Completing the hypusine pathway in Plasmodium.

In searching for new targets for antimalarials we investigated the biosynthesis of hypusine present in eukaryotic initiation factor-5A (eIF-5A) in Plasmodium. Here, we describe the cloning and expression of deoxyhypusine hydroxylase (DOHH), which completes the modification of eIF-5A through hydroxylation of deoxyhypusine. The dohh cDNA sequence revealed an ORF of 1236 bp encoding a protein of 412 amino acids with a calculated molecular mass of 46.45 kDa and an isoelectric point of 4.96. Interestingly, DOHH from Plasmodium has a FASTA SCORE of only 27 compared with its human ortholog and contains several matches similar to E-Z-type HEAT-like repeat proteins (IPR004155 (InterPro), PF03130 (Pfam), SM00567 (SMART) present in the phycocyanin lyase subunits of cyanobacteria. Purified DOHH protein displayed hydroxylase activity in a novel in vitro DOHH assay, but phycocyanin lyase activity was absent. dohh is present as a single-copy gene and is transcribed in the asexual blood stages of the parasite. A signal peptide at the N-terminus might direct the protein to a different cellular compartment. During evolution, Plasmodium falciparum acquired an apicoplast that lost its photosynthetic function. It is possible that plasmodial DOHH arose from an E/F-type phycobilin lyase that gained a new role in hydroxylation. Structured digital abstract: * MINT-7255047: DHS (uniprotkb:P49366) enzymaticly reacts (MI:0414) with eIF-5A (uniprotkb:Q710D1) by enzymatic studies (MI:0415) * MINT-7255326: DOHH (uniprotkb:Q8I701) enzymaticly reacts (MI:0414) with eIF-5A (uniprotkb:Q710D1) by enzymatic studies (MI:0415).

Cloning, expression and functional activity of deoxyhypusine synthase from Plasmodium vivax.

BACKGROUND: Plasmodium vivax is the most widespread human malaria parasite. However, genetic information about its pathogenesis is limited at present, due to the lack of a reproducible in vitro cultivation method. Sequencing of the Plasmodium vivax genome suggested the presence of a homolog of deoxyhypusine synthase (DHS) from P. falciparum, the key regulatory enzyme in the first committed step of hypusine biosynthesis. DHS is involved in cell proliferation, and thus a valuable drug target for the human malaria parasite P. falciparum. A comparison of the enzymatic properties of the DHS enzymes between the benign and severe Plasmodium species should contribute to our understanding of the differences in pathogenicity and phylogeny of both malaria parasites. RESULTS: We describe the cloning of a 1368 bp putative deoxyhypusine synthase gene (dhs) sequence from genomic DNA of P. vivax PEST strain Salvador I (Accession number AJ549098) after touchdown PCR. The corresponding protein was expressed and functionally characterized as deoxyhypusine synthase by determination of its specific activity and cross-reactivity to human DHS on a Western blot. The putative DHS protein from P. vivax displays a FASTA score of 75 relative to DHS from rodent malaria parasite, P. yoelii, and 74 relative to that from the human parasite, P. falciparum strain 3D7. The ORF encoding 456 amino acids was expressed under control of IPTG-inducible T7 promoter, and expressed as a protein of approximately 50 kDa (theoretically 52.7 kDa) in E. coli BL21 DE3 cells. The N-terminal histidine-tagged protein was purified by Nickel-chelate affinity chromatography under denaturing conditions. DHS with a theoretical pI of 6.0 was present in both eluate fractions. The specific enzymatic activity of DHS was determined as 1268 U/mg protein. The inhibitor, N-guanyl-1, 7-diaminoheptane (GC7), suppressed specific activity by 36-fold. Western blot analysis performed with a polyclonal anti-human DHS antibody revealed cross-reactivity to DHS from P. vivax, despite an amino acid identity of 44% between the proteins. CONCLUSION: We identify a novel DHS protein in the more benign malaria parasite,P. vivax, on the basis of specific enzymatic activity, cross-reactivity with a polyclonal antibody against human DHS, and amino acid identity with DHS homologs from the rodent malaria parasite, P. yoelii, and human P. falciparum strains.

Spermidine metabolism in parasitic protozoa--a comparison to the situation in prokaryotes, viruses, plants and fungi.

Targeting polyamines of parasitic protozoa in chemotherapy has attracted attention because polyamines might reveal novel drug targets for antiparasite therapies (Müller et al. 2001). The biological function of the triamine spermidine in parasitic protozoa has not been studied in great detail although the results obtained mainly imply three different functions, i.e., cell proliferation, cell differentiation, and biosynthesis of macromolecules. Sequence information from the malaria genome project databases and inhibitor studies provide evidence that the current status of spermidine research has to be extended since enzymes of spermidine metabolism are present in the parasite (Kaiser et al. 2001). Isolation and characterisation of these enzymes, i.e., deoxyhypusine synthase (EC 1.1.1.249) (DHS) and homospermidine synthase (EC 2.5.1.44) (HSS) might lead to valuable new targets in drug therapy. Currently research on spermidine metabolism is based on the deposition of the deoxyhypusine synthase nucleic acid sequence in GenBank while the activity of homospermidine synthase was deduced from inhibitor studies. Spermidine biosynthesis is catalyzed by spermidine synthase (EC 2.5.1.16) which transfers an aminopropyl moiety from decarboxylated S-adenosylmethionine to putrescine. Spermidine is also an important precursor in the biosynthesis of the unusual amino acid hypusine (Wolff et al. 1995) and the uncommon triamine homospermidine in eukaryotes, in particular in pyrrolizidine alkaloid-producing plants (Ober and Hartmann 2000). Hypusine is formed by a two-step enzymatic mechanism starting with the transfer of an aminobutyl moiety from spermidine to the epsilon-amino group of one of the lysine residues in the precursor protein of eukaryotic initiation factor eIF5A by DHS (Lee and Park 2000). The second step of hypusinylation is completed by deoxyhypusine hydroxylase (EC 1.14.9929) (Abbruzzese et al. 1985). Homospermidine formation in eukaryotes parallels deoxyhypusine formation in the way that in an NAD(+)-dependent reaction an aminobutyl moiety is transferred from spermidine. In the case of homospermidine synthase, however the acceptor is putrescine. Thus the triamine homospermidine consists of two symmetric aminobutyl moieties while there is one aminobutyl and one aminopropyl moiety present in spermidine. Here, we review the metabolism of the triamine spermidine with particular focus on the biosynthesis of hypusine and homospermidine in parasitic protozoa, i.e., Plasmodium, Trypanosoma and Leishmania, compared to that in prokaryotes i.e., Escherichia coli, a phytopathogenic virus and pyrrolizidine alkaloid-producing plants (Asteraceae) and fungi.