Completing the hypusine pathway in Plasmodium Deoxyhypusine hydroxylase is an E-Z type HEAT repeat protein David Frommholz1, Peter Kusch1, Robert Blavid1, Hugo Scheer2, Jun-Ming Tu2, Katrin Marcus3, Kai-Hong Zhao4,5, Veronica Atemnkeng1, Jana Marciniak1 and Annette E Kaiser1 Hochschule Bonn-Rhein-Sieg, Rheinbach, Germany Department of Biologie I-Botanik, Universitat Munchen, Germany ă ă Medizinisches Proteom Center, Ruhr-Universitat Bochum, Germany ă State Key Laboratory of Agricultural Microbiology, Huazhong Agricultural University, Wuhan, China College of Life Science and Technology, Huazhong University of Science and Technology, Wuhan, China Keywords deoxyhypusine hydroxylase; hypusine; malaria; phycocyanin lyase; Plasmodium Correspondence A E Kaiser, Hochschule Bonn-Rhein-Sieg, Von Liebig Strasse 20, D-53356 Rheinbach, Germany Fax: +49 2241 8586 Tel: +49 2241 865 586 E-mail: kaiser@microbiology-bonn.de (Received 24 May 2009, revised July 2009, accepted 10 August 2009) doi:10.1111/j.1742-4658.2009.07272.x 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 l MINT-7255047: DHS (uniprotkb:P49366) enzymaticly reacts (MI:0414) with eIF-5A (uniprotkb:Q710D1) by enzymatic studies (MI:0415) l MINT-7255326: DOHH (uniprotkb:Q8I701) enzymaticly reacts (MI:0414) with eIF-5A (uniprotkb:Q710D1) by enzymatic studies (MI:0415) Introduction The life-cycle of the malaria parasite is complex It consists of a succession of developmental stages in which cell proliferation oscillates between cell-cycle arrest (as in the sporozoites in the salivary glands of the mosquito vector) and intense cell multiplication (as in the erythrocytic stages of the vertebrate human Abbreviations DHS, deoxyhypusine synthase; DOHH, deoxyhypusine hydroxylase; eIF-5A (Dhp), deoxyhypusinylated eIF-5A; eIF-5A, eukaryotic initiation factor; MCF, methyl chloroformate; PCB, phycocyanobilin; PVB, phycoviolobilin FEBS Journal 276 (2009) 5881–5891 ª 2009 The Authors Journal compilation ª 2009 FEBS 5881 Deoxyhypusine hydroxylase from Plasmodium D Frommholz et al host) [1] Completion of the parasitic life-cycle requires rapid changes in its environment such as the stimulation and inhibition of cell division An important issue facing global health is the need for new, effective and affordable drugs against malaria, particularly in resource-poor countries Moreover, the currently available antimalarials are limited by factors ranging from parasite resistance to safety, compliance and cost Innovations in medicinal chemistry are presently lacking Plasmodium falciparum and Plasmodium vivax belong to the order Apicocomplexa and are characterized by the presence of an apicoplast that is essential for the parasite to invade its host Thus, the apicoplast appears to be an excellent target for antimalarial drugs The apicoplast is thought to be the relic of a chloroplast derived from an ingested red alga Such chloroplasts, in turn, are thought to be of cyanobacterial (prokaryotic) origin Although the apicoplast has lost all photosynthetic capacity [2] it retains some metabolic pathways of the chloroplast which are therefore potential targets for antimalarial drugs Consistent with the view that the apicoplast is a chloroplast relic of cyanobacterial origin, we have discovered that DOHH from P falciparum contains several matches to E-Z-type HEAT-like repeat proteins present in the phycocyanin lyase subunits of cyanobacteria and red algae These heterodimeric proteins attach linear tetrapyrrolic chromophores (bilins) covalently to their apoproteins, which then organize into phycobilisomes, the light-harvesting supercomplexes of cyanobacteria and red algae Attachment of the apoproteins to the bilin chromophores is only partly understood; there are several lyases characterized that serve different binding sites The conserved Cys-a84 site of phyco(erythro)cyanins is served by E ⁄ F-type lyases [3], which have been studied in some detail They either attach the chromophore to the D3,31 double bond by thiol addition, or catalyze the attachment by simultaneous isomerization of the chromophore [4] Both E ⁄ F lyase subtypes are characterized by the aforementioned HEAT-like repeats The triamine spermidine [5] is essential for proliferation of the parasite and is an essential substrate in the biosynthesis of hypusine [N(epsilon)-(4-amino-2-hydroxybutyl) lysine], a novel amino acid present in eukaryotic initiation factor-5A (eIF-5A) Hypusine is formed in a post-translational modification that involves two sequential