BACKGROUND
plasmodium vivax is the most widely distributed human malaria parasite and infects 70– <dig> million individuals per year worldwide.
in most malaria endemic regions, p. falciparum and p. vivax infections coexist, and mixed infections are prevalent.
interactions between the two parasites determine the outcome of the disease and future therapeutic perspectives with regard to infection control.
however, there is limited knowledge about the biology of the p. vivax parasite at present, due to the lack of an in vitro culture system.
to accelerate drug discovery for the more benign p. vivax, genome sequencing of the malaria parasite was performed to facilitate its annotation  <cit> .
a transcriptome approach  <cit>  was applied to study gene expression in the mixed erythrocytic stages of a p. vivax field isolate.
transcribed genes  displayed significant homology to those involved in metabolism and ribosomal proteins consistent with the trophozoite stage of the field strain.
over recent years, increasing resistance of malaria parasites to therapy, and in particular, re-emergence of p. vivax infections, has increased the need to develop strategies of identifying novel drug targets.
the polyamine pathway has been exploited for antiparasitic chemotherapy.
our research is focused on the formation of the novel amino acid hypusine, which involves two steps.
in the first step, deoxyhypusine synthase   transfers an aminobutyl moiety from the triamine spermidine to a specific lysine residue in the eif-5a  precursor protein to form deoxyhypusine.
in the second step, deoxyhypusine hydroxylase   completes hypusine biosynthesis through hydroxylation.
deoxyhypusine modification is important for the viability of eukaryotic cells  <cit> .
we have recently cloned two genes involved in hypusine biosynthesis from the malaria parasites p. falciparum and p. vivax, specifically, deoxyhypusine synthase  and eukaryotic initiation factor 5a .
to elucidate the phylogenetic relationship between deoxyhypusine synthase  proteins from both parasitic species, we clone the putative dhs gene from p. vivax, and determine its specific enzymatic activity.
RESULTS
nucleotide sequence of a putative dhs gene from p. vivax
the dhs amino acid sequence of p. falciparum strain nf  <dig>  <cit>  was used to screen a library of expressed sequence  tags from p. vivax strain pest salvador i. based on the 5' and 3' ends of dhs, two primers were constructed to amplify the  <dig> bp sequence encoding  <dig> amino acids from the genomic dna of plasmodium vivax strain pest salvador i. the purified fragment was subcloned into pstblue-1acceptor vector, and sequenced .
the dhs gene has an at content of 52%, and a comparable gc content of 48%.
the deduced amino acid sequence of the p. vivax protein was aligned with different putative dhs sequences from other plasmodium species, i. e., p. falciparum dd <dig> and 3d <dig> strains , rodent malaria parasite p. yoelii , and anopheles gambiae .
the p. vivax dhs amino acid sequence displayed fasta scores of  <dig> and  <dig> relative to the p. falciparum strains dd <dig> and 3d <dig>  respectively.
notably, p. vivax dhs exhibits 66% amino acid identity between positions 1– <dig> and 91% identity between positions 276– <dig> with the p. falciparum dd <dig> strain.
there are several gaps in the amino acid stretches at positions  <dig>   <dig>   <dig> and  <dig> in the p. vivax dhs protein, which are absent in the p. falciparum homolog .
the p. vivax dhs protein has a fasta score of  <dig> to its homolog from the rodent malaria parasite p. yoelii, while amino acid identity to the human dhs protein  is only 44%.
the lowest fasta score of  <dig> was obtained between p. vivax and anopheles gambiae dhs proteins
expression of p. vivax dhs protein in e. coli cells and affinity purification
the purified p. vivax dhs protein has a calculated molecular size of  <dig> kda , and a pi value of  <dig> .
initially, primers# <dig> and # <dig> containing ndei and bamhi restriction sites, respectively, were used for amplification of dhs, which was cloned into the pet15b vector for protein expression.
protein extracts prepared after  <dig> h of induction with iptg exhibited a prominent band of approximately  <dig> kda on an sds-page gel .
in contrast, no band was detectable in the uninduced control culture  the recombinant protein was expressed with a six-histidine tag at the amino-terminus, allowing purification by nickel-chelate affinity chromatography.
