FRINK Linked Data Fragments server

Matches in SemOpenAlex for { <https://semopenalex.org/work/W2072863297> ?p ?o ?g. }

• W2072863297 endingPage "16874" @default.
• W2072863297 startingPage "16863" @default.
• W2072863297 abstract "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 ϵ-proteobacteria, suggesting that both of these genes were likely transferred by lateral gene transfer from an ancestor of ϵ-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 γ-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. 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 ϵ-proteobacteria, suggesting that both of these genes were likely transferred by lateral gene transfer from an ancestor of ϵ-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 γ-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) 1The abbreviations used are: Fe-S, iron-sulfur; NIF, nitrogen fixation; ISC, iron-sulfur cluster; SUF, mobilization of sulfur; PLP, pyridoxal-5′-phosphate; ORF, open reading frame; rEhNifS, recombinant EhNifS; rEhNifU, recombinant EhNifU; DTT, dithiothreitol; GST, glutathione S-transferase; CS, cysteine synthase; MGL, methionine γ-lyase. 1The abbreviations used are: Fe-S, iron-sulfur; NIF, nitrogen fixation; ISC, iron-sulfur cluster; SUF, mobilization of sulfur; PLP, pyridoxal-5′-phosphate; ORF, open reading frame; rEhNifS, recombinant EhNifS; rEhNifU, recombinant EhNifU; DTT, dithiothreitol; GST, glutathione S-transferase; CS, cysteine synthase; MGL, methionine γ-lyase. 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 (1Beinert H. Holm R.H. Munck E. Science. 1997; 277: 653-659Crossref PubMed Scopus (1497) Google Scholar). 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 (2Zheng L. White R.H. Cash V.L. Jack R.F. Dean D.R. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 2754-2758Crossref PubMed Scopus (498) Google Scholar, 3Zheng L. White R.H. Cash V.L. Dean D.R. Biochemistry. 1994; 33: 4714-4720Crossref PubMed Scopus (352) Google Scholar, 4Zheng L. Cash V.L. Flint D.H. Dean D.R. J. Biol. Chem. 1998; 273: 13264-13272Abstract Full Text Full Text PDF PubMed Scopus (573) Google Scholar, 5Agar J.N. Krebs C. Frazzon J. Huynh B.H. Dean D.R. Johnson M.K. Biochemistry. 2000; 39: 7856-7862Crossref PubMed Scopus (384) Google Scholar, 6Tokumoto U. Takahashi Y. J. Biochem. (Tokyo). 2001; 130: 63-71Crossref PubMed Scopus (214) Google Scholar, 7Takahashi Y. Nakamura M. J. Biochem. (Tokyo). 1999; 126: 917-926Crossref PubMed Scopus (226) Google Scholar, 8Takahashi Y. Tokumoto U. J. Biol. Chem. 2002; 277: 28380-28383Abstract Full Text Full Text PDF PubMed Scopus (350) Google Scholar). 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 (2Zheng L. White R.H. Cash V.L. Jack R.F. Dean D.R. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 2754-2758Crossref PubMed Scopus (498) Google Scholar, 3Zheng L. White R.H. Cash V.L. Dean D.R. Biochemistry. 1994; 33: 4714-4720Crossref PubMed Scopus (352) Google Scholar). NifS is a homodimer of two identical subunits of a pyridoxal-5′-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 (3Zheng L. White R.H. Cash V.L. Dean D.R. Biochemistry. 1994; 33: 4714-4720Crossref PubMed Scopus (352) Google Scholar) and nucleophilic attack of the reactive cysteine by a conserved histidine residue near the active site (9Kaiser J.T. Clausen T. Bourenkow G.P. Bartunik H.D. Steinbacher S. Huber R. J. Mol. Biol. 2000; 297: 451-464Crossref PubMed Scopus (125) Google Scholar), 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 (10Yuvaniyama P. Agar J.N. Cash V.L. Johnson M.K. Dean D.R. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 599-604Crossref PubMed Scopus (271) Google Scholar). The NifU protein possesses two distinct types of iron-binding sites (10Yuvaniyama P. Agar J.N. Cash V.L. Johnson M.K. Dean D.R. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 599-604Crossref PubMed Scopus (271) Google Scholar). One of these binding sites, located in the central third of the NifU protein, binds a stable permanent [2Fe-2S]2+ cluster per subunit as shown in A. vinelandii (11Fu W. Jack R.F. Morgan T.V. Dean D.R. Johnson M.K. Biochemistry. 1994; 33: 13455-13463Crossref PubMed Scopus (129) Google Scholar). The second type of site, located within the amino-terminal third of the NifU protein, binds a labile mononuclear iron and/or labile cluster (11Fu W. Jack R.F. Morgan T.V. Dean D.R. Johnson M.K. Biochemistry. 1994; 33: 13455-13463Crossref PubMed Scopus (129) Google Scholar). Site-directed mutagenesis of the cluster-ligated cysteine residues of NifU from A. vinelandii has shown that the permanent [2Fe-2S]2+ clusters play an essential role in the maturation of nitrogenase (12Agar J.N. Yuvaniyama P. Jack R.F. Cash V.L. Smith A.D. Dean D.R. Johnson M.K. J. Biol. Inorg. Chem. 2000; 5: 167-177Crossref PubMed Scopus (107) Google Scholar). The NIF system has been identified in only nitrogen-fixing bacteria and non-diazotrophic ϵ-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 (13Olson J.W. Agar J.N. Johnson M.K. Maier R.J. Biochemistry. 2000; 39: 16213-16219Crossref PubMed Scopus (86) Google Scholar). 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 (4Zheng L. Cash V.L. Flint D.H. Dean D.R. J. Biol. Chem. 1998; 273: 13264-13272Abstract Full Text Full Text PDF PubMed Scopus (573) Google Scholar, 6Tokumoto U. Takahashi Y. J. Biochem. (Tokyo). 2001; 130: 63-71Crossref PubMed Scopus (214) Google Scholar, 7Takahashi Y. Nakamura M. J. Biochem. (Tokyo). 1999; 126: 917-926Crossref PubMed Scopus (226) Google Scholar, 14Schwartz C.J. Djaman O. Imlay J.A. Kiley P.J. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 9009-9014Crossref PubMed Scopus (257) Google Scholar, 15Kato S. Mihara H. Kurihara T. Takahashi Y. Tokumoto U. Yoshimura T. Esaki N. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 5948-5952Crossref PubMed Scopus (115) Google Scholar, 16Flint D.H. J. Biol. Chem. 1996; 271: 16068-16074Abstract Full Text Full Text PDF PubMed Scopus (59) Google Scholar). 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 (NifIscA) and responsible for binding labile Fe-S clusters (5Agar J.N. Krebs C. Frazzon J. Huynh B.H. Dean D.R. Johnson M.K. Biochemistry. 2000; 39: 7856-7862Crossref PubMed Scopus (384) Google Scholar, 17Wu G. Mansy S.S. Hemann C. Hille R. Surerus K.K. Cowan J.A. J. Biol. Inorg. Chem. 2002; 7: 526-532Crossref PubMed Scopus (70) Google Scholar) and its transfer to apoproteins (18Ollagnier-de-Choudens S. Mattioli T. Takahashi Y. Fontecave M. J. Biol. Chem. 2001; 276: 22604-22607Abstract Full Text Full Text PDF PubMed Scopus (171) Google Scholar). 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 (19Hoff K.G. Silberg J.J. Vickery L.E. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 7790-7795Crossref PubMed Scopus (200) Google Scholar). Protein-protein interaction between each of the components of the ISC system has also been demonstrated (18Ollagnier-de-Choudens S. Mattioli T. Takahashi Y. Fontecave M. J. Biol. Chem. 2001; 276: 22604-22607Abstract Full Text Full Text PDF PubMed Scopus (171) Google Scholar, 20Tokumoto U. Nomura S. Minami Y. Mihara E. Shin-ichiro K. Kurihara T. Esaki N. Kanazawa H. Matsubara H. Takahashi Y. J. Biochem. (Tokyo). 