enzymatic steps catalyzed by deoxyhypusine synthase (DHS; EC 1.11.2249) and deoxyhypusine hydroxylase (DOHH; EC 1.14.9929) [6] Whereas DHS catalyzes transfer of the aminobutyl moiety to a specific lysine residue in the eIF-5A 5882 precursor protein, DOHH activity completes hypusine biosynthesis via hydroxylation and thereby completes eIF-5A formation Three different dohh genes have been functionally analyzed from Saccharomyces cerevisiae [7], human [7] and bovine [8] sources The predicted DOHH protein structure from human and yeast revealed that it is a HEAT-repeat-containing metalloenzyme [8] consisting of eight tandem repeats of an a-helical pair (HEAT motif) organized in a symmetrical dyad Although the structure is unrelated to Fe(II)-dependent dioxygenases, four strictly conserved histidine–glutamate metalcoordination sites have been identified [7] In the fission yeast Schizosaccharomyces pombe, the homolog of the dohh gene, Mmd1, was recently reported to be important for normal mitochondrial morphology and distribution [9] By contrast, the DOHH protein is not essential for proliferation in S cerevisiae A S cerevisiae knockout mutant showed only a slower growth rate in the presence of the accumulated deoxyhypusinylated form of eIF-5A [9] Although eIF-5A and DHS have proven to be potential targets of antitumor [10] and anti-HIV-1 therapy [11], no enzyme-specific noniron chelating inhibitors of purified DOHH have been reported to date Over recent years, we have investigated the biosynthesis of hypusine present in eIF-5A of different human malaria parasites, such as P falciparum and P vivax The cloning, expression and inhibition of DHS from these parasites showed that this enzyme is involved in cell proliferation [12] These findings suggested that DHS is a valuable drug target [13,14] because P falciparum and P vivax DHS share 48 and 44% amino acid identity to the human homolog, respectively Experiments with alkyl 4-oxo-piperidine-3-carboxylates derived from mimosine as a lead structure had the most efficient antiplasmodial effect in vivo and in vitro [14] To complete elucidation of the hypusine pathway in P falciparum for further target evaluation the dohh gene was cloned from the parasite Based on the nucleic acid sequence of the yeast and human dohh genes we identified the ORF encoding the DOHH protein from P falciparum and characterized the purified enzyme in vitro Results Cloning and characterization of the dohh gene from P falciparum strain NF54 Based on the published DOHH amino acid sequences from yeast and human sources, we performed a FEBS Journal 276 (2009) 5881–5891 ª 2009 The Authors Journal compilation ª 2009 FEBS D Frommholz et al bio-informatics screening of the P falciparum genome [15] From the nucleic acid sequence obtained, we constructed two gene-specific primers for the 5¢- and 3¢-ends and amplified a 1236 bp fragment encoding a protein of 412 amino acids We identified an ORF on chromosome 13 encoding a protein with HEATlike repeat domains that is homologous to an E ⁄ F-type phycobilin lyase The putative dohh gene from P falciparum strain NF54 had an AT content of 74.2%, significantly surpassing the GC content of 25.8% The deduced amino acid sequence of human DOHH (Fig 1) showed that each domain of four HEAT-like repeats (i.e HEAT-like repeats 2, 3, and 7) contains a highly significant histidine–glutamate (HE) motif corresponding to the characteristic metal-chelating sites By contrast, DOHH of P falciparum has five E-Z-type HEAT-like repeat domains (amino acid positions 94–123, 127–156, 278–307, 311– 340 and 344–385, labeled in blue) with different homologies to phycobilin-lyases from different species, and two stretches of HEAT-like repeats of phycoerythrocyanin subunits located between amino acid positions 76–179 and 47–184 (i.e HEAT-like repeats and 2, single amino acids labeled in green and red in Fig 1) For better alignment of the HEAT-like repeats, we enclosed the amino acid sequences of CpcE from Synechococcus elongatus (11% amino acid identity with Plasmodium) and of PecA the apo a-subunit of phycoerythrocyanin from Nostoc spec PCC7120 (4% amino acid identity with Plasmodium) (Fig 1) In comparison with human DOHH, the histidine-glutamate motifs are also highly conserved The amino acid identity between Plasmodium and the human ortholog was 27% [7] Schizosaccharomyces and Saccharomyces dohh genes are very closely related sharing an amino acid identity of 50% Total cellular RNA from P falciparum strain NF54 at