purified protein was analyzed by sds-page .
the protein was detected at  <dig> kda in both eluate fractions .
modification of p. vivax eif-5a precursor protein by p. vivax deoxyhypusine synthase
for the deoxyhypusine synthase assay, the purified eif-5a precursor protein and dhs enzyme from p. vivax were applied  <cit> .
the incorporation of radioactively labeled spermidine into the substrate precursor protein was assayed using a filter paper assay  <cit> .
purified samples were immediately used to determine specific enzymatic dhs activity.
the average specific enzymatic activity from two experiments was  <dig> u/mg protein suggesting that dhs protein from p. vivax is able to modify the eif-5a precursor protein .
in a control experiment, no specific enzymatic activity was detected when water was used instead of dhs .
the specific enzymatic activity of p. vivax dhs was suppressed 36-fold with the inhibitor n-guanyl- <dig> -diaminoheptane  .
plasmodium vivax deoxyhypusine synthase shows cross-reactivity to its human homolog
to determine whether a polyclonal antibody against human dhs recognized the corresponding protein from p. vivax, the nickel-chelate purified protein was subjected to western blot analysis .
as a positive control, we used purified human dhs protein .
the antibody specifically recognized human dhs protein with a molecular size of  <dig> kda , and cross-reacted with the plasmodium vivax dhs protein .
discussion
reduced in vitro susceptibility  <cit>  of field isolates from plasmodium falciparum strains to artemeter derivatives is an alarm signal necessitating the rapid deployment of drug combinations.
there is an urgent need to elucidate the genome of human malaria parasites.
in  <dig>  after the completion of the p. falciparum genome project, the sequencing of expressed sequence tags  from the human malaria parasite p. vivax of salvador pest strain i, isolated from a malaria patient from thailand, was initiated.
no information about this parasite is available to date.
in contrast to p. falciparum, it infects reticulocytes, which are young immature erythrocytes.
due to the lack of a reproducible continuous in vitro culture system  <cit> , detailed molecular analyses were not possible.
recently, we exploited the biosynthetic pathway of the generation of the novel amino acid, hypusine, in the human malaria parasite p. falciparum  <cit> .
two proteins, i.e., dhs and eukaryotic initiation factor 5a  involved in the formation of deoxyhypusine, the first committed step in the hypusine biosynthetic pathway, are potential new drug targets in plasmodium falciparum  <cit> .
to identify the phylogenetic homologs of the dhs gene in different plasmodium species, a library of plasmodium vivax expressed sequence tags was screened for a dhs homolog from p. falciparum.
the putative dhs sequence from p. vivax is encoded by a  <dig> bp nucleic acid sequence, with the most significant fasta scores of  <dig> to p. yoelii and  <dig> to p. falciparum strain 3d <dig>  these results are in agreement with initial comparative analyses of the p. vivax genome with the other related plasmodium species, which revealed significant conservation of gene synteny between the different species within chromosome cores, in contrast to subtelomeric regions displaying reduced homology  <cit> .
p. vivax dhs displays striking amino acid sequence identity to its homolog from p. falciparum.
notably, 89% amino acid identity is observed between p. vivax dhs protein  and the human homolog between residues 290– <dig>  .
however, several gaps appear in the p. vivax protein sequence at positions  <dig>   <dig>   <dig> and  <dig>  which are absent in the plasmodium falciparum homolog.
the most significant differences between human and plasmodium dhs homologs are evident in the spermidine binding site , although the active site is highly conserved.
the active site includes a conserved region of six amino acids from glu  <dig> to lys  <dig>    <cit> .
asp  <dig>  one of the charged amino acids at the bottom of the active site tunnel responsible for binding to spermidine and the gc <dig> inhibitor, is highly conserved in dhs proteins in plasmodium  <cit> .
moreover, in both dhs proteins, gly  <dig> is within hydrogen-binding distance of the guanidinium group  <cit> .
the nad-binding site  is strongly conserved between the different plasmodium species and their human homologs.
however, a stretch of  <dig> amino acids mainly comprising asparagine in this site is missing in dhs homologs from plasmodium.
