An intestinal parasitic protist, Entamoeba histolytica, possesses a non-redundant nitrogen fixation-like system for iron-sulfur cluster assembly under anaerobic conditions.

We have characterized the iron-sulfur (Fe-S) cluster formation in an anaerobic amitochondrial protozoan parasite, Entamoeba histolytica, in which Fe-S proteins play an important role in energy metabolism and electron transfer. A genomewide search showed that E. histolytica apparently possesses a simplified and non-redundant NIF (nitrogen fixation)-like system for the Fe-S cluster formation, composed of only a catalytic component, NifS, and a scaffold component, NifU. Amino acid alignment and phylogenetic analyses revealed that both amebic NifS and NifU (EhNifS and EhNifU, respectively) showed a close kinship to orthologs from epsilon-proteobacteria, suggesting that both of these genes were likely transferred by lateral gene transfer from an ancestor of epsilon-proteobacteria to E. histolytica. The EhNifS protein expressed in E. coli was present as a homodimer, showing cysteine desulfurase activity with a very basic optimum pH compared with NifS from other organisms. Eh-NifU protein existed as a tetramer and contained one stable [2Fe-2S]2+ cluster per monomer, revealed by spectroscopic and iron analyses. Fractionation of the whole parasite lysate by anion exchange chromatography revealed three major cysteine desulfurase activities, one of which corresponded to the EhNifS protein, verified by immunoblot analysis using the specific EhNifS antibody; the other two peaks corresponded to methionine gamma-lyase and cysteine synthase. Finally, ectopic expression of the EhNifS and EhNifU genes successfully complemented, under anaerobic but not aerobic conditions, the growth defect of an Escherichia coli strain, in which both the isc and suf operons were deleted, suggesting that EhNifS and EhNifU are necessary and sufficient for Fe-S clusters of non-nitrogenase Fe-S proteins to form under anaerobic conditions. This is the first demonstration of the presence and biological significance of the NIF-like system in eukaryotes.

Iron-sulfur (Fe-S) 1 clusters are cofactors of proteins probably present in all living organisms. The Fe-S clusters play various important roles in electron transfer, redox regulation, nitrogen fixation, and sensing for regulatory processes (1). Despite the importance of Fe-S proteins, little is known about the biochemical mechanisms of Fe-S cluster assembly in vivo. Recent studies using genetic and biochemical methods have unveiled the complex mechanism of the assembly in vitro and in vivo (2)(3)(4)(5)(6)(7)(8). These studies led to the identification of three distinct systems, namely nitrogen fixation (NIF), iron-sulfur cluster (ISC), and mobilization of sulfur (SUF). Two to six components have been shown to participate in the formation of Fe-S clusters, depending upon the system. For instance, in the NIF system, two genes (nifS and nifU) and their encoded proteins have been shown to be involved in the assembly of Fe-S clusters of the nitrogenase proteins in Azotobacter vinelandii (2,3). NifS is a homodimer of two identical subunits of a pyridoxal-5Ј-phosphate (PLP)-dependent cysteine desulfurase, which catalyzes the formation of L-alanine and elemental sulfur from L-cysteine. The catalysis is initiated by the formation of a Schiff base between the amino group of cysteine and PLP cofactor (3) and nucleophilic attack of the reactive cysteine by a conserved histidine residue near the active site (9), which in turn mobilizes elemental sulfur. NifU is a scaffold protein for the transient assembly of Fe-S clusters, which are transferred to target apoproteins (10). The NifU protein possesses two distinct types of iron-binding sites (10). One of these binding sites, located in the central third of the NifU protein, binds a stable permanent [2Fe-2S] 2ϩ cluster per subunit as shown in A. vinelandii (11). The second type of site, located within the amino-terminal third of the NifU protein, binds a labile mononuclear iron and/or labile cluster (11). Site-directed mutagenesis of the cluster-ligated cysteine residues of NifU from A. vinelandii has shown that the permanent [2Fe-2S] 2ϩ clusters play an essential role in the maturation of nitrogenase (12). The NIF system has been identified in only nitrogen-fixing bacteria and nondiazotrophic ⑀-proteobacteria including Campylobacter jejuni and Helicobacter pylori, and appears to be involved in Fe-S assembly of the nitrogenase proteins in bacteria that belong to the former group and of non-nitrogenase Fe-S proteins in the latter (13). In contrast to the NIF system, the ISC system is well conserved from bacteria to a wide range of eukaryotes including Saccharomyces, Arabidopsis, Caenorhabditis, Drosophila, and Homo, and thus, assumed to play more general housekeeping roles for Fe-S cluster assembly. The components of the ISC system are more complex than those of the NIF system: at least six proteins, encoded in a single operon (isc-SUA-hscBA-fdx) in prokaryotes, e.g. Escherichia coli, are involved in the process (4, 6, 7, 14 -16). IscS bears sequence identity with NifS and shares function as cysteine desulfurase. IscU is similar to the amino-terminal domain of NifU and shares function as a scaffold for intermediate Fe-S clusters. IscA is closely related to its NIF counterpart ( Nif IscA) and responsible for binding labile Fe-S clusters (5,17) and its transfer to apoproteins (18). HscA and HscB are chaperones that belong to the DnaK and DnaJ proteins families, respectively; but their roles in Fe-S cluster biogenesis remain unclear (19). Protein-protein interaction between each of the components of the ISC system has also been demonstrated (18,20). The SUF system, a third bacterial system for the assembly of Fe-S clusters, is encoded in the suf operon (sufABCDSE) and widely present in Eubacteria, Archaea, and plastids (8). Disruption of the E. coli suf operon alone did not cause any major defect, whereas lethality was observed when both the isc and suf operons were inactivated (8). It has also been shown that the SUF system plays a role for Fe-S cluster assembly and/or repair under oxidative stress conditions (21,22) and iron starvation (23). In addition, the SUF system has been shown to be necessary for virulence of Gram-negative bacterium Erwinia chrysanthemi, causing soft-rot disease in plants (21,24). In addition to the catalytic component SufS, the SUF system requires at least five additional proteins including SufA, a scaffold component, SufC, an unorthodox ATPase of the ABC superfamily, SufB and SufD, which are associated with SufC (24), and SufE, which interacts with SufS and stimulates its cysteine desulfurase activity (25).