different developmental stages (i.e trophozoites and schizonts) was used in RT experiments The dohh gene transcript (1236 bp) was distributed equally in trophozoites and schizonts (Fig 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] Predictions from different databases and plasmoDB identified dohh as a single-copy gene on chromosome 13 in the Plasmodium genome Expression profiles of the intra-erythrocytic phase with the Plasmodium strain 3D7 detected an expression of 80% in asexual blood stages (http://plasmodb.org/plasmo/) Deoxyhypusine hydroxylase from Plasmodium Expression, purification and functional analysis of P falciparum deoxyhypusine hydroxylase Expression of the histidine-tagged dohh constructs in either pET-15b or in pET-28a was performed in Escherichia coli BL21 (DE3) cells harboring the T7 RNA polymerase under control of the T7 promotor Expression and purification of the DOHH protein by nickelchelate-affinity chromatography (Fig 2) under native conditions showed a protein of 42 kDa which eluted in one of the eluate fractions (Fig 2, lane 5) To investigate a potential E ⁄ F-type phycobilin lyase activity of the enzyme (Fig 3), the DOHH gene was introduced into E coli strains capable of synthesizing the chromophore, phycocyanobilin (PCB) and the His6-tagged acceptor proteins (CpcA or PecA) [4] Controls were the respective strain without DOHH, and the strain expressing, in addition, the genes for the nonisomerizing lyase, cpcE ⁄ F, and the isomerizing lyase, pecE ⁄ F, respectively Addition of the apoprotein to the chromophore was followed by absorption spectroscopy of the cells (not shown) and of the acceptor protein purified by Ni2+-chelating chromatography In the absence of the lyase, no chromophore was attached to the acceptor protein, irrespective of the absence or presence of DOHH (Fig 3A,C) In the control experiment with lyase subunits, the chromophore was properly attached; here the presence of DOHH either had no influence (PecE ⁄ F; Fig 3D) or was somewhat inhibitory (CpcE ⁄ F; Fig 3B) We therefore conclude that DOHH has no phycobilin lyase activity under these conditions, and may even be inhibitory This was also supported in vitro; the data with PecA as acceptor protein are shown in Fig S2 Under these conditions there is a residual, spontaneous (nonenzymatic) addition of the PecA to ring A of the chromophore [17], which generates a small background absorption at 645 nm Crude extracts with expressed (green lane) and nonexpressed (red lane) DOHH protein showed a reduced background reaction, and also, in small yield, the addition of PecA at the central methine bridge of the chromophore which generates a bilirubin (k  430 nm) The isomerized product of the phycoerythrocyanin lyase, phycoviolobilin (PVB), is characterized by absorption at  565 nm but the minute absorption, at 562 nm, formed with the crude extracts, was not significantly different when compared with the control without expressed DOHH protein (Fig S2, red lane) Moreover, a 10–100-fold higher absorption would be generated in the presence of a lyase, either around 640 or 565 nm depending on the lyase subtype (Fig 3) To analyze the hydroxylase activity, a nonradioactive system was established First, we modified the FEBS Journal 276 (2009) 5881–5891 ª 2009 The Authors Journal compilation ª 2009 FEBS 5883 Deoxyhypusine hydroxylase from Plasmodium D Frommholz et al Fig Multiple amino acid alignment of DOHH proteins from four different eukaryotes (Saccharomyces cerevisiae, Schizosaccharomyces pombe, Homo sapiens and Plasmodium falciparum strain NF54, the homolog of a biliprotein lyase (CpcE) from Synechococcus elongatus, and an a-subunit of a cyanobacterial biliprotein (PecA from Nostoc sp PCC7120) The five individual E-Z-type HEAT repeat domains from P falciparum are numbered and shown above the alignment Amino acids with blue capital letters show various degrees of homology to E-Z-type HEAT repeats present in proteins involved in energy metabolism and conversion The most significant amino acid identity to HEAT repeats in CpcE from Synechococcus elongates is found in E-Z-type HEAT repeat domains and (amino acid positions 76–179 and 47–184) Identical amino acids are marked in red Histidine– glutamate motifs are highlighted in purple The secondary structure prediction above the alignment presents H for the a helix, E for an extended structure, T for a b turn and C for the remainder and was obtained using JPRED v 3.0 and SCRATCH [28] Gaps (-) were introduced to