2002; 131: 713-719Crossref PubMed Scopus (97) Google Scholar). 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 (8Takahashi Y. Tokumoto U. J. Biol. Chem. 2002; 277: 28380-28383Abstract Full Text Full Text PDF PubMed Scopus (350) Google Scholar). 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 (8Takahashi Y. Tokumoto U. J. Biol. Chem. 2002; 277: 28380-28383Abstract Full Text Full Text PDF PubMed Scopus (350) Google Scholar). It has also been shown that the SUF system plays a role for Fe-S cluster assembly and/or repair under oxidative stress conditions (21Nachin L. EL Hassouni M. Loiseau L. Expert D. Barras F. Mol. Microbiol. 2001; 39: 960-972Crossref PubMed Scopus (153) Google Scholar, 22Zheng M. Wang X. Templeton L.J. Smulski D.R. LaRossa R.A. Storz G. J. Bacteriol. 2001; 183: 4562-4570Crossref PubMed Scopus (653) Google Scholar) and iron starvation (23Patzer S.I. Hantke K. J. Bacteriol. 1999; 181 (3307): 3307Crossref PubMed Google Scholar). 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 (21Nachin L. EL Hassouni M. Loiseau L. Expert D. Barras F. Mol. Microbiol. 2001; 39: 960-972Crossref PubMed Scopus (153) Google Scholar, 24Nachin L. Loiseau L. Expert D. Barras F. EMBO J. 2003; 22: 427-437Crossref PubMed Scopus (219) Google Scholar). 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 (24Nachin L. Loiseau L. Expert D. Barras F. EMBO J. 2003; 22: 427-437Crossref PubMed Scopus (219) Google Scholar), and SufE, which interacts with SufS and stimulates its cysteine desulfurase activity (25Loiseau L. Ollagnier-de-Choudens S. Nachin L. Fontecave M. Barras F. J. Biol. Chem. 2003; 278: 38352-38359Abstract Full Text Full Text PDF PubMed Scopus (171) Google Scholar). 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 (26Strain J. Lorenz C.R. Bode J. Garland S. Smolen G.A. Ta D.T. Vickery L.E. Culotta V.C. J. Biol. Chem. 1998; 273: 31138-31144Abstract Full Text Full Text PDF PubMed Scopus (168) Google Scholar). IscS homologs are produced in the cytosol and transported to mitochondria (27Nakai Y. Yoshihara Y. Hayashi H. Kagamiyama H. FEBS Lett. 1998; 433: 143-148Crossref PubMed Scopus (50) Google Scholar) and the nucleus in yeasts and mammals (28Schilke B. Voisine C. Beinert H. Craig E. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 10206-10211Crossref PubMed Scopus (268) Google Scholar, 29Tong W.H. Jameson G.N.L. Huynh B.H. Rouault T.A. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 9762-9767Crossref PubMed Scopus (182) Google Scholar). An independent study also revealed that mitochondria play a crucial role in the cluster formation of extramitochondrial Fe-S proteins (30Kispal G. Csere P. Prohl C. Lill R. EMBO J. 1999; 18: 3981-3989Crossref PubMed Scopus (585) Google Scholar). 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 (31Tong W.H. Rouault T. EMBO J. 2000; 19: 5692-5700Crossref PubMed Scopus (168) Google Scholar, 32Muhlenhoff U. Lill R. Biochim. Biophys. Acta. 2000; 1459: 370-382Crossref PubMed Scopus (181) Google Scholar). 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 (33Ellis K.E.S. Clough B. Saldanha J.W. Wilson R.J.M. Mol. Microbiol. 2001; 41: 973-981Crossref PubMed Scopus (87) Google Scholar), and two anaerobic protozoa, Giardia lamblia and Trichomonas vaginalis (34Tachezy J. Sanchez L.B. Muller M. Mol. Biol. Evol. 2001; 18: 1919-1928Crossref PubMed Scopus (140) Google Scholar). 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 (35Martin W. Muller M. Nature. 1998; 392: 37-41Crossref PubMed Scopus (895) Google Scholar). 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 (36Tovar J. Leon-Avila G. Sanchez L.B. Sutak R. Tachezy J. Van Der Giezen M. Hernandez M. Muller M. Lucocq J.M. Nature. 