Recent studies have demonstrated that a mitochondrial IscS homolog in yeasts (Nfs1) is involved in Fe-S assembly of aconitase and succinate dehydrogenase and thus is essential for mitochondrial function (26). IscS homologs are produced in the cytosol and transported to mitochondria (27) and the nucleus in yeasts and mammals (28,29). An independent study also revealed that mitochondria play a crucial role in the cluster formation of extramitochondrial Fe-S proteins (30). Thus, the assembly of Fe-S clusters is more complex in eukaryotes than prokaryotes and apparently occurs in mitochondria, cytoplasm, and nucleus, suggesting organelle-specific Fe-S cluster assembly in eukaryotes (31,32). Thus, the presence or absence, i.e. ubiquity and specificity, of these three distinct systems for Fe-S assembly among organisms, together with their specific function in each organism, remains largely unknown, especially in parasitic protozoa.
IscS has been demonstrated in an aerobic protozoan parasite, Plasmodium falciparum (33), and two anaerobic protozoa, Giardia lamblia and Trichomonas vaginalis (34). The latter two parasites belong, together with another enteric parasitic protist Entamoeba histolytica, to a group of amitochondrial eukaryotes. Amitochondrial eukaryotes can be divided into two metabolic types (35). Type I organisms including G. lamblia and Entamoeba lack organelles involved in core energy metabolism. Instead, Fe-S protein (i.e. pyruvate:ferredoxin oxidoreductase)-mediated metabolism of pyruvate, substrate-level phosphorylation, and ATP synthesis takes place in the cytosol. In contrast, type II organisms including Trichomonas, some ciliates, and chytrid fungi harbor a double-membrane limited organelle, hydrogenosome, which represents a site of the abovementioned core energy metabolism. The fact that the scaffold component IscU is also present in Trichomonas and Giardia (36) supported the premise that the machinery for Fe-S cluster assembly in these amitochondrial anaerobic protists shares common features with the ISC system in other organisms. In contrast, the machinery for the Fe-S cluster assembly in E. histolytica is largely unknown. The present study demonstrates that E. histolytica possesses the NIF-like system as a sole pathway for the biosynthesis of Fe-S clusters. We describe here for the first time the molecular identification of NifS and NifU from E. histolytica and provide evidence for the possible horizontal transfer of these genes from an ancestor of ⑀-proteobacteria. We also show biochemical properties distinct from those of other organisms including bacteria and mammals. In addition, we demonstrate that the amebic NifS and NifU are necessary and sufficient for the Fe-S cluster formation under anaerobic conditions by heterologous complementation of an isc/suf-lacking mutant of E. coli.
Search of the E. histolytica Genome Database-The E. histolytica genome databases (contigs and singletons) at The Institute for Genomic Research 2 and Sanger Institute 3 were searched using the TBLASTN algorithm with protein sequences corresponding to the catalytic component of Fe-S cluster formation (NifS, IscS, and SufS) from a variety of species. We also searched for homologs of the Nif-or Isc-specific scaffold component (NifU or IscU, respectively) from A. vinelandii and H. pylori and components shared by both the Isc and Suf systems (IscA and SufA) of E. coli, A. vinelandii, and P. falciparum, or components unique to the Suf system (SufB, C, D, and E) from E. coli, Bacillus subtilis, Methanococcus jannashii, Mycobacterium tuberculosis, and P. falciparum.
Amino Acid Alignments and Phylogenetic Analysis-The sequences of NifS, IscS, NifU, IscU, and related proteins showing similarity in amino acid sequence to EhNifS and EhNifU were obtained from the National Center for Biotechnology Information 4 using the BLASTP search. Alignment and phylogenetic analysis were performed with CLUSTAL W version 1.81 (39) using the neighbor-joining method with the Blosum matrix. A rooted or an unrooted neighbor-joining tree composed of the amino acid sequences of 25 NifS/IscS or 20 NifU/IscU from various organisms was constructed using 352 or 120 shared amino acid positions, respectively, after removing gaps. The most distal members of NifS/IscS that belong to group II including E. coli cysteine sulfinate desulfinase (CsdA) and SufS (also known as CsdB or selenocysteine lyase) were used as the out-group to obtain a rooted tree. Trees were drawn by Tree ViewPPC ver.1.6.6 (40). Branch lengths and bootstrap values (1000 replicates) were derived from the neighbor-joining analysis.