obtain maximum alignment Asterisks label amino acid identities, colons (:) and dots (.) label amino acid similarities 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 Microcon-YM 100 and 30 kDa In Fig 4, lane the first size-exclusion chromatography step with the Microcon-YM100 is presented showing that both proteins are recovered in the eluate Subsequent size-exclusion 5884 chromatography with the Microcon-YM30 column cut-off DHS (Fig 4, lane 3) and enriched eIF-5A (Dhp), although no proteins could be detected in the flow-through of the YM 30 columns (Fig 4, lane 1) These results were confirmed in a western blot analysis of the eluate and flow-through fractions after the different steps of size-exclusion chromatography with anti-(eIF-5A) and anti-DHS Ig (Fig 5, eIF-5A, lane B) Anti-(eIF-5A) Ig detected purified eIF-5A protein FEBS Journal 276 (2009) 5881–5891 ª 2009 The Authors Journal compilation ª 2009 FEBS D Frommholz et al Deoxyhypusine hydroxylase from Plasmodium 0.15 43 kDa 42 kDa 0.10 0.05 29 kDa 20 kDa Absorption 55 kDa A 1.2 B 0.8 M 0.4 Fig Expression and purification of DOHH (A) Purification of histidine-tagged recombinant DOHH by nickel-chelate affinity chromatography under native conditions M, Roti standard protein marker Lane 1, lyzed crude cell extract; 2, flow-through; and 4, wash fractions; 5, eluate fraction containing recombinant DOHH 6) second eluate faction C 0.12 0.06 Absorption in the complete DHS assay (Fig 5, eIF-5A, lane A) and in the eluate (Fig 5, eIF-5A, lane E) after Microcon-YM 100 size-exclusion chromatography EIF-5A protein was present in the eluate after subsequent size exclusion with Microcon-YM 30 but absent in the flow-through (Fig 5, FT-YM 30 and E-YM 30) DHS antibody detected the DHS protein in the complete DHS assay with associated eIF-5A and in the eluate after the Microcon-YM100 size-exclusion chromatography (Fig S3) DHS protein was absent in the eluate and flow-through after size-exclusion chromatography with the Microcon-YM30 column (Fig S3) eIF-5A (Dhp) was analyzed by peptide hydrolysis for deoxyhypusine modification in a typical DHS assay Figure 6A shows the characteristic GC ⁄ MS spectrum of the formed deoxyhypusine after derivatization with methyl chloroformate [18] which esterifies reactive side chains and carboxyl groups In addition to the molecular ion [M]+• at m ⁄ z 347, several prominent fragments of deoxyhypusine were detected, i.e [M-NH-C(O)OCH3] at [M-74]+, [M-C(O)OCH3] with [M-59]+, [M-2(NH-C(OO)CH3)+] with [M-2Ỉ74]+ and [M-2ỈNHC(O)OCH3-C(O)OCH3-OCH3] with [M-2Ỉ7459-31]+ In order to assay the activity of recombinant DOHH derived from P falciparum, the nonradioactively modified eIF-5A (Dhp) was incubated with purified recombinant DOHH from P falciparum In the DHS assay, deoxyhypusine, but not hypusine, could be detected (Fig 6A) By contrast, hypusine was found in the assay with purified DOHH enzyme (Fig 6B) together with small amounts of deoxyhyusine We identified the molecular ion [M]+• at 377 for hypusine and, in contrast to deoxyhypusine, a molecular fragment of [M-OCH3] with [M-31]+ 0.18 2.1 D 1.4 0.7 400 500 600 λ (nm) 700 800 Fig Assay of DOHH for phycocyanin C-a84 lyase (A,B) and phycoerythrocyanin C-a84 lyase ⁄ isomerase (C,D) activities Absorption spectra of acceptor proteins, CpcA and PecA, after treatment with PCB, and purification by Ni2+ affinity chromatography (A) Assay for the attachment of PCB to CpcA in the presence (——) and absence (- - -) of DOHH (B) Control in the additional presence of the lyase, CpcE ⁄ F (C) Assay for the attachment of PCB to PecA and isomerization to PVB in the presence (——) and absence (- - -) of DOHH (D) Control in the additional presence of the isomerizing lyase, CpcE ⁄ F All reactions were carried out in E coli (see Materials and methods for details) Discussion Here, we have described cloning of the dohh gene from P falciparum strain NF54, its expression in E coli and its hydroxylation activity of deoxyhypusinylated eIF-5A The data demonstrate that a complete hypusine biosynthetic pathway is present in Plasmodium DOHH is encoded by an ORF of 412 amino acids in P falciparum with a molecular mass of 42 kDa DOHH has certain peculiar features: for example, the occurrence of five E-Z HEAT-like repeat motifs in contrast to four present in the human enzyme [7] Referring to predictions from the Pfam database, the HEAT-like repeats in Plasmodium DOHH form a multi-helical fold comprised of two FEBS Journal 276 (2009) 5881–5891 ª 2009 The Authors