to confirm the function of p. vivax dhs, we determined its specific enzymatic activity in modification of eukaryotic translation initiation factor, eif-5a, by incorporation of the aminobutyl-moiety of 14-spermidine as a substrate .
specific enzymatic activity was determined as  <dig> u/mg protein, which was 9-fold less than that calculated by molitor and colleagues   <cit> , who used human dhs protein for the modification of eif-5a from p. falciparum.
in a control experiment with water instead of dhs from p. vivax, no specific enzymatic activity was detected.
in a parallel inhibition experiment, the most commonly used competitive inhibitor of dhs, n1-guanyl- <dig> -diaminoheptane  , was employed.
gc <dig> inhibits dhs in a competitive manner by binding to its active site  <cit> , as observed in crystallized form ii that was recently obtained for the complex of dhs and gc <dig>  this inhibitor reduced specific enzymatic activity by 36-fold .
while the amino acid identity between human and plasmodium vivax dhs is only 44%, we detected cross-reactivity between a polyclonal antibody raised against the human dhs enzyme and the expressed nickel chelate-purified protein  from p. vivax, suggesting that the anti-dhs antibody recognizes specific epitopes present in both proteins.
CONCLUSIONS
the putative p. vivax dhs protein consists of  <dig> amino acids with significant fasta scores of  <dig> to p. yoelii and  <dig> to p. falciparum strain 3d <dig> dhs proteins.
a specific dhs enzymatic activity assay with 14c-labeled spermidine as the substrate and inhibition by n-guanyl- <dig> -diaminoheptane  confirmed functional activity.
moreover, the dhs protein from p. vivax cross-reacted with a polyclonal antibody against anti-human dhs.
further biochemical characterization analyses of the p. vivax dhs protein are currently underway.

 plasmodium vivax is the most widely distributed human malaria parasite and infects 70– million individuals per year worldwide. in most malaria endemic regions, p. falciparum and p. vivax infections coexist, and mixed infections are prevalent. interactions between the two parasites determine the outcome of the disease and future therapeutic perspectives with regard to infection control. however, there is limited knowledge about the biology of the p. vivax parasite at present, due to the lack of an in vitro culture system. to accelerate drug discovery for the more benign p. vivax, genome sequencing of the malaria parasite was performed to facilitate its annotation. a transcriptome approach was applied to study gene expression in the mixed erythrocytic stages of a p. vivax field isolate. transcribed genes displayed significant homology to those involved in metabolism and ribosomal proteins consistent with the trophozoite stage of the field strain. over recent years, increasing resistance of malaria parasites to therapy, and in particular, re-emergence of p. vivax infections, has increased the need to develop strategies of identifying novel drug targets. the polyamine pathway has been exploited for antiparasitic chemotherapy. our research is focused on the formation of the novel amino acid hypusine, which involves two steps. in the first step, deoxyhypusine synthase transfers an aminobutyl moiety from the triamine spermidine to a specific lysine residue in the eif-5a precursor protein to form deoxyhypusine. in the second step, deoxyhypusine hydroxylase completes hypusine biosynthesis through hydroxylation. deoxyhypusine modification is important for the viability of eukaryotic cells. we have recently cloned two genes involved in hypusine biosynthesis from the malaria parasites p. falciparum and p. vivax, specifically, deoxyhypusine synthase and eukaryotic initiation factor 5a. to elucidate the phylogenetic relationship between deoxyhypusine synthase proteins from both parasitic species, we clone the putative dhs gene from p. vivax, and determine its specific enzymatic activity.  nucleotide sequence of a putative dhs gene from p. vivax the dhs amino acid sequence of p. falciparum strain nf was used to screen a library of expressed sequence tags from p. vivax strain pest salvador i. based on the 5' and 3' ends of dhs, two primers were constructed to amplify the bp sequence encoding amino acids from the genomic dna of plasmodium vivax strain pest salvador i. the purified fragment was subcloned into pstblue-1acceptor vector, and sequenced. the dhs gene has an at content of 52%, and a comparable gc content of 48%. the deduced amino acid sequence of the p. vivax protein was aligned with different putative dhs sequences from other plasmodium species, i. e., p. falciparum dd and 3d strains, rodent malaria parasite p. yoelii, and anopheles gambiae. the p. vivax dhs amino acid sequence displayed fasta scores of and relative to the p. falciparum strains dd and 