Cloning of E. histolytica NifS and NifU-On the basis of the nucleotide sequences of the protein-encoding region of the putative amebic nifS and nifU genes (EhNifS and EhNifU) in the genome database, two sets of primers, shown below, were designed to amplify the open reading frames (ORFs) using a cDNA library (41) as a template to construct plasmids to produce EhNifS and EhNifU fusion proteins with a histi-dine tag or glutathione S-transferase at the amino terminus, respectively. The EhNifS ORF was amplified with a sense (5Ј-CCTGGATCC-GATGCAAAGTACAAAATCAGT-3Ј) and an antisense (5Ј-CCAGGATC-CTTAAGCATATGTTGATGATAATTGTC-3Ј) primer, where BamHI sites are underlined and the translation initiation and termination codons are italicized. The initial step, denaturation at 94°C for 2 min with platinum pfx DNA polymerase (Invitrogen), was followed by the 30 cycles of denaturation at 94°C for 15 s, annealing at 55°C for 30 s, and elongation at 68°C for 2 min, and a final extension for 10 min at 68°C. The ϳ1.2-kb PCR fragment was digested with BamHI, electrophoresed, purified with Geneclean kit II (BIO 101, Vista, CA), and cloned into BamHI-digested pET-15b (Novagen) in the same orientation as the T7 promoter. The EhNifU ORF was amplified using a sense (5Ј-ATGTCA-AAGAATAAATTAATTGGTGGAGC-3Ј) and an antisense (5Ј-CTAATC-TTTCTTTTTGATATTTAAGGT-3Ј) primer from a cDNA library (the translation initiation and termination sites are italicized). A PCR fragment containing EhNifU was end-blunted and cloned into the EcoRIdigested and end-filled site of pGEX2T (Amersham Biosciences). The final constructs were designated pHisEhNifS and pGSTEhNifU, respectively.
Expression and Purification of Recombinant EhNifS and EhNifU Proteins-To express the recombinant proteins in E. coli, pHisEhNifS or pGSTEhNifU was introduced into BL21 (DE3) or DH5␣ cells, respectively. Expression of the recombinant EhNifS (rEhNifS) and EhNifU (rEhNifU) fusion proteins was induced with 1 mM isopropyl-␤-thiogalactoside at 30°C for 5-6 h. The rEhNifS and EhNifU fusion proteins were purified using a Ni 2ϩ -NTA column (Novagen) or glutathione-Sepharose 4B column (Amersham Biosciences), respectively, according to the manufacturer's instructions with a few modifications. The bacterial cells were washed, sonicated in the lysis buffer (50 mM Tris-HCl (pH 8.0), 500 mM NaCl, 5 mM 2-mercaptoethanol, and 10 mM imidazole) containing 0.1% Triton X-100 (v/v), 100 g/ml of lysozyme, and Complete Mini EDTA free protease inhibitor mixture (Roche, Tokyo, Japan), and centrifuged at 24,000 ϫ g for 15 min at 4°C. The histidine-tagged rEhNifS protein was eluted from the Ni 2ϩ -NTA column with 100 mM imidazole in 50 mM Tris-HCl (pH 8.0), 300 mM NaCl, and 0.1% Triton X-100 (v/v) and extensively dialyzed in 50 mM Tris-HCl, 300 mM NaCl (pH 8.0), and 0.1% Triton X-100 (v/v) containing 10% glycerol (v/v) and the protease inhibitors. To obtain the rEhNifU, bacterial cells were lysed in 100 mM sodium phosphate buffer (pH 7.4), 2 mM DTT, 0.1% Triton X-100 (v/v), 1 mM phenylmethylsulfonyl fluoride, and 100 g/ml of lysozyme. After the GST-EhNifU fusion protein was bound to the glutathione-Sepharose 4B column, the rEhNifU was obtained by digestion of GST-EhNifU fusion proteins with thrombin (Amersham Biosciences) in the column or outside of the column, followed by elution from the column. Thrombin was removed by passing through a HiTrap-benzamidine column (Amersham Biosciences), and rEhNifU was extensively dialyzed at 4°C with 100 mM sodium phosphate buffer (pH 7.4) containing 2 mM DTT.
The purified rEhNifS and rEhNifU proteins were presumed to contain additional 25 (MGSSHHHHHHSSGLVPRGSHMLEDP) or 5 (GSPGI) amino acids at the amino terminus, respectively. The purified enzymes were stored at Ϫ 80°C with 50% glycerol before use. No decrease in the enzyme activity of rEhNifS was observed under these conditions for at least 3 months. Protein concentrations were determined with the Coomassie Brilliant Blue assay (Nacalai Tesque, Inc., Kyoto, Japan) with bovine serum albumin as the standard.
Enzyme Assays-For the cysteine desulfurase assay, rEhNifS protein was reconstituted with PLP by incubating 1 mg/ml of EhNifS with 0.1 mM PLP for 1 h on ice, followed by dialysis for 5-6 h against 100 -200fold volumes of 50 mM N-[tris(hydroxymethyl)methyl]-3-aminopropanesulfonic acid/NaOH (pH 9.0) with two buffer exchanges, as described previously for the Synechocystis enzyme (42). The cysteine desulfurase activity of EhNifS was monitored based on the production of sulfide using L-cysteine as the substrate (43). The standard EhNifS reaction was performed in 200 l of reaction mixture (50 mM 3-(cyclohexylamino)-1-propanesulfonic acid/NaOH buffer (pH 10.0) containing 0.02 mM PLP, 1 mM DTT, 10 mM MgCl 2 , 0.5 mM substrate (L-cysteine), and appropriate amounts (25-50 g) of purified EhNifS protein). In experiments to determine pH optima, the following buffers were used: 50 mM 2-(N-morpholino)ethanesulfonic acid/NaOH for pH 5.5, 6.0, and 6.5; HEPES/NaOH for pH 7.0, 7.5, and 8.0; N-[tris(hydroxymethyl)methyl]-3-aminopropanesulfonic acid/NaOH for pH 8.5 and 9.0; and 3-(cyclohexylamino)-1-propanesulfonic acid for pH 9.7, 10.0, 10.5, and 11.0. The reaction was terminated by adding 20 l of 20 mM N,N-dimethyl-pphenylenediamine sulfate in 7.2 N HCl and 20 l of 30 mM FeCl 3 in 1.2 N HCl. After further incubation in the dark for 30 min, the protein precipitate was removed by centrifugation, and then the absorption at 670 nm (A 670 ) of the supernatant was measured. Na 2 S (0-100 M) was used as the standard. Elemental sulfur (S 0 ) was measured by the cyanolysis method (2, 44) with minor modifications. The alanine production was monitored by measuring pyruvate formed by deamination of alanine using L-alanine aminotransferase in a coupling reaction as described previously (2). The rEhNifS protein was stable in the presence of 10% glycerol, and 90 -95% of the initial activity was retained when stored at 4 or Ϫ 20°C for 24 h. However, it was reduced to 25-35% when stored without any additive at room temperature, 4, or Ϫ 20°C for 24 h.