2003; 426: 172-176Crossref PubMed Scopus (409) Google Scholar) 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 ϵ-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. Chemicals and Reagents—All chemicals of analytical grade were purchased from Wako (Tokyo, Japan) unless stated otherwise. l-cysteine, l-cystine, O-acetyl serine, N-acetyl cysteine, dl-homocysteine, d-cysteine, sodium sulfide, O-phenanthroline, hydroxylamine, N,N-dimethyl-p-phenylenediamine sulfate, ferrous ammonium sulfate, PLP, 2-(N-morpholino) ethanesulfonic acid, HEPES, N-[tris(hydroxymethyl-)methyl]-3-aminopropanesulfonic acid, 3-(cyclohexylamino)-1-propane-sulfonic acid, ampicillin, and carbenicillin disodium salt were purchased from Sigma-Aldrich. Microorganisms and Cultivation—Trophozoites of E. histolytica strain HM-1:IMSS cl-6 (37Diamond L.S. Mattern C.F. Bartgis I.L. J. Virol. 1972; 9: 326-341Crossref PubMed Google Scholar) were maintained axenically in Diamond's BI-S-33 medium (38Diamond L.S. Harlow D.R. Cunnick C.C. Trans. R. Soc. Trop. Med. Hyg. 1978; 72: 431-432Abstract Full Text PDF PubMed Scopus (1565) Google Scholar) at 35.5 °C. Trophozoites were harvested in the late-logarithmic growth phase 2-3 days after inoculation of 1/12 to 1/6 of the total culture volume and washed twice with ice-cold phosphate-buffered saline, pH 7.4, at 4 °C. E. coli strains BL21 (DE3) and DH5α were purchased from Novagen (Madison, WI) and Invitrogen, respectively. Search of the E. histolytica Genome Database—The E. histolytica genome databases (contigs and singletons) at The Institute for Genomic Research 2Internet address: www.tigr.org/tdb/. and Sanger Institute 3Internet address: www.sanger.ac.uk/Projects/E_histolytica/. 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 4Internet address: www.ncbi.nih.gov/. using the BLASTP search. Alignment and phylogenetic analysis were performed with CLUSTAL W version 1.81 (39Thompson J.D. Higgins D.G. Gibson T.J. Nucleic Acids Res. 1994; 22: 4673-4680Crossref PubMed Scopus (55399) Google Scholar) 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 (40Page R.D. Comput. Appl. Biosci. 1996; 12: 357-358PubMed Google Scholar). 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 (41Nozaki T. Asai T. Kobayashi S. Ikegami F. Noji M. Saito K. Takeuchi T. Mol. Biochem. Parasitol. 1998; 97: 33-44Crossref PubMed Scopus (82) Google Scholar) as a template to construct plasmids to produce EhNifS and EhNifU fusion proteins with a histidine tag or glutathione S-transferase at the amino terminus, respectively. The EhNifS ORF was amplified with a sense (5′-CCTGGATCCGATGCAAAGTACAAAATCAGT-3′) and an antisense (5′-CCAGGATCCTTAAGCATATGTTGATGATAATTGTC-3′) 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 ∼1.2-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 (5′-ATGTCAAAGAATAAATTAATTGGTGGAGC-3′) and an antisense (5′-CTAATCTTTCTTTTTGATATTTAAGGT-3′) 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 EcoRI-digested 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 DH5α cells, respectively. Expression of the recombinant EhNifS (rEhNifS) and EhNifU (rEhNifU) fusion proteins was induced with 1 mm isopropyl-β-thiogalactoside at 30 °C for 5-6 h. The rEhNifS and EhNifU fusion proteins were purified using a Ni2+-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 μg/ml of lysozyme, and Complete Mini EDTA free protease inhibitor mixture (Roche, Tokyo, Japan), and centrifuged at 24,000 × g for 15 min at 4 °C. The histidine-tagged rEhNifS protein was eluted from the Ni2+-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 μg/ml of lysozyme. After the GSTEhNifU 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 - 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 100-200-fold volumes of 50 mmN-[tris(hydroxymethyl)methyl]-3-aminopropanesulfonic acid/NaOH (pH 9.0) with two buffer exchanges, as described previously for the Synechocystis enzyme (42Jaschkowitz K. Seidler A. Biochemistry. 