all known Fe-S proteins are found in the mitochondria, but at least one is found in the cytosol. Here we report cloning of the human homologs to IscU and NifU, iron-binding proteins that play a critical role in Fe-S cluster assembly in bacteria. In human cells, alternative splicing of a common pre-mRNA results in synthesis of two proteins that differ at the N-terminus and localize either to the cytosol (IscU1) or to the mitochondria (IscU2). Biochemical analyses demonstrate that IscU proteins specifically associate with IscS, a cysteine desulfurase that is proposed to sequester inorganic sulfur for Fe-S cluster assembly. Protein complexes containing IscU and IscS can be found in the mitochondria as well as in the cytosol, implying that Fe-S cluster assembly takes place in multiple subcellular compartments in mammalian cells. The possible roles of the IscU proteins in mammalian cells and the potential implications of compartmentalization of Fe-S cluster assembly are discussed.     

Identification of IscU residues critical for de novo iron–sulfur cluster assembly

IscU is a central component of the ISC machinery and serves as a scaffold for the de novo assembly of iron–sulfur (Fe–S) clusters prior to their delivery to target apo‐Fe–S proteins. However, the molecular mechanism is not yet fully understood. In this study, we have conducted mutational analysis of E. coli IscU using the recently developed genetic complementation system of a mutant that can survive without Fe–S clusters. The Fe–S cluster ligands (C37, C63, H105, C106) and the proximal D39 and K103 residues are essential for in vivo function of IscU and could not be substituted with any other amino acids. Furthermore, we found that substitution of Y3, a strictly conserved residue among IscU homologs, abolished in vivo functions. Surprisingly, a second‐site suppressor mutation in IscS (A349V) reverted the defect caused by IscU Y3 substitutions. Biochemical analysis revealed that IscU Y3 was crucial for functional interaction with IscS and sulfur transfer between the two proteins. Our findings suggest that the critical role of IscU Y3 is linked to the conformational dynamics of the flexible loop of IscS, which is required for the ingenious sulfur transfer to IscU.     

Iron–sulfur cluster biosynthesis: characterization of IscU–IscS complex formation and a structural model for sulfide delivery to the [2Fe–2S] assembly site

Recent work on the bacterial iron–sulfur cluster (isc) family of gene products, and eukaryotic homologs, has advanced the molecular understanding of cellular mechanisms of iron–sulfur cluster biosynthesis. Members of the IscS family are pyridoxyl-5′-phosophate dependent proteins that deliver inorganic sulfide during assembly of the [2Fe–2S] cluster on the IscU scaffold protein. Herein it is demonstrated through calorimetry, fluorescence, and protein stability measurements that Thermotoga maritima IscS forms a 1:1 complex with IscU in a concentration-dependent manner (K _D varying from 6 to 34 μM, over an IscS concentration range of approximately 2–50 μM). Docking simulations of representative IscU and IscS proteins reveal critical contact surfaces at the N-terminal helix of IscU and a C-terminal loop comprising a chaperone binding domain. Consistent with the isothermal titration calorimetry results described here, an overall dominant contribution of charged surfaces with a change in the molar heat capacity of binding, ΔC _p ~ 199.8 kcal K⁻¹ mol⁻¹, is observed that accounts for approximately 10% of the total accessible surface area at the binding interface. Both apo and holo IscUs and homologs were found to bind to IscS in an enthalpically driven reaction with comparable K _D values. Both helix and loop regions are highly conserved among phylogenetically diverse organisms from a pool of archael, bacterial, fungal, and mammalian representatives.     

Network of protein-protein interactions among iron-sulfur cluster assembly proteins in Escherichia coli

The assembly of iron-sulfur (Fe-S) clusters is mediated by complex machinery which, in Escherichia coli, is encoded by the iscRSUA-hscBA-fdx-ORF3 gene cluster. Here, we demonstrate the network of protein-protein interactions among the components involved in the machinery. We have constructed (His)(6)-tagged versions of the components and identified their interacting partners that were co-purified from E. coli extracts with a Ni-affinity column. Direct associations of the defined pair of proteins were further examined in yeast cells using the two-hybrid system. In accord with the previous in vitro binding and kinetic experiments, interactions were observed for the combinations of IscS and IscU, IscU and HscB, IscU and HscA, and HscB and HscA. In addition, we have identified previously unreported interactions between IscS and Fdx, IscS and ORF3, IscA and HscA, and HscA and Fdx. We also found, by site-directed mutational analysis combined with the two-hybrid system, that two cysteine residues in IscU are essential for binding with HscB but not with IscS. Despite the complex network of interactions in various combinations of components, heteromultimeric complexes were not observed in our experiments except for the putative oligomeric form of IscU-IscS-ORF3. Thus, the sequential association and dissociation among the IscS, IscU, IscA, HscB, HscA, Fdx, and ORF3 proteins may be a critical process in the assembly of Fe-S clusters.     

