Modification of eukaryotic initiation factor 5A from Plasmodium vivax by a truncated deoxyhypusine synthase from Plasmodium falciparum: An enzyme with dual enzymatic properties

Kaiser, Annette; Hammels, Ina; Gottwald, A; Marwa, Nassar; Zaghloul, Mai A. Saad; Motaal, Basma Abdal; Hauber, Joachim; Hoerauf, Achim

doi:10.1016/j.bmc.2007.06.026

Cited by 23 publications

(17 citation statements)

References 34 publications

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“…S1) suggesting its presence in the asexual blood stage of the parasite. These results paralleled those obtained from previous RT-PCR experiments on eIF-5A and dhs genes in different developmental stages within the infected erythrocyte [16].…”

Section: Resultssupporting

confidence: 89%

“…The radioactive filter assay is rather inaccurate because of unspecific binding of [ 14 C]labeled spermidine [25]. We combined eIF-5A from P. vivax and human DHS for the synthesis of deoxyhypusine because the enzymatic activity of the human ortholog is significantly higher [16]. Modified eIF-5A was enriched by two sequential steps of size-exclusion chromatography which removed the DHS enzyme ( Fig.…”

Section: Discussionmentioning

confidence: 99%

“…First, we modified the eIF-5A protein from P. vivax to the deoxyhypusinylated form, i.e. eIF-5A (Dhp) using human DHS, which has a much higher specific enzymatic activity than the parasitic enzyme [16]. eIF-5A (Dhp) was isolated using two size-exclusion chromatography steps, i.e.…”

Section: Expression Purification and Functional Analysis Of P Falcimentioning

confidence: 99%

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Completing the hypusine pathway in Plasmodium

et al. 2009

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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 http://mint.bio.uniroma2.it/mint/search/interaction.do?interactionAc=MINT-7255047: DHS (uniprotkb:http://www.ebi.uniprot.org/entry/P49366) enzymaticly reacts (http://www.ebi.ac.uk/ontology-lookup/?termId=MI:0414) with eIF‐5A (uniprotkb:http://www.ebi.uniprot.org/entry/Q710D1) by enzymatic studies (http://www.ebi.ac.uk/ontology-lookup/?termId=MI:0415) http://mint.bio.uniroma2.it/mint/search/interaction.do?interactionAc=MINT-7255326: DOHH (uniprotkb:http://www.ebi.uniprot.org/entry/Q8I701) enzymaticly reacts (http://www.ebi.ac.uk/ontology-lookup/?termId=MI:0414) with eIF‐5A (uniprotkb:http://www.ebi.uniprot.org/entry/Q710D1) by enzymatic studies (http://www.ebi.ac.uk/ontology-lookup/?termId=MI:0415)

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Section: Resultssupporting

confidence: 89%

Section: Discussionmentioning

confidence: 99%

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Completing the hypusine pathway in Plasmodium

et al. 2009

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“…To facilitate the first step of eIF-5A modification, purified recombinant eIF-5A from P. vivax was modified with recombinant human DHS, which has low substrate affinity but higher specific enzyme activity than the parasitic enzyme (Kaiser et al 2007), and achieves the same modification. Both human DHS and P. vivax eIF-5A were N-terminally histidine tagged fusion proteins in recombinant pET-15b vectors and successfully expressed in E. coli BL21.…”

Section: Enzymatic Synthesis Of Hypusinementioning

confidence: 99%

New insights into novel inhibitors against deoxyhypusine hydroxylase from plasmodium falciparum: compounds with an iron chelating potential