2000; 39: 3416-3423Crossref PubMed Scopus (40) Google Scholar). The cysteine desulfurase activity of EhNifS was monitored based on the production of sulfide using l-cysteine as the substrate (43Siegel L.M. Anal. Biochem. 1965; 11: 126-132Crossref PubMed Scopus (370) Google Scholar). The standard EhNifS reaction was performed in 200 μl 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 MgCl2, 0.5 mm substrate (l-cysteine), and appropriate amounts (25-50 μg) 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 μl of 20 mmN,N-dimethyl-p-phenylenediamine sulfate in 7.2 n HCl and 20 μl of 30 mm FeCl3 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 (A670) of the supernatant was measured. Na2S (0-100 μm) was used as the standard. Elemental sulfur (S0) was measured by the cyanolysis method (2Zheng L. White R.H. Cash V.L. Jack R.F. Dean D.R. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 2754-2758Crossref PubMed Scopus (498) Google Scholar, 44Wood J.L. Methods Enzymol. 1987; 143: 25-29Crossref PubMed Scopus (168) Google Scholar) 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 (2Zheng L. White R.H. Cash V.L. Jack R.F. Dean D.R. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 2754-2758Crossref PubMed Scopus (498) Google Scholar). 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 - 20 °C for 24 h. However, it was reduced to 25-35% when stored without any additive at room temperature, 4, or - 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 (13Olson J.W. Agar J.N. Johnson M.K. Maier R.J. Biochemistry. 2000; 39: 16213-16219Crossref PubMed Scopus (86) Google Scholar). The EhNifU samples were acidified by the addition of 3-5 μl 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 μl 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 (A512) was measured with 0-100 μm of ferrous ammonium sulfate as the standard. Anion-Exchange Chromatography of the Native and Recombinant EhNifS—Approximately 2 × 107E. 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 μg/ml of trans-epoxysuccinyl-l-leucylamido-(4-guanidino)butane and t" @default.
• W2072863297 created "2016-06-24" @default.
• W2072863297 creator A5013624532 @default.
• W2072863297 creator A5030734026 @default.
• W2072863297 creator A5038826116 @default.
• W2072863297 creator A5043332865 @default.
• W2072863297 creator A5057061617 @default.
• W2072863297 date "2004-04-01" @default.
• W2072863297 modified "2023-10-13" @default.
• W2072863297 title "An Intestinal Parasitic Protist, Entamoeba histolytica, Possesses a Non-redundant Nitrogen Fixation-like System for Iron-Sulfur Cluster Assembly under Anaerobic Conditions" @default.
• W2072863297 cites W1532421291 @default.
• W2072863297 cites W1630260213 @default.
• W2072863297 cites W1655025181 @default.
• W2072863297 cites W1960048856 @default.
• W2072863297 cites W1969490516 @default.
• W2072863297 cites W1970359761 @default.
• W2072863297 cites W1972222847 @default.
• W2072863297 cites W1972789197 @default.
• W2072863297 cites W1984882823 @default.
• W2072863297 cites W1985844569 @default.
• W2072863297 cites W1994264299 @default.
• W2072863297 cites W1995950751 @default.
• W2072863297 cites W1998134754 @default.
• W2072863297 cites W1998142761 @default.