Journal compilation ª 2009 FEBS 5885 Deoxyhypusine hydroxylase from Plasmodium D Frommholz et al 212 kDa 118 kDa 66 kDa 43 kDa DHS 29 kDa elF-5A 20 kDa FT 30 E 100 E 30 Fig Separation of modified eIF-5A on SDS ⁄ PAGE after sizeexclusion chromatography with a Microcon-YM 100 kDa and a Microcon-YM-30 kDa column 1, Flow-through after the MicroconYM-30 kDa column; 2, eluate of eIF-5A (Dhp) obtained after the Microcon-YM 100 kDa column; 3, DHS cut-off by the Microcon-YM 30 kDa column EIF - 5A 69 kDa 29 kDa eIF-5A 20 kDa FT E E A M YM 100 YM 30 YM 30 Fig Western blot experiment after size-exclusion chromatography of modified eIF-5A (Dhp); : 1000 diluted anti-(eIF-5A) polyclonal serum was applied (A) Complete DHS assay; FT, flow-through; E, eluate obtained with YM-30 kDa or YM-100 kDa columns curved layers of a helices arranged in a regular righthanded superhelix with the repeats arranged about a common axis [19] These superhelical structures pres5886 ent an extensive solvent-accessible surface that is well suited to binding of proteins or nucleic acids This topology has been found in the armadillo repeat (found in b-catenins and a-importins such as the b-subunit of karyopherin) The structural domains of plasmodial DOHH resemble those found originally in phycocyanin lyase subunits of the E ⁄ F type [4], which prompted us to test it for lyase activity These lyases attach phycocyanin via a thioether bond to the apoprotein, in this case CpcA or PecA [3], in some cases with a concomitant isomerization [3]; the binding can be followed chromatographically using a His6-tagged apoprotein and the resulting increase in absorption and band-shift can be followed spectroscopically Phycocyanin can also add to the acceptor protein spontaneously, generating a weak unspecific background, therefore an E coli system has been established that lacks this background signal [4] In our tests with DOHH, there was, neither in vitro nor in E coli, a signal observed that was indicative of a lyase function of DOHH, it may even be somewhat inhibitory It seems likely that DOHH was originally recruited from phycocyanin lyase of cyanobacteria [3] with an original function in the biosynthesis of phycobiliprotein-type light-harvesting complexes, but subsequently adapted to a new role as a hydroxylase during evolution Because the dohh gene is not part of the apicoplast genome, this would imply a gene transfer to the nucleus A structural annotation in MADIBA [20] for selection of putative target proteins in the malaria parasite predicted gene homology of Plasmodium DOHH to orthologs in the rice and Arabidopsis genome This observation is even more supported by the occurrence of conserved motifs (i.e CGATT or TAGCC) in promoter regions which are found in chlorophyll a ⁄ b binding proteins [21] dohh is present as a single-copy gene on chromosome 13 in P falciparum and is transcribed in asexual blood stages (http://plasmodb.org/plasmo/) Inhibition of spermidine synthase [5] depletes hypusine formation and parasite proliferation in vitro In this context, it would be of considerable interest for the future to study the phenotype of a dohh knockout mutant by targeted gene disruption that progresses through the malaria life-cycle of a Plasmodium berghei rodent model with impaired function [22] These experiments have recently being performed for the Plasmodium protein UIS4 (the upregulated infective sporozoites gene 4), which is critical for complete liver stage development One interesting feature, according to the prediction of the PlasmoAP bioinformatic tool [23] is the FEBS Journal 276 (2009) 5881–5891 ª 2009 The Authors Journal compilation ª 2009 FEBS D Frommholz et al Deoxyhypusine hydroxylase from Plasmodium A OCH3 O Abundance – 59 32 000 30 000 28 000 26 000 24 000 22 000 20 000 18 000 16 000 14 000 12 000 10 000 8000 6000 4000 2000 NH – 74 79.1 NH – 74 HN – 59 O 52.1 – 59 253.0 H3CO OCH3 O 331.1 346 119.0 158.1 195.0 222.9 281.0 401.1 20 40 60 80 100 120 140 160 180 200 220 240 260 280 300 320 340 360 380 400 m/z > 7000 Abundance 6000 346.0 [M]+ 5000 4000 3000 347.0 2000 348.1 1000 349.1 342 343 344 345 346 347 m/z > 348 349 350 351 B 405.1 Abundance 120 000 110 000 Abundance 156.1 70 000 60 000 50 000 40 000 30 000 20 000 10 000 Fig (A) Identification of deoxyhypusine by GC ⁄ MS analysis after a typical DHS assay obtained from a peptide hydrolysate of modified eIF-5A (Dhp) after derivatization with methyl chloroformate The molecular ion [M]+• at 347 is shown for deoxyhypusine (B) Ion