3d respectively. notably, p. vivax dhs exhibits 66% amino acid identity between positions 1– and 91% identity between positions 276– with the p. falciparum dd strain. there are several gaps in the amino acid stretches at positions and in the p. vivax dhs protein, which are absent in the p. falciparum homolog. the p. vivax dhs protein has a fasta score of to its homolog from the rodent malaria parasite p. yoelii, while amino acid identity to the human dhs protein is only 44%. the lowest fasta score of was obtained between p. vivax and anopheles gambiae dhs proteins expression of p. vivax dhs protein in e. coli cells and affinity purification the purified p. vivax dhs protein has a calculated molecular size of kda, and a pi value of. initially, primers# and # containing ndei and bamhi restriction sites, respectively, were used for amplification of dhs, which was cloned into the pet15b vector for protein expression. protein extracts prepared after h of induction with iptg exhibited a prominent band of approximately kda on an sds-page gel. in contrast, no band was detectable in the uninduced control culture the recombinant protein was expressed with a six-histidine tag at the amino-terminus, allowing purification by nickel-chelate affinity chromatography. purified protein was analyzed by sds-page. the protein was detected at kda in both eluate fractions. modification of p. vivax eif-5a precursor protein by p. vivax deoxyhypusine synthase for the deoxyhypusine synthase assay, the purified eif-5a precursor protein and dhs enzyme from p. vivax were applied. the incorporation of radioactively labeled spermidine into the substrate precursor protein was assayed using a filter paper assay. purified samples were immediately used to determine specific enzymatic dhs activity. the average specific enzymatic activity from two experiments was u/mg protein suggesting that dhs protein from p. vivax is able to modify the eif-5a precursor protein. in a control experiment, no specific enzymatic activity was detected when water was used instead of dhs. the specific enzymatic activity of p. vivax dhs was suppressed 36-fold with the inhibitor n-guanyl- -diaminoheptane. plasmodium vivax deoxyhypusine synthase shows cross-reactivity to its human homolog to determine whether a polyclonal antibody against human dhs recognized the corresponding protein from p. vivax, the nickel-chelate purified protein was subjected to western blot analysis. as a positive control, we used purified human dhs protein. the antibody specifically recognized human dhs protein with a molecular size of kda, and cross-reacted with the plasmodium vivax dhs protein. discussion reduced in vitro susceptibility of field isolates from plasmodium falciparum strains to artemeter derivatives is an alarm signal necessitating the rapid deployment of drug combinations. there is an urgent need to elucidate the genome of human malaria parasites. in after the completion of the p. falciparum genome project, the sequencing of expressed sequence tags from the human malaria parasite p. vivax of salvador pest strain i, isolated from a malaria patient from thailand, was initiated. no information about this parasite is available to date. in contrast to p. falciparum, it infects reticulocytes, which are young immature erythrocytes. due to the lack of a reproducible continuous in vitro culture system, detailed molecular analyses were not possible. recently, we exploited the biosynthetic pathway of the generation of the novel amino acid, hypusine, in the human malaria parasite p. falciparum. two proteins, i.e., dhs and eukaryotic initiation factor 5a involved in the formation of deoxyhypusine, the first committed step in the hypusine biosynthetic pathway, are potential new drug targets in plasmodium falciparum. to identify the phylogenetic homologs of the dhs gene in different plasmodium species, a library of plasmodium vivax expressed sequence tags was screened for a dhs homolog from p. falciparum. the putative dhs sequence from p. vivax is encoded by a bp nucleic acid sequence, with the most significant fasta scores of to p. yoelii and to p. falciparum strain 3d these results are in agreement with initial comparative analyses of the p. vivax genome with the other related plasmodium species, which revealed significant conservation of gene synteny between the different species within chromosome cores, in contrast to subtelomeric regions displaying reduced homology. p. vivax dhs displays striking amino acid sequence identity to its homolog from p. falciparum. notably, 89% amino acid identity is observed between p. vivax dhs protein and the human homolog between residues 290–. however, several gaps appear in the p. vivax protein sequence at positions and which are absent in the plasmodium falciparum homolog. the most significant differences