• W2072863297 cites W2000755989 @default.
• W2072863297 cites W2000959843 @default.
• W2072863297 cites W2002593242 @default.
• W2072863297 cites W2003418336 @default.
• W2072863297 cites W2003531759 @default.
• W2072863297 cites W2006178088 @default.
• W2072863297 cites W2007826649 @default.
• W2072863297 cites W2014601814 @default.
• W2072863297 cites W2015661405 @default.
• W2072863297 cites W2024152854 @default.
• W2072863297 cites W2028890624 @default.
• W2072863297 cites W2046347557 @default.
• W2072863297 cites W2047906206 @default.
• W2072863297 cites W2051317564 @default.
• W2072863297 cites W2059831206 @default.
• W2072863297 cites W2060121377 @default.
• W2072863297 cites W2061510311 @default.
• W2072863297 cites W2062424661 @default.
• W2072863297 cites W2064756788 @default.
• W2072863297 cites W2072207453 @default.
• W2072863297 cites W2072238174 @default.
• W2072863297 cites W2077752559 @default.
• W2072863297 cites W2083359532 @default.
• W2072863297 cites W2087921651 @default.
• W2072863297 cites W2088723535 @default.
• W2072863297 cites W2089518921 @default.
• W2072863297 cites W2090079874 @default.
• W2072863297 cites W209726389 @default.
• W2072863297 cites W2102454051 @default.
• W2072863297 cites W2102489917 @default.
• W2072863297 cites W2103205373 @default.
• W2072863297 cites W2106882534 @default.
• W2072863297 cites W2123296298 @default.
• W2072863297 cites W2134701031 @default.
• W2072863297 cites W2142477354 @default.
• W2072863297 cites W2143453049 @default.
• W2072863297 cites W2153371171 @default.
• W2072863297 cites W2167495150 @default.
• W2072863297 cites W4255324595 @default.
• W2072863297 doi "https://doi.org/10.1074/jbc.m313314200" @default.
• W2072863297 hasPubMedId "https://pubmed.ncbi.nlm.nih.gov/14757765" @default.
• W2072863297 hasPublicationYear "2004" @default.
• W2072863297 type Work @default.
• W2072863297 sameAs 2072863297 @default.
• W2072863297 citedByCount "113" @default.
• W2072863297 countsByYear W20728632972012 @default.
• W2072863297 countsByYear W20728632972013 @default.
• W2072863297 countsByYear W20728632972014 @default.
• W2072863297 countsByYear W20728632972015 @default.
• W2072863297 countsByYear W20728632972016 @default.
• W2072863297 countsByYear W20728632972017 @default.
• W2072863297 countsByYear W20728632972018 @default.
• W2072863297 countsByYear W20728632972019 @default.
• W2072863297 countsByYear W20728632972021 @default.
• W2072863297 countsByYear W20728632972022 @default.
• W2072863297 countsByYear W20728632972023 @default.
• W2072863297 crossrefType "journal-article" @default.
• W2072863297 hasAuthorship W2072863297A5013624532 @default.
• W2072863297 hasAuthorship W2072863297A5030734026 @default.
• W2072863297 hasAuthorship W2072863297A5038826116 @default.
• W2072863297 hasAuthorship W2072863297A5043332865 @default.
• W2072863297 hasAuthorship W2072863297A5057061617 @default.
• W2072863297 hasBestOaLocation W20728632971 @default.
• W2072863297 hasConcept C104317684 @default.
• W2072863297 hasConcept C164866538 @default.
• W2072863297 hasConcept C178790620 @default.
• W2072863297 hasConcept C181199279 @default.
• W2072863297 hasConcept C181440489 @default.
• W2072863297 hasConcept C185592680 @default.
• W2072863297 hasConcept C199360897 @default.
• W2072863297 hasConcept C2776867272 @default.
• W2072863297 hasConcept C2777852564 @default.
• W2072863297 hasConcept C2778330419 @default.
• W2072863297 hasConcept C41008148 @default.

• next