mass spectrum of hypusine identified in a DOHH activity assay after peptide hydrolysis and subsequent MCF derivatization In case of hypusine the molecular ion [M]+• at 377 and a molecular fragment of [M-OCH3] with [M-31]+ representing the hydroxyl group were identified The most characteristic fragment ions are presented in the structure of the hypusine derivative Deoxyhypusine is also present 327.1 100 000 90 000 80 000 79.1 253.0 36.0 195.0 119.1 297.0 223.0 375.1 20 40 60 80 100 120 140 160 180 200 220 240 260 280 300 320 340 360 380 400 420 m/z > 44 000 42 000 40 000 38 000 36 000 34 000 32 000 30 000 28 000 26 000 24 000 22 000 20 000 18 000 16 000 14 000 12 000 10 000 8000 6000 4000 2000 343.1 [M-31]+ 344.1 345.1 337 339.0 338 339 FEBS Journal 276 (2009) 5881–5891 ª 2009 The Authors Journal compilation ª 2009 FEBS 341.1 342.1 340 341 342 346.1 347.0 343 344 m/z > 345 348.0 346 348 347 349 5887 Deoxyhypusine hydroxylase from Plasmodium D Frommholz et al occurrence of a signal peptide with a cleavage site at the N-terminal position 26 in the dohh gene In photosynthetic plants, and in Plasmodia which contain the nonphotosynthetic apicoplasts, transit peptides [23,24], direct proteins to the apicoplast that has certain plantlike-metabolic pathways No common structural elements or consensus sequences have been identified for transit peptides Recent experiments for the malaria parasite suggest that a net basic charge and a chaperone binding site are critical for accurate targeting [24]; however, the N-terminus of the DOHH protein is not hydrophobic Targeting to a different compartment might be possible in the case of smaller molecular mass Plasmodium DOHH We also describe a novel, nonradioactive assay for the analysis of hypusine modification in eIF-5A from P falciparum The radioactive filter assay is rather inaccurate because of unspecific binding of [14C]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 4) before hydrolysis Methyl chloroformate derivatives [18] were analyzed by GC ⁄ MS applying lysine and hydroxylysine as internal reference standards (data not shown) Purified hypusine was applied as a control In addition to hypusine, we identified deoxyhypusine in the DOHH activity assay To investigate how the additional E-Z-type HEAT repeat present in DOHH from Plasmodium may influence hydroxylase activity, a quantitative assay with nonradioactively labeled eIF-5A [26] and purified DOHH enzyme from human and the parasite is currently underway Materials and methods Isolation of cellular RNA from P falciparum strain NF54 Cellular RNA from P falciparum NF54 was isolated according to a protocol from Qiagen (Hilden, Germany) RNeasy Mini plant isolation kit Red blood cells with a parasitemia of 8.9% were applied The concentration of cellular RNA was calculated to be 0.9 lg per lL PCR amplification of the dohh gene from Plasmodium strain NF54 by reverse transcription PCR amplification of the dohh gene was performed according to a protocol with the access RT-PCR system from Promega 5888 (Madison, WI, USA) A final PCR volume of 50 lL contained: 33 lL of nuclease-free water, AMV ⁄ Tfl 5· reaction buffer 10 lL, dNTP Mix (10 mm each dNTP) 0.2 mm, upstream primer DOHH forward: 5¢-ATGGGAGAAAATAA CGACAAC-3¢ lm, downstream primer DOHH reverse 5¢CTAGTGAACCTCTATAGATATAA-3¢ lm, 25 mm MgSO4, mm, AMV reverse transcriptase 0.1 lL)1 and a proof-reading ReproFast Taq polymerase (Genaxxon, Ulm, Germany) 0.1 lL)1 and 4.5 lg total cellular RNA from P falciparum strain NF54 First-strand synthesis was performed at 45 °C for 45 AMV ⁄ RT was inactivated at 94 °C for The following program was applied for second-strand synthesis and PCR amplification: 94 °C for 30 s, 60 °C for min, 68 °C for (40 cycles) The final extension was performed for at 68 °C The resulting DNA fragment of 1236 bp was sequenced by MWG (Munich, Germany) After purification, the blunt-ended PCR fragment was modified with Taq DNA polymerase and dATP to obtain A-tailed fragments which were subcloned into pST-Acceptor vector (Novagen, Madison, WI, USA) and resequenced Expression of the dohh gene in pET-15b and pET-28a vector in E coli BL21 (DE3) cells and subsequent purification by nickel-chelate chromatography E coli BL21 (DE3) cells containing the recombinant dohh plasmid were grown for expression with pET-15b vector in ampicillin (30 lgỈmL)1) and kanamycin (15 lgỈmL)1) was used for expression in pET-28a One milliliter samples from the expressing strain was taken and centrifuged at 13 000 rpm for