et al. 2015

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Deoxyhypusine hydroxylase (DOHH) is a dinuclear iron enzyme required for hydroxylation of the aminobutyl side chain of deoxyhypusine in eukaryotic translation initiation factor 5A (eIF-5A), the second step in hypusine biosynthesis. DOHH has been recently identified in P. falciparum and P. vivax. Both enzymes have very peculiar features including E-Z type HEAT-like repeats and a diiron centre in their active site. Both proteins share only 26 % amino acid identity to the human paralogue. Hitherto, no X-ray structure exists from either enzyme. However, structural predictions based on the amino acid sequence of the active site in comparison to the human enzyme show that four conserved histidine and glutamate residues provide the coordination sites for chelating the ferrous iron ions. Recently, we showed that P. vivax DOHH is inhibited by zileuton (N-[1-(1-benzothien-2-yl)ethyl]-N-hydroxyurea), a drug that is known for inhibiting human 5-lipoygenase (5-LOX) by the complexation of ferrous iron. A novel discovery program was launched to identify inhibitors of the P. falciparum DOHH from the Malaria Box, consisting of 400 chemical compounds, which are highly active in the erythrocytic stages of Malaria infections. In a first visual selection for potential ligands of ferrous iron, three compounds from different scaffold classes namely the diazonapthyl benzimidazole MMV666023 (Malaria Box plate A, position A03), the bis-benzimidazole MMV007384 (plate A, position B08), and a 1,2,5,-oxadiazole MMV665805 (plate A, position C03) were selected and subsequently evaluated in silico for their potential to complex iron ions. As a proof of principle, a bioanalytical assay was performed and the inhibition of hypusine biosynthesis was determined by GC-MS. All tested compounds proved to be active in this assay and MMV665805 exhibited the strongest inhibitory effect. Notably, the results were in accordance with the preliminary quantum-mechanical calculations suggesting the strongest iron complexation capacity for MMV665805. This compound might be a useful tool as well as a novel lead structure for inhibitors of P. falciparum DOHH.

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“…To evaluate Plasmodium DHS (Kaiser et al 2007) as a potential target in malaria therapy, we investigated the inhibitory effect of CNI-1493 (formerly known as semapimod) in different concentrations after Ni-affinity purification of the truncated protein (Table 1), because DHS expression was only possible after truncation of a signal peptide similar structure as previously described (Kaiser et al 2007). Three-fold inhibition of DHS activity was determined with 2 μM CNI-1493 (lower concentrations did not inhibit).…”

Section: Cni-1493 Inhibits P Falciparum Dhsmentioning

confidence: 99%

The guanylhydrazone CNI-1493: an inhibitor with dual activity against malaria—inhibition of host cell pro-inflammatory cytokine release and parasitic deoxyhypusine synthase

Specht¹,

Sarite²,

Hauber

et al. 2008

Parasitol Res

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Malaria is still a major cause of death in the tropics. There is an urgent need for new anti-malarial drugs because drug-resistant plasmodia frequently occur. Over recent years, we elucidated the biosynthesis of hypusine, a novel amino acid contained in eukaryotic initiation factor 5A (eIF-5A) in Plasmodium. Hypusine biosynthesis involves catalysis of deoxyhypusine synthase (DHS) in the first step of post-translational modification. In a screen for new inhibitors of purified plasmodium DHS, CNI-1493, a novel selective pro-inflammatory cytokine inhibitor used in clinical phase II for the treatment of Crohn's disease, inhibited the enzyme of the parasite 3-fold at a concentration of 2 microM. In vitro experiments with 200 microM CNI-1493 in Plasmodium-infected erythrocytes, which lack nuclei and DHS protein, showed a parasite clearance within 2 days. This can presumably be attributed to an anti-proliferating effect because of the inhibition of DHS by the parasite. The determined IC50 of CNI-1493 was 135.79 microM after 72 h. In vivo application of this substance in Plasmodium berghei ANKA-infected C57BL/6 mice significantly reduced parasitemia after dosage of 1 mg/kg or 4 mg/kg/body weight and prevented death of mice with cerebral malaria. This effect was paralleled by a decrease in serum TNF levels of the mice. We suggest that the new mechanism of CNI-1493 is caused by a decrease in modified eIF-5A biosynthesis with a downstream effect on the TNF synthesis of the host. From the current data, we consider CNI-1493 to be a promising drug for anti-malarial therapy because of its combined action, i.e., the decrease in eIF-5A biosynthesis of the parasite and host cell TNF biosynthesis.

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Modification of eukaryotic initiation factor 5A from Plasmodium vivax by a truncated deoxyhypusine synthase from Plasmodium falciparum: An enzyme with dual enzymatic properties

Cited by 23 publications

References 34 publications

Completing the hypusine pathway in Plasmodium

Completing the hypusine pathway in Plasmodium

New insights into novel inhibitors against deoxyhypusine hydroxylase from plasmodium falciparum: compounds with an iron chelating potential

The guanylhydrazone CNI-1493: an inhibitor with dual activity against malaria—inhibition of host cell pro-inflammatory cytokine release and parasitic deoxyhypusine synthase

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