半胱氨酸脱硫酶突变体H104Q,E156Q,D180G,Q183E和K206A通过改变对磷酸吡哆醛的结合影响酶活性

大肠杆菌半胱氨酸脱硫酶（cysteine desulfurase,IscS）是一类依赖磷酸吡哆醛（pyridoxal phosphate,PLP）的同质二聚体的酶.IscS能催化游离底物L-半胱氨酸脱硫,生成L-丙氨酸和单质硫.在此催化过程中,可形成与酶结合的半胱氨酸过硫化物中间物,并出现了7种具有不同特征性吸收峰的中间反应物.为了研究PLP的结合及中间反应物的形成及累积,对IscS中与PLP结合相关,及IscS半胱氨酸活性口袋中特定氨基酸残基位点（His104,Glu156,Asp180,Gln183和Lys206）进行定点突变,结果发现：1）IscS突变体H104Q、D180G、Q183E、K206A对PLP的结合能力具有不同程度的减弱,酶的活性明显降低甚至消失,PLP与蛋白结合的特异吸收峰消失,或发生明显偏移并出现新的吸收峰,且这些新出现的吸收峰又与蛋白形成的各种中间反应物的吸收峰一致|2）IscS突变体E156Q的活性增高,PLP与蛋白结合的吸收峰明显增加.这些结果都表明,IscS氨基酸残基可通过影响PLP的结合及质子转移引起催化过程中不同中间反应物的形成及累积,同时提高或降低蛋白的活性.     

Thiosulfate Transfer Mediated by DsrE/TusA Homologs from Acidothermophilic Sulfur-oxidizing Archaeon Metallosphaera cuprina

Conserved clusters of genes encoding DsrE and TusA homologs occur in many archaeal and bacterial sulfur oxidizers. TusA has a well documented function as a sulfurtransferase in tRNA modification and molybdenum cofactor biosynthesis in Escherichia coli, and DsrE is an active site subunit of the DsrEFH complex that is essential for sulfur trafficking in the phototrophic sulfur-oxidizing Allochromatium vinosum. In the acidothermophilic sulfur (S⁰)- and tetrathionate (S₄O₆²⁻)-oxidizing Metallosphaera cuprina Ar-4, a dsrE3A-dsrE2B-tusA arrangement is situated immediately between genes encoding dihydrolipoamide dehydrogenase and a heterodisulfide reductase-like complex. In this study, the biochemical features and sulfur transferring abilities of the DsrE2B, DsrE3A, and TusA proteins were investigated. DsrE3A and TusA proved to react with tetrathionate but not with NaSH, glutathione persulfide, polysulfide, thiosulfate, or sulfite. The products were identified as protein-Cys-S-thiosulfonates. DsrE3A was also able to cleave the thiosulfate group from TusA-Cys¹⁸-S-thiosulfonate. DsrE2B did not react with any of the sulfur compounds tested. DsrE3A and TusA interacted physically with each other and formed a heterocomplex. The cysteine residue (Cys¹⁸) of TusA is crucial for this interaction. The single cysteine mutants DsrE3A-C⁹³S and DsrE3A-C¹⁰¹S retained the ability to transfer the thiosulfonate group to TusA. TusA-C¹⁸S neither reacted with tetrathionate nor was it loaded with thiosulfate with DsrE3A-Cys-S-thiosulfonate as the donor. The transfer of thiosulfate, mediated by a DsrE-like protein and TusA, is unprecedented not only in M. cuprina but also in other sulfur-oxidizing prokaryotes. The results of this study provide new knowledge on oxidative microbial sulfur metabolism.     

The identification of a novel protein involved in molybdenum cofactor biosynthesis in Escherichia coli

In the second step of the molybdenum cofactor (Moco) biosynthesis in Escherichia coli, the l-cysteine desulfurase IscS was identified as the primary sulfur donor for the formation of the thiocarboxylate on the small subunit (MoaD) of MPT synthase, which catalyzes the conversion of cyclic pyranopterin monophosphate to molybdopterin (MPT). Although in Moco biosynthesis in humans, the thiocarboxylation of the corresponding MoaD homolog involves two sulfurtransferases, an l-cysteine desulfurase, and a rhodanese-like protein, the rhodanese-like protein in E. coli remained enigmatic so far. Using a reverse approach, we identified a so far unknown sulfurtransferase for the MoeB-MoaD complex by protein-protein interactions. We show that YnjE, a three-domain rhodanese-like protein from E. coli, interacts with MoeB possibly for sulfur transfer to MoaD. The E. coli IscS protein was shown to specifically interact with YnjE for the formation of the persulfide group on YnjE. In a defined in vitro system consisting of MPT synthase, MoeB, Mg-ATP, IscS, and l-cysteine, YnjE was shown to enhance the rate of the conversion of added cyclic pyranopterin monophosphate to MPT. However, YnjE was not an enhancer of the cysteine desulfurase activity of IscS. This is the first report identifying the rhodanese-like protein YnjE as being involved in Moco biosynthesis in E. coli. We believe that the role of YnjE is to make the sulfur transfer from IscS for Moco biosynthesis more specific because IscS is involved in a variety of different sulfur transfer reactions in the cell.