Giardia intestinalis (syn. lamblia) is one of the most widespread intestinal protozoan pathogens worldwide, causing hundreds of thousands of cases of diarrhoea each year. Giardia is a member of the diplomonads, often described as an ancient protist group whose primitive nature is suggested by the lack of typical eukaryotic organelles (for example, mitochondria, peroxisomes), the presence of a poorly developed endomembrane system and by their early branching in a number of gene phylogenies. The discovery of nuclear genes of putative mitochondrial ancestry in Giardia and the recent identification of mitochondrial remnant organelles in amitochondrial protists such as Entamoeba histolytica and Trachipleistophora hominis suggest that the eukaryotic amitochondrial state is not a primitive condition but is rather the result of reductive evolution. Using an in vitro protein reconstitution assay and specific antibodies against IscS and IscU--two mitochondrial marker proteins involved in iron-sulphur cluster biosynthesis--here we demonstrate that Giardia contains mitochondrial remnant organelles (mitosomes) bounded by double membranes that function in iron-sulphur protein maturation. Our results indicate that Giardia is not primitively amitochondrial and that it has retained a functional organelle derived from the original mitochondrial endosymbiont.

Pyridoxal-5'-phosphate-dependent cysteine desulfurase (IscS) is an essential enzyme in the assembly of FeS clusters in bacteria as well as in the mitochondria of eukaryotes. Although FeS proteins are particularly important for the energy metabolism of amitochondrial anaerobic eukaryotes, there is no information about FeS cluster formation in these organisms. We identified and sequenced two IscS homologs of Trichomonas vaginalis (TviscS-1 and TviscS-2) and one of Giardia intestinalis (GiiscS). TviscS-1, TviscS-2, and GiiscS possess the typical conserved regions implicated in cysteine desulfurase activity. N-termini of TviscS-1 and TviscS-2 possess eight amino acid extensions, which resemble the N-terminal presequences that target proteins to hydrogenosomes in trichomonads. No presequence was evident in GiiscS from Giardia, an organism that apparently lacks hydrogenosmes or mitochondria. Phylogenetic analysis showed a close relationship among all eukaryotic IscS genes including those of amitochondriates. IscS of proteobacteria formed a sister group to the eukaryotic clade, suggesting that isc-related genes were present in the proteobacterial endosymbiotic ancestor of mitochondria and hydrogenosomes. NifS genes of nitrogen-fixing bacteria, which are IscS homologs required for specific formation of FeS clusters in nitrogenase, formed a more distant group. The phylogeny indicates the presence of a common mechanism for FeS cluster formation in mitochondriates as well as in amitochondriate eukaryotes. Furthermore, the analyses support a common origin of Trichomonas hydrogenosomes and mitochondria, as well as secondary loss of mitochondrion/hydrogenosome-like organelles in Giardia.

The gene coding for the acetyl-CoA synthetase (ADPforming) from the amitochondriate eukaryote Giardia lamblia has been expressed in Escherichia coli. The recombinant enzyme exhibited the same substrate specificity as the native enzyme, utilizing acetyl-CoA and adenine nucleotides as preferred substrates and less efficiently, propionyl-and succinyl-CoA. N-and C-terminal parts of the G. lamblia acetyl-CoA synthetase sequence were found to be homologous to the ␣-and ␤-subunits, respectively, of succinyl-CoA synthetase. Sequence analysis of homologous enzymes from various bacteria, archaea, and the eukaryote, Plasmodium falciparum, identified conserved features in their organization, which allowed us to delineate a new superfamily of acyl-CoA synthetases (nucleoside diphosphateforming) and its signature motifs. The representatives of this new superfamily of thiokinases vary in their domain arrangement, some consisting of separate ␣-and ␤-subunits and others comprising fusion proteins in ␣-␤ or ␤-␣ orientation. The presence of homologs of acetylCoA synthetase (ADP-forming) in such human pathogens as G. lamblia, Yersinia pestis, Bordetella pertussis, Pseudomonas aeruginosa, Vibrio cholerae, Salmonella typhi, Porphyromonas gingivalis, and the malaria agent P. falciparum suggests that they might be used as potential drug targets. ATP or GTP formation by substrate level phosphorylation of ADP or GDP at the expense of the free energy of the thioester bond of acyl-CoA is a well known and much studied biochemical process (1). The formation of NTP in this reaction leads to the liberation of CoA and a free organic acid (Equation 1).

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The enteric protist parasites Entamoeba histolytica and Entamoeba dispar possess a cysteine biosynthetic pathway, unlike their mammalian host, and are capable of de novo production of L-cysteine. We cloned and characterized cDNAs that encode the regulated enzyme serine acetyltransferase (SAT) in this pathway from these amoebae by genetic complementation of a cysteine-auxotrophic Escherichia coli strain with the amoebic cDNA libraries. The deduced amino acid sequences of the amoebic SATs exhibited, within the most conserved region, 36 -52% identities with the bacterial and plant SATs. The amoebic SATs contain a unique insertion of eight amino acids, also found in the corresponding region of a plasmid-encoded SAT from Synechococcus sp., which showed the highest overall identities to the amoebic SATs. Phylogenetic reconstruction also revealed a close kinship of the amoebic SATs with cyanobacterial SATs. Biochemical characterization of the recombinant E. histolytica SAT revealed several enzymatic features that distinguished the amoebic enzyme from the bacterial and plant enzymes: 1) inhibition by L-cysteine in a competitive manner with L-serine; 2) inhibition by Lcystine; and 3) no association with cysteine synthase. Genetically engineered amoeba strains that overproduced cysteine synthase and SAT were created. The cysteine synthase-overproducing amoebae had a higher level of cysteine synthase activity and total thiol content and revealed increased resistance to hydrogen peroxide. These results indicate that the cysteine biosynthetic pathway plays an important role in antioxidative defense of these enteric parasites.The cysteine biosynthetic pathway plays an important role in incorporation of inorganic sulfur into organic compounds. In bacteria and plants, L-cysteine is the precursor of most sulfurcontaining metabolites including methionine and glutathione. Extracellular sulfate is first imported by specific transporters. Intracellular sulfate is then activated by ATP sulfurylase and adenosine-5Ј-phosphosulfate kinase to form adenosine-5Ј-phosphosulfate and 3Ј-phosphoadenosine 5Ј-phosphosulfate, respectively. These activated sulfates are further reduced to sulfide. Sulfide then reacts with O-acetylserine, which is produced from serine and acetyl-CoA by serine acetyltransferase (SAT, 1 EC 2.3.1.30). This final reaction forming L-cysteine, by transfer of the alanyl moiety of O-acetylserine to sulfide, is catalyzed by L-cysteine synthase (CS; O-acetyl-L-serine (thiol)-lyase, EC 4.2.99.8). In contrast to bacteria and plants, animals are presumed to lack the sulfur assimilation pathway and thus require exogenous methionine as a sulfur source. Biochemical studies using purified (1-4) and recombinant enzymes (5), as well as a genetic approach using a yeast two-hybrid system, revealed that CS and SAT form a heteromeric complex. SAT activity and O-acetylserine availability are the major regulatory factors in the control of the L-cysteine production in plants (6, 7). Cytosolic isoforms of the SAT from Citrullus vulgaris and A...