Báo cáo khoa học: Completing the hypusine pathway in Plasmodium Deoxyhypusine hydroxylase is an E-Z type HEAT repeat protein docx

Completing the hypusine pathway in Plasmodium
Deoxyhypusine hydroxylase is an E-Z type HEAT repeat protein
David Frommholz
1
, Peter Kusch
1
, Robert Blavid
1
, Hugo Scheer
2
, Jun-Ming Tu
2
, Katrin Marcus
3
,
Kai-Hong Zhao
4,5
, Veronica Atemnkeng
1
, Jana Marciniak
1
and Annette E. Kaiser
1
1 Hochschule Bonn-Rhein-Sieg, Rheinbach, Germany
2 Department of Biologie I-Botanik, Universita
¨
tMu
¨
nchen, Germany
3 Medizinisches Proteom Center, Ruhr-Universita
¨

t Bochum, Germany
4 State Key Laboratory of Agricultural Microbiology, Huazhong Agricultural University, Wuhan, China
5 College of Life Science and Technology, Huazhong University of Science and Technology, Wuhan, China
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
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:
(Received 24 May 2009, revised 9 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 biosyn-
thesis of hypusine present in eukaryotic initiation factor-5A (eIF-5A) in
Plasmodium. Here, we describe the cloning and expression of deoxyhypu-
sine 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 (uni-
protkb:
Q710D1)byenzymatic studies (MI:0415)
l
MINT-7255326: DOHH (uniprotkb:Q8I701) enzymaticly reacts (MI:0414) with eIF-5A (uni-
protkb:
Q710D1)byenzymatic studies (MI:0415)
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
host) [1]. Completion of the parasitic life-cycle requires
rapid changes in its environment such as the stimula-
tion 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 pres-
ently lacking.
Plasmodium falciparum and Plasmodium vivax
belong to the order Apicocomplexa and are character-
ized 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 cyanobacte-
rial (prokaryotic) origin. Although the apicoplast has
lost all photosynthetic capacity [2] it retains some met-
abolic 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 cyanobac-
teria and red algae. These heterodimeric proteins
attach linear tetrapyrrolic chromophores (bilins) cova-
lently to their apoproteins, which then organize into
phycobilisomes, the light-harvesting supercomplexes of
cyanobacteria and red algae. Attachment of the apo-
proteins 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 D3,3
1
dou-
ble 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 prolifera-
tion of the parasite and is an essential substrate in the
biosynthesis of hypusine [N(epsilon)-(4-amino-2-hy-
droxybutyl) 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 de-
oxyhypusine hydroxylase (DOHH; EC 1.14.9929) [6].
Whereas DHS catalyzes transfer of the aminobutyl
moiety to a specific lysine residue in the eIF-5A
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 dioxygenas-
es, four strictly conserved histidine–glutamate metal-
coordination 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.AS. cerevisiae knockout mutant showed
only a slower growth rate in the presence of the accu-
mulated deoxyhypusinylated form of eIF-5A [9].
Although eIF-5A and DHS have proven to be poten-
tial 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 biosyn-
thesis 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 sug-
gested 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-carboxy-
lates 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 pro-
tein 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
Deoxyhypusine hydroxylase from Plasmodium D. Frommholz et al.
5882 FEBS Journal 276 (2009) 5881–5891 ª 2009 The Authors Journal compilation ª 2009 FEBS
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 HEAT-
like 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, 6
and 7) contains a highly significant histidine–gluta-
mate (HE) motif corresponding to the characteristic
metal-chelating sites. By contrast, DOHH of P. falci-
parum 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 phy-
coerythrocyanin subunits located between amino acid
positions 76–179 and 47–184 (i.e. HEAT-like repeats
1 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 histi-
dine-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 troph-
ozoites 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 ( />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 Esch-
erichia coli BL21 (DE3) cells harboring the T7 RNA
polymerase under control of the T7 promotor. Expres-
sion and purification of the DOHH protein by nickel-
chelate-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] Con-
trols were the respective strain without DOHH, and the
strain expressing, in addition, the genes for the noniso-
merizing 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