Iron Assay-The iron content of EhNifU was determined by the O-phenanthroline method as described, except that the volume of the reaction mixture was reduced (13). The EhNifU samples were acidified by the addition of 3-5 l of concentrated HCl and then diluted with buffer or distilled water to 0.2 ml. The mixtures were heated to 80°C for 10 min and cooled down to room temperature; then 0.6 ml of water, 40 l of 10% hydroxylamine hydrochloride, and 0.2 ml of 0.1% O-phenanthroline were added. The mixtures were further incubated at room temperature for 30 min, and then absorbance at 512 nm (A 512 ) was measured with 0 -100 M of ferrous ammonium sulfate as the standard.
Anion-Exchange Chromatography of the Native and Recombinant EhNifS-Approximately 2 ϫ 10 7 E. histolytica trophozoites (250 -300 mg) was resuspended in 1.0 ml of 100 mM Tris-HCl (pH 8.0), 1.0 mM EDTA, 2.0 mM DTT, and 15% glycerol containing 10 g/ml of transepoxysuccinyl-L-leucylamido-(4-guanidino)butane and the protease inhibitor mixture. After sonication, the lysate was centrifuged at 45,000 ϫ g for 15 min at 4°C, filtered through a 0.45 m cellulose acetate membrane, and dialyzed with the binding buffer (100 mM Tris-HCl (pH 8.0), 1 mM DTT, 1 mM EDTA, 0.1 mM phenylmethylsulfonyl fluoride, 1 g/ml of trans-epoxysuccinyl-L-leucylamido-(4-guanidino)butane and 10% glycerol) using a Centricon YM-10 (Millipore). The sample (1 ml) containing ϳ5 mg of soluble lysate protein or recombinant EhNifS was applied to a Mono Q 5/5 HR column preequilibrated with the binding buffer on the AKTA Explorer 10S system (Amersham Biosciences). After the column was extensively washed with the binding buffer, bound proteins were eluted with a linear gradient of 0 -0.7 M NaCl. All of the fractions (0.3 ml) were analyzed for cysteine desulfurase activity as described above and subjected to immunoblot analyses as described below.
Heterologous Expression of the Amebic NifS and NifU Genes in the isc/suf-Mutant E. coli Strain-A plasmid to coexpress EhNifS and Eh-NifU in E. coli was constructed. The EhNifS and EhNifU ORF were PCR-amplified using a sense (5Ј-CTCTCTAGAGATGCAAAGTACAAA-TCAGT-3Ј) and an antisense (5Ј-GGTACATGTTTAaaactcctcAGCATA-TGTTGATGATAATTGTC-3Ј) primer (EhNifS); a sense (5Ј-GCACCAT-GGCAAAGAATAAATTAATTGGTGGAG-3Ј) and an antisense (5Ј-GCTGCTAGCCTAATCTTTCTTTTTGATATTTAAGG-3Ј) primer (Eh-NifU), where XbaI, PciI, NcoI, and NheI sites are underlined, the translation initiation and termination codons are italicized, and the ribosome binding sequence is shown in lowercase. Because of the engineered ribosome-binding sequence, the resulting EhNifS protein has three additional amino acids (EEF) at the carboxyl terminus. The PCR fragments of EhNifS and EhNifU were digested with appropriate enzymes and cloned in tandem in the XbaI and NheI double-digested pRKNMC (7) to produce pRKEhNifSU. The pRKISC or pRKSUF, which

Identification and Features of EhNifS and EhNifU Genes
and Their Encoded Proteins-We identified putative NifS/IscS/ SufS and NifU/IscU gene homologs in the E. histolytica genome database as described in "Experimental Procedures." Putative EhNifS and EhNifU genes contained an ORF of 1173 and 1047 bp encoding a protein of 390 and 348 amino acids with predicted molecular masses of 42.8 and 38.9 kDa and a pI of 5.9 and 5.6, respectively. Neither the PSORT II program, which predicts protein localization in cells 5 nor a Kite-Doolittle hydropathy plot suggested any possible cellular localization other than a cytosolic distribution for both EhNifS and EhNifU. A protein alignment of 26 NifS/IscS/SufS homologs from Archaea, bacteria, fungi, protists, plants, and metazoa revealed (an alignment of only representative members is shown in Fig. 1) that EhNifS showed greatest homology (52-55% identity) to NifS from ⑀-proteobacteria Campylobacter jejuni and Helicobacter pylori; 37-42% identity to NifS from nitrogen-fixing Eubacteria including A. vinelandii, Klebsiella pneumoniae, and cyanobacteria; and 32-33% identity to IscS from nitrogen-fixing and non-fixing Eubacteria, fungi, and other protists including G. lamblia, T. vaginalis, P. yoelii, and Cryptosporidium parvum, and metazoa. EhNifS also showed limited homology (21-24% identity) to other (group II) NifS/IscS/SufS homologs including CsdA (cysteine sulfinate desulfinase) and SufS (CsdB or selenocysteine lyase) from E. coli, a hypothetical protein from Synechocystis, and a chloroplast homolog from Arabidop-sis thaliana (data not shown; see also "phylogenetic analyses" and Fig. 3A). All of the residues of the active site and amino acids implicated in the cysteine desulfurase activity were conserved: (i) His 106 (numbered according to EhNifS), which is involved in the initial deprotonation of the substrate (9); (ii) the