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 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.
Highlights
The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EBI Data Bank with accession number(s) AB112427
A protein alignment of 26 NifS/IscS/SufS homologs from Archaea, bacteria, fungi, protists, plants, and metazoa revealed 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
All of the residues of the active site and amino acids implicated in the cysteine desulfurase activity were conserved: (i) His106, which is involved in the initial deprotonation of the substrate [9]; (ii) the PLP-binding site with the Schiff base-forming amino acids Lys208, Asp182, and Gln185 that bind the pyridine nitrogen and the phenolate oxygen of PLP, respectively; (iii) Thr76, His207, Ser/Thr205, and Thr243, 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 Cys330, which provides a reactive cysteinyl residue [3], as well as Arg356, Asn157, and Asn35, which anchor the cysteine with a salt bridge and hydrogen bond [9]
Summary
Fe-S, iron-sulfur; NIF, nitrogen fixation; ISC, iron-sulfur cluster; SUF, mobilization of sulfur; PLP, pyridoxal-5Ј-phosphate; ORF, open reading frame; rEhNifS, recombinant EhNifS; rEhNifU, recombinant EhNifU; DTT, dithiothreitol; GST, glutathione S-transferase; CS, cysteine synthase; MGL, methionine ␥-lyase. 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. The fact that the scaffold component IscU is 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. 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
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