Cells were lysed with 400 lL lysis buffer (50 mm Tris ⁄ HCl, pH 8.0, mm EDTA), centrifuged, resuspended in lysis buffer and sonicated twice at °C for 30 s (tip at 50% using a Branson sonifier) After centrifugation for 10 at 16 000 rpm at °C, samples were diluted : in loading buffer (20 mm Tris, pH 6.8, 2% w ⁄ v SDS, mm EDTA, 20% v ⁄ v glycerol, 0.3% bromphenol blue) heated at 100 °C and run on a 10% SDS polyacrylamide gel at 100 V Protein purification was performed by nickel-chelate affinity chromatography under native conditions according to the Qiagen protocol with some variations A pellet derived from a mL culture of dohh expressing E coli BL21 (DE3) cells was resuspended in 630 lL lysis buffer containing pH 8.0 Lysozyme stock solution (70 lL of 10 mgỈmL)1) and mL)1 culture volume BenzonasNuclease were added The suspension was incubated on ice for 15–30 Centrifugation was applied at 12 000 g for 30 at °C A Ni-NTA spin column was equilibrated with 600 lL lysis buffer containing 10 mm imidazole Centrifugation for at 890 g followed Six hundred microliters of the cleared lysate containing the 6· His-tagged protein was loaded onto the pre-equilibrated FEBS Journal 276 (2009) 5881–5891 ª 2009 The Authors Journal compilation ª 2009 FEBS D Frommholz et al Ni-NTA spin column and centrifuged for at 270 g The Ni-NTA spin column was washed twice with 600 lL washing buffer containing 5.0 mm NaH2PO4, 300 mm NaCl, mm imidazole, pH 8.0 and centrifuged for at 890 g DOHH was eluted from the column with 300 lL elution buffer 50 mm NaH2PO4, 300 mm NaCl, 500 mm imidazole, pH 8.0 in two fractions Subcloning of the dohh gene into histidine tagged pET-28a and pET-15b expression vectors Amplification of the dohh gene was performed from genomic DNA of P falciparum strain NF54 using primers with restriction enzymes for NotI (recognition site is underlined) DOHH expressforw 5¢-TTAAGCGGCCGCATGGGAGAA AATAACGA-3¢ and BamHI DOHH expressrev 5¢-AAAAG GATCCCTAGTGAACCTCTATAGATAT-3¢ The resulting fragment of 1236 bp was digested with NotI and BamHI and ligated into NotI ⁄ BamHI-digested pET-28a vector and re-sequenced For subcloning into pET-15b, which was digested with NdeI and BamHI, the amplification was performed with primers DOHH expressfor 5¢AAAAAACATATGGAGAAAATAAC-3¢ and DOHH expressrev 5¢-AAAAGGATCCCTAGTGAACCTCTATAG ATATT-3¢ The restriction sites for NdeI and BamHI are underlined Expression of the dohh gene in E coli capable of biosynthesis of a-subunits of biliproteins, C-phycocyanin and phycoerythrocyanin The parent strains producing PCB, and the His6-tagged acceptor protein (CpcA or PecA), plus or minus the respective lyases, CpcE ⁄ F or PecE ⁄ F, are described elsewhere [4;28] The dohh gene in the abforementioned expression plasmid was transformed into the BL21 (DE3) strain containing the respective plasmids After induction of the cells, extraction and purification of the acceptor protein by chelating chromatography, the spectroscopic assay were done as before [27] Nonradioactive preparation of deoxyhypusine as a substrate for deoxyhypusine hydroxylase activity assay and its identification by GC/MS The N-terminal histidine tagged fusion proteins of eIF-5A and DHS in recombinant pET-15b were expressed in E coli BL21 (DE3) and purified by nickel-chelate affinity chromatography under native conditions Buffer exchange was performed with a Sephadex-G25 column before the incubation of DHS activity A reaction mixture of mL containing spermidine, eIF-5A from P vivax (40 lm each), 0.5 mm NAD+, and 25 lg purified DHS from human was incubated at 30 °C for h The un-modified and modified eIF-5A Deoxyhypusine hydroxylase from Plasmodium precursor protein was recovered by a Microcon-YM 100 kDa column (Amicon, Millipore, Schwalbach, Germany), retaining DHS A subsequent application of a Microcon-YM 30 kDa column enriched both forms of eIF-5A Protein hydrolysis was performed under nitrogen in m HCl at 120 °C for 24 h The eluate was evaporated to dryness, derivatized with methyl chloroformate according to the protocol by Husek [18] and subsequently analyzed by GC ⁄ MS DOHH activity assay DOHH substrate, i.e eIF-5A (Dhp) [16], was prepared as described in the Results section A typical assay contained DOHH purified by nickel-chelate chromatography from P falciparum NF54 strain (7.5 lg), 50 mm NaCl ⁄ Pi pH 7.4, mm NAD, mm dithiothreitol and  20 lg eIF-5A (Dhp) in a reaction volume of 600 lL Incubation was performed for h at 37 °C The modified eIF-5A protein was recovered by