between human and plasmodium dhs homologs are evident in the spermidine binding site, although the active site is highly conserved. the active site includes a conserved region of six amino acids from glu to lys. asp one of the charged amino acids at the bottom of the active site tunnel responsible for binding to spermidine and the gc inhibitor, is highly conserved in dhs proteins in plasmodium. moreover, in both dhs proteins, gly is within hydrogen-binding distance of the guanidinium group. the nad-binding site is strongly conserved between the different plasmodium species and their human homologs. however, a stretch of amino acids mainly comprising asparagine in this site is missing in dhs homologs from plasmodium. to confirm the function of p. vivax dhs, we determined its specific enzymatic activity in modification of eukaryotic translation initiation factor, eif-5a, by incorporation of the aminobutyl-moiety of 14-spermidine as a substrate. specific enzymatic activity was determined as u/mg protein, which was 9-fold less than that calculated by molitor and colleagues, who used human dhs protein for the modification of eif-5a from p. falciparum. in a control experiment with water instead of dhs from p. vivax, no specific enzymatic activity was detected. in a parallel inhibition experiment, the most commonly used competitive inhibitor of dhs, n1-guanyl- -diaminoheptane, was employed. gc inhibits dhs in a competitive manner by binding to its active site, as observed in crystallized form ii that was recently obtained for the complex of dhs and gc this inhibitor reduced specific enzymatic activity by 36-fold. while the amino acid identity between human and plasmodium vivax dhs is only 44%, we detected cross-reactivity between a polyclonal antibody raised against the human dhs enzyme and the expressed nickel chelate-purified protein from p. vivax, suggesting that the anti-dhs antibody recognizes specific epitopes present in both proteins.  the putative p. vivax dhs protein consists of amino acids with significant fasta scores of to p. yoelii and to p. falciparum strain 3d dhs proteins. a specific dhs enzymatic activity assay with 14c-labeled spermidine as the substrate and inhibition by n-guanyl- -diaminoheptane confirmed functional activity. moreover, the dhs protein from p. vivax cross-reacted with a polyclonal antibody against anti-human dhs. further biochemical characterization analyses of the p. vivax dhs protein are currently underway. 
sequencing of the plasmodium vivax genome suggested the presence of a homolog of deoxyhypusine synthase from p. falciparum, the key regulatory enzyme in the first committed step of hypusine biosynthesis. the putative dhs protein from p. vivax displays a fasta score of relative to dhs from rodent malaria parasite, p. yoelii, and relative to that from the human parasite, p. falciparum strain 3d the orf encoding amino acids was expressed under control of iptg-inducible t promoter, and expressed as a protein of approximately kda in e. coli bl de cells. the specific enzymatic activity of dhs was determined as u/mg protein. 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. 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. we describe the cloning of a bp putative deoxyhypusine synthase gene sequence from genomic dna of p. vivax pest strain salvador i after touchdown pcr. 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. 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. dhs with a theoretical pi of was present in both eluate fractions. the n-terminal histidine-tagged protein was purified by nickel-chelate affinity chromatography under denaturing conditions. dhs is involved in cell proliferation, and thus a valuable drug target for the human malaria parasite p. falciparum. plasmodium vivax is the most widespread human malaria parasite.
 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 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.  we describe the cloning of a bp putative deoxyhypusine synthase gene sequence from genomic dna of p. vivax pest strain salvador i 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 relative to dhs from rodent malaria parasite, p. yoelii, and relative to that from the human parasite, p. falciparum strain 3d the orf encoding amino acids was expressed under control of iptg-inducible t promoter, and expressed as a protein of approximately kda in e. coli bl de cells. the n-terminal histidine-tagged protein was purified by nickel-chelate affinity chromatography under denaturing conditions. dhs with a theoretical pi of was present in both eluate fractions. the specific enzymatic activity of dhs was determined as u/mg protein. the inhibitor, n-guanyl- 7-diaminoheptane, 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.  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. 