PLP-binding site with the Schiff base-forming amino acids Lys 208 , Asp 182 , and Gln 185 that bind the pyridine nitrogen and the phenolate oxygen of PLP, respectively; (iii) Thr 76 , His 207 , Ser/Thr 205 , and Thr 243 , involved in the formation of an additional six hydrogen bonds anchoring the phosphate group of PLP (2,9); and (iv) the substrate-binding site including Cys 330 , which provides a reactive cysteinyl residue (3), as well as Arg 356 , Asn 157 , and Asn 35 , which anchor the cysteine with a salt bridge and hydrogen bond (9). A carboxyl-terminal extension consisting of 20 -21 amino acids including the consensus sequence SPL(W/Y)(E/D)(M/L)X(K/Q)XG(I/V)D(L/I)XX(/V)X-WXXX was previously proposed to differentiate proteobacterial and eukaryotic IscS from homologs of all other organisms (34) but also is present in nitrogen-fixing bacteria and cyanobacteria (Fig. 1). However, this extension is absent in the NifS homologs from E. histolytica and ⑀-proteobacteria.
A protein alignment of 21 NifU/IscU/Nfu1p homologs from Archaea, bacteria, fungi, protists, plants, and metazoa revealed (an alignment of only representative members is shown in Fig.  2) that EhNifU showed, similar to NifS, greatest homology (55-56% identity) to NifU from ⑀-proteobacteria and less homology (32-35% identity) to NifU from diazotrophic Eubacteria throughout the proteins. The amino-terminal half of EhNifU (residues 15-151) also exhibited 28 -37% identity to IscU from non-diazotrophic bacteria, fungi, other protists, plants, and metazoa. The amino terminus of EhNifU showed insertions of five and six amino acids at two positions (38 -42 and 118 -123), which are also observed in ⑀-proteobacteria. In addition, the 5 Internet address: psort.ims.u-tokyo.ac.jp/. carboxyl-terminal half of EhNifU (178 -348) showed limited homology (12-14% identity) to Nfu1p homologs from S. cerevisiae and A. thaliana. Nine of 12 cysteine residues of EhNifU (Cys 54 , Cys 81 , Cys 131 , Cys 161 , Cys 163 , Cys 196 , Cys 199 , Cys 291 , and Cys 294 ) were conserved. These residues include three cysteines (Cys 54 , Cys 81 , and Cys 131 ) in the amino terminus, which constitute a binding site for iron and transient Fe-S cluster (5) and are essential for the function of IscU, Isu1p, and NifU (12,47). In contrast, Cys 95 was conserved only in EhNifU and among NifU proteins from ⑀-proteobacteria. The central portion of EhNifU contains four conserved cysteine residues, Cys 161 , Cys 163 , Cys 196 , and Cys 199 , which were shown to be responsible for permanent [2Fe-2S] 2ϩ cluster binding as described in a mutational study on A. vinelandii NifU (12). Two additional cysteines (Cys 291 and Cys 294 ) at the carboxyl-terminal region that are present in mammalian Nfu and have been shown to function as a scaffold protein for one transient [4Fe-4S] 2ϩ cluster per two Nfu monomers (29) are also conserved in EhNifU. Asp 56 was conserved in all species and was implicated in the release of the NifU-bound transient Fe-S cluster (10).
Phylogenetic Analysis of EhNifS and EhNifU-NifS/IscS/ SufS homologs were shown previously to form two distinct groups, I and II (48). Distal members of the homologs that belong to group II (SufS and CsdA) were used as the outgroup to examine the phylogenetic relationship of EhNifS. IscS from a wide range of non-diazotrophic Eubacteria other than ⑀-proteobacteria, fungi, metazoa, and parasitic protists (G. lamblia, T. vaginalis, P. yoelii, and C. parvum) and NifS from nitrogenfixing Eubacteria formed independent clades that were well supported by high bootstrap values (99.8 -100%) (Fig. 3A). The amebic NifS formed a separate clade (also well supported by a 100% bootstrap value) with ⑀-proteobacteria H. pylori and C. jejuni. This, together with high amino acid identities among this group, reinforces a close kinship of EhNifS with homologs from ⑀-proteobacteria.
The phylogenetic relationship of EhNifU with 20 other NifU/ IscU homologs from Archaea, bacteria, fungi, protists, plants, and metazoa was presented as a radial unrooted tree as an appropriate outgroup was not available (Fig. 3B). IscU from nitrogen-fixing and non-fixing bacteria, fungi, protists except for E. histolytica, plants, and metazoa and NifU from nitrogenfixing bacteria, cyanobacteria, ⑀-proteobacteria, and E. histolytica form two independent clades, well supported by high bootstrap values (92-96%). EhNifU showed strong affinity for homologs from ⑀-proteobacteria as shown in the case of EhNifS and represents an independent group well separated from the group of NifU from nitrogen-fixing bacteria. This, together with the high amino acid identities and common insertions, clearly supported a close association of E. histolytica NifU with NifU from ⑀-proteobacteria.
Expression and Purification of rEhNifS and rEhNifU Proteins-The rEhNifS protein was purified at high yield, ϳ100 mg/l of E. coli culture. Cells, cell extracts, and purified EhNifS were yellowish in color. The rEhNifS protein revealed an apparently homogeneous band of 45 kDa on SDS-PAGE (Fig. 4A), which was consistent with the predicted size of the deduced EhNifS protein with the extra 25 amino acids added at the amino terminus. The purified rEhNifS protein was evaluated ϳ98% pure by densitometric measurement of the Coomassiestained SDS-PAGE gel. The cell pellet of the E. coli that overexpressed the GST-EhNifU fusion protein was brown in color. The purified recombinant GST-EhNifU protein also showed a slight brownish color, characteristic of Fe-S proteins. The recombinant GST-EhNifU protein showed an apparently homogeneous band of 65 kDa on SDS-PAGE run under reducing or non-reducing conditions (Fig. 4B, data for under the non-reduc-ing conditions not shown), which was consistent with the predicted size of the deduced monomeric EhNifU protein with the extra 26-kDa GST fused at the amino terminus. This also indicates that GST-EhNifU does not form an inter-molecule disulfide bridge. After final purification, rEhNifU protein was evaluated ϳ98% pure. The final yield of purified rEhNifU was lower than rEhNifS (3 mg/l of E. coli culture). The antibody raised against either rEhNifS or rEhNifU recognized an apparently single protein in the E. histolytica lysate, which is similar in size to rEhNifS or rEhNifU, respectively (Fig. 4, A and B,  right panels), confirming the molecular mass of native E. histolytica proteins. We roughly estimated from immunoblots of a series of diluted recombinant proteins that the ameba contains a comparable amount of EhNifS and EhNifU, which consists of ϳ0.1% of the total soluble protein (data not shown).
Biochemical Characterization of Recombinant EhNifS-The purified rEhNifS protein showed an absorption spectrum typ- ical of a PLP-binding protein, a characteristic peak at 370 -380 nm (Fig. 5A). Incubation with 0.5 mM L-cysteine shifted the peak to 348 nm, with a broad shoulder at 410 nm (Fig. 5A), similar to NifS from H. pylori (13). In contrast, A. vinelandii NifS showed major and minor peaks at 416 and 370 nm, respectively, in the presence of L-cysteine (2). The spectral change was observed previously for NifS-like protein from T. maritima and proposed to be attributable to deprotonation/protonation of aldimine, leading to the formation of either germinal diamine or enolimine species (49), which occurs in conjunction with persulfide formation (9). The whole reaction required 10 -15 min to complete. At 30 min after the addition of cysteine, no further spectral change was observed. The optimum pH for EhNifS was 10 -10.5; the half-maximum activity was reached at about pH 9.0 (Fig. 5B). The rEhNifS protein showed cysteine desulfurase activity, as measured in sulfide production, of ϳ9.5 nmol/min/mg pure protein with 0.5 mM cysteine, which is comparable to NifS from H. pylori (9.9 nmol/min/mg with 50 mM cysteine) (13) and lower than other bacterial homologs (A. vinelandii NifS, 89.4 nmol/min/mg with 0.5 mM cysteine (2); A. vinelandii IscS, 67.6 nmol/min/mg with 2.5 mM cysteine (4); E. coli IscS, 78 nmol/min/mg with 2.5 mM cysteine (16); E. coli CSD, 3.4 mol/min/mg (V max ) (48); and E. coli SufS, 0.019 or 0.9 mol/min/mg (V max ) in the absence or presence of SufE, respectively (25)). Among a variety of possible substrates, Eh-NifS was shown to be specific for L-cysteine and L-cystine. No sulfide production was observed when D-cysteine, N-acetylcysteine, DL-homocysteine, DL-selenocysteine, cysteine sulfinic acid, or L-cysteic acid was used at up to 5 mM. rEhNifS showed 40 -50% cysteine desulfurase activity toward L-cystine (4.0 nmol sulfide/min/mg) in the presence of DTT. Both alanine and cyanolysis assays also showed the comparable cysteine desulfurase activity of rEhNifS (9.1 nmol alanine/min/mg and 8.0 nmol sulfur/min/mg, respectively) with 0.5 mM cysteine, whereas rEhNifS showed the specific activity of 3.0 nmol alanine/min/mg against L-cystine by alanine assay.
Biochemical Characterization of Recombinant EhNifU-Spectrophotometric analysis of the purified recombinant Eh-NifU revealed peaks at 330, 420, and 460 nm and a shoulder at 550 nm, which indicates the presence of a [2Fe-2S] 2ϩ cluster (Fig. 6), as described for NifU of A. vinelandii (11). Extinction coefficients of EhNifU per 38-kDa subunit were also consistent with the premise that EhNifU contains a [2Fe-2S] 2ϩ center: the peaks at 330 nm (extinction coefficient, 11700 M Ϫ1 cm Ϫ1 ), 420 nm (6800 M Ϫ1 cm Ϫ1 ), and 460 nm (7000 M Ϫ1 cm Ϫ1 ), a shoulder at ϳ550 nm (4700 M Ϫ1 cm Ϫ1 ), and a ratio between 280 and 335 nm (A 335 /A 280 ) of 0.27. The dithionite-reduced rEhNifU showed, in contrast, a relatively featureless spectrum, which increased in intensity at shorter wavelengths because of dithionite. Effects of Fe chelators on the cluster structure were assessed by treating oxidized and reduced rEhNifU with a 40-fold excess of 2,2Ј-dipyridyl or EDTA. No change of A 520 was observed over 2 h (results not shown), indicating that the Fe-S cluster is either stable or inaccessible to solvent, which is also similar to A. vinelandii NifU (11). The iron analysis indicates that EhNifU contains two irons per subunit (2.1 Ϯ 0.084 Fe/ subunit from three independent EhNifU preparations).
Determination of Multimeric Structure of EhNifS and Eh-NifU-The cysteine desulfurase activity of rEhNifS was eluted at the predicted molecular mass of 85-95 kDa (Fig. 7) by gel filtration chromatography. This is consistent with the notion that rEhNifS exists as a dimer of two identical subunits (42.5 kDa plus 2.6 kDa corresponding to the histidine tag), which is similar to all other organisms reported earlier. EhNifU was eluted at 150 -170 kDa; GST-EhNifU was also eluted at 260 -280 kDa (Fig. 7). These results show clearly that EhNifU is a tetramer irrespective of the presence or absence of a fusion partner. Elution patterns of EhNifU remained unchanged when EhNifU was pretreated with 2 mM DTT and fractionated in the presence of DTT (results not shown).
Anion-Exchange Chromatographic Separation of Native and Recombinant Cysteine Desulfurase Activity-To correlate native cysteine desulfurase activity in the E. histolytica lysate with the recombinant enzyme, the lysate from the trophozoites and rEhNifS were subjected to chromatographic separation on a Mono Q anion exchange column and analyzed by cysteine desulfurase assay and also immunoblotting using anti-EhCS, EhMGL, and EhNifS antibodies. E. histolytica trophozoites possess two isotypes of CS (CS1 and CS2) (41) and two isotypes of MGL (MGL1 and MGL2) (46), both of which showed substantial cysteine desulfurase activity. 6 Thus, we attempted to separate native NifS from the CS and MGL isotypes. The E. histolytica lysate showed three major peaks of cysteine desulfurase activity (Fig. 8A). Fractions corresponding to the last and largest cysteine desulfurase peak (fractions 20 -27) contained the protein that reacted well with the anti-EhNifS and anti-CS antibodies (Fig. 8B). In contrast, fractions corresponding to the first and second cysteine desulfurase peaks did not react with anti-EhNifS antibody but reacted with anti-MGL and anti-CS antibodies, respectively. rEhNifS was fractionated under the same conditions and showed a single peak eluted at a slightly lower ionic strength (0.5 ml earlier elution volume) than the native EhNifS (Fig. 8C), which is consistent with the fact that recombinant histidine-tagged EhNifS has a slightly higher pI (6.15) than the native protein (5.9). These results suggest that the third dominant cysteine desulfurase peak of the parasite lysate represents activity mainly attributable to native EhNifS.
Heterologous Expression of the Amebic NifS and NifU in the isc/suf-Mutant E. coli Strain-To assess the in vivo role of EhNifS and EhNifU, we attempted heterologous complementation of an E. coli mutant UT109 in which the chromosomal isc and suf operons were deleted. This strain was lethal unless we provided the suf or isc operon in a complementing plasmid carrying a temperature-sensitive replicative origin. 7 At the restrictive temperature, introduction of the E. coli isc or suf operon in a complementing plasmid carrying a temperatureinsensitive replicative origin complemented the growth defects of the UT109 strain under both the aerobic and anaerobic conditions (Fig. 9). In contrast, coexpression of EhNifS and EhNifU rescued the growth of UT109 only under the anaerobic, not aerobic, conditions at the restrictive temperature. Thus, the amebic NifS and NifU are apparently necessary and sufficient for the Fe-S cluster formation under anaerobic conditions in this heterologous system. Although CS and MGL showed cysteine desulfurase activity in vitro, coexpression of CS1 or MGL2, together with EhNifU, did not complement the growth defects of UT109 under either aerobic or anaerobic conditions (data not shown). DISCUSSION We have identified and characterized two necessary and sufficient components, NifS and NifU, of the NIF-like system for the assembly of Fe-S clusters in a human intestinal proto-zoan anaerobe. As far as we are aware, this is the first demonstration of the NIF-like system in eukaryotes. Despite a thorough search of the E. histolytica genome database, no other proteins that were shown to be involved in ISC/SUF systems of other organisms were found, suggesting that this parasite possesses the NIF-like system as a sole and non-redundant system for the biosynthesis of all Fe-S proteins. Because E. histolytica 6 V. Ali and T. Nozaki, unpublished data. 7 U. Tokumoto and Y. Takahashi, manuscript in preparation. does not possess nitrogenase and is incapable of nitrogen fixation, the presence of the NIF-like system and the lack of other systems in this organism reinforce the premise that the NIF system is not specific for the Fe-S cluster formation of nitrogenase but is involved in the Fe-S cluster assembly for nitrogenase and non-nitrogenase proteins, as proposed for the NIFlike system in H. pylori (13). Our in vivo complementation of a temperature-dependent growth defect of the E. coli isc/suf mutant strain by expression of a heterologous NIF-like system indicates that the NIF-like system plays an interchangeable role in the Fe-S cluster assembly of non-nitrogenase proteins with the ISC or SUF system under anaerobic conditions. Despite common catalytic and scaffold mechanisms shared by the NIF, ISC, and SUF systems, there are a number of differences among these systems (10); the ISC and SUF systems appear to be considerably more complex than the NIF system. For example, heat-shock-cognate (Hsc) proteins have been suggested to have chaperone-like functions in the ISC system for the formation of transient Fe-S clusters or insertion into various target proteins in bacteria and yeast (7,26,30,50,51). Our in vivo complementation study revealed that the NIF-like system does not require any additional component other than NifS and NifU for Fe-S cluster assembly under anaerobic conditions. However, it is also possible that as yet unidentified proteins that remain in the isc/suf-mutant strain of E. coli function together with the exogenous NifS and NifU. It is also conceivable that the NIF-like system is shared by other anaerobic parasitic organisms. However, two other well-characterized anaerobic protozoan parasites G. lamblia and T. vaginalis appear to possess the ISC and lack the NIF system (32,34). Thus, the presence of the NIF-like system is likely unique to E. histolytica, which might be attributable to a rare horizontal gene transfer from a NIF-like-containing prokaryotic organism (see below).
Several lines of evidence support close kinship between the amebic NifS and NifU and their homologs from ⑀-proteobacteria. First, phylogenetic analyses indicate that E. histolytica and ⑀-proteobacteria represent an independent clade well separated from other NifS and NifU homologs. Second, this close phylogenetic association is also supported by the common insertions and deletions of amino acids shared by NifS and NifU from these organisms. Third, the multimeric structure of the amebic and H. pylori NifU is identical (i.e. tetramer), whereas NifU from A. vinelandii and IscU from E. coli and yeast form a dimer. Fourth, UV/visible absorption spectra and specific activity of EhNifS were comparable to the H. pylori NifS but notably different from A. vinelandii. These data suggest that amebic nif-like genes were likely obtained from an ancestral organism currently represented by ⑀-proteobacteria by lateral gene transfer, as suggested for other metabolic enzymes that are proposed to have been transferred from Archaea and/or bacteria by a similar mechanism (52)(53)(54).
Fractionation of the crude extract of E. histolytica by anionexchange chromatography revealed that at least three groups of enzymes, CS, MGL, and NifS, contributed to cysteine desulfurase activity, i.e. activity to mobilize the sulfur or sulfide from L-cysteine, and thus possibly provide sulfur for Fe-S cluster synthesis. Although both CS and MGL showed cysteine desulfurase activity 6 (see also "Results" and Ref. 16 for E. coli CS) and also in vitro activity to convert an apo form of recombinant amebic [4Fe-4S] 2ϩ ferredoxin (53, 55) into a holo form, 6 we argue that EhNifS is the sole protein that functions in Fe-S cluster biosynthesis in vivo. Expression of CS or MGL (i.e. EhCS1 or EhMGL2), when coexpressed with EhNifU, did not complement the Fe-S cluster formation of the isc/suf-mutant strain of E. coli (data not shown). This reinforces the previous observation on E. coli cysteine synthase A, B, and ␥-cystathionase (16) and indicates that the in vitro conversion assay of apo Fe-S protein does not reflect in vivo function.
We did not demonstrate in the present study the species of the temporal Fe-S cluster (i.e. [2Fe-2S] 2ϩ or [4Fe-4S] 2ϩ ) that formed on EhNifU in addition to the stable [2Fe-2S] 2ϩ cluster shown in Fig. 6. However, it is conceivable, by analogy to NifU from A. vinelandii involved in the Fe-S cluster formation of nitrogenase, that amebic NifU functions as an intermediate site for the transient [2Fe-2S] 2ϩ cluster assembly (12). The putative labile [2Fe-2S] 2ϩ , if present, was likely lost during purification under aerobic conditions. We also speculate that the transition of Fe-S clusters on EhNifU may occur under anaerobic conditions; one [4Fe-4S] 2ϩ cluster may be formed from two [2Fe-2S] ϩ2 clusters, as shown for Azotobacter IscU (5). Because the amebic NifU forms a tetramer, the mechanism of the [4Fe-4S] 2ϩ cluster formation from [2Fe-2S] 2ϩ on NifU may differ from Azotobacter NifU, which exists as a dimer. Because FIG. 9. Functional complementation of the isc/suf-mutant E. coli strain by coexpression of EhNifS and EhNifU under anaerobic conditions. pRKEhNifSU, pRKISC, or pRKSUF, which contains E. histolytica EhNifS and EhNifU, E. coli iscSUA-hscBA-fdx, or E. coli sufABCDSE operon, respectively, in pRKNMC, or pRKNMC alone was introduced into E. coli strain UT109 [⌬(iscSUA-hscBA)::Km r , ⌬(sufABCDSEU)::Gm r ] as described in "Experimental Procedures." The transformants were cultivated at the restrictive temperature under either aerobic or anaerobic conditions. E. histolytica is not a nitrogen-fixing organism, the NIF-like system is not necessarily specific for nitrogenase proteins, but they are also involved in Fe-S cluster assembly for other Fe-S proteins, as shown previously for H. pylori (13). Although all characterized or putative Fe-S proteins in the genome database of E. histolytica, including ferredoxins and pyruvate:ferredoxin oxidoreductase, likely contain 2[4Fe-4S] 2ϩ clusters, the fact that the amebic NIF-like system functions in the formation of the [2Fe-2S] 2ϩ cluster in E. coli indicates that the amebic NIF-like system is involved in the biosynthesis of all forms of Fe-S clusters.