size-exclusion chromatography and hydrolyzed in m HCl at 120 °C for 24 h Hypusine was isolated as deoxyhypusine and derivatized by methylchloroformate and determined by GC ⁄ MS [19] GC/MS GC ⁄ MS measurements were made using a 7890A gas chromatograph and a 5975C quadrupole mass spectrometer (Agilent Technologies, Santa Clara, CA, USA) operated in electron impact ionization mode The fused silica capillary column, 30 m long, 0.25 mm (ID) was used with HP-5MS (Agilent Technologies) as stationary phase and film thickness 0.25 lm The temperature of the column was programmed from 60 °C (1 hold) and increased by °CỈmin)1 to 280 °C (50 hold) A constant helium flow of 0.928 cm3Ỉmin)1 was used The temperature of the split ⁄ splitless injector was 250 °C The electron impact ion source with the energy of 70 eV was kept at 230 °C The quadrupole temperature was 150 °C, and the mass range was m ⁄ z 30–750 In vitro Phycoerythrocyanin lyase/isomerase activity assay The apo-a-subunit of phycoerythrocyanin, PecA, was dissolved in Tris ⁄ HCl buffer (50 mm, pH 6.5) containing mercaptoethanol (5 mm) PCB in dimethylsulfoxide (1 mm) and the expressed DOHH protein were added so that the final concentration of dimethylsulfoxide was 1% and the final phycobilin concentration in the reconstitution mixture was 10 lm After incubation in the dark at ambient temperature (details presented in the Result), the mixture was centrifuged for 15 at 15 000 g to remove any particulate matter and the supernatant was investigated by UV ⁄ Vis absorption and light-induced absorption changes [4] FEBS Journal 276 (2009) 5881–5891 ª 2009 The Authors Journal compilation ª 2009 FEBS 5889 Deoxyhypusine hydroxylase from Plasmodium D Frommholz et al Acknowledgements We thank Professor Dr J Hauber (Heinrich-PetteInstitut, Hamburg, Germany) and Dr R J Porra (CSIRO, Canberra, Australia) for critical reading of the manuscript We are grateful to Drs M Park and E Wolff for pure hypusine This work was supported in part by the bioinnovation award to AK and the Deutsche Forschungsgemeinschaft JMT acknowledges a fellowship from the Chinese scholarship Council, HD support from the Deutsche Forschungsgemeinschaft (SFB 553) and KHZ support from the National Natural Science Foundation of China (grants30670489 and 30870541) References 11 12 13 Dorin D, Semblat JP, 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Parasitol 137, 65–74 26 Kaiser A, Gottwald A, Maier W & Seitz HM (2003) Targeting enzymes involved in spermidine metabolism of parasitic protozoa – a possible new strategy for antiparasitic treatment Parasitol Res 91, 508–516 27 Blot N, Wu XJ, Thomas JC, Zhang J, Garczarek L, Bohm S, Tu JM, Zhou M, Ploscher M, Eichacker L ă ă et al (2009) Phycourobilin in trichromatic phycocyanin from oceanic cyanobacteria is formed post-translation- Deoxyhypusine hydroxylase from Plasmodium ally by a phycoerythrobilin lyase-isomerase J Biol Chem 284, 9290–9298 28 Cuff JA, Clamp ME, Siddiqui AS, Finlay M & Barton GJ (1998) JPred: a consensus secondary structure prediction server Bioinformatics 14, 892–893 Supporting information The following supplementary material is available: Fig S1 RT-PCR of the dohh gene from Plasmodium falciparum with subcellular, total RNA Fig S2 Phycoerythrocyanin lyase ⁄ isomerase activity assay Fig S3 Western blot experiment after size-exclusion chromatography of modified eIF-5A (Dhp) This supplementary material can be found in the online article Please note: As a service to our authors and readers, this journal provides supporting information supplied by the authors Such materials are peer-reviewed and may be re-organized for online delivery, but are not copy-edited or typeset Technical support issues arising from supporting information (other than missing files) should be addressed to the authors FEBS Journal 276 (2009) 5881–5891 ª 2009 The Authors Journal compilation ª 2009 FEBS 5891 ... present in proteins involved in energy metabolism and conversion The most significant amino acid identity to HEAT repeats in CpcE from Synechococcus elongates is found in E-Z- type HEAT repeat domains... [3], in some cases with a concomitant isomerization [3]; the binding can be followed chromatographically using a His6-tagged apoprotein and the resulting increase in absorption and band-shift can... preparation of deoxyhypusine as a substrate for deoxyhypusine hydroxylase activity assay and its identification by GC/MS The N-terminal histidine tagged fusion proteins of eIF-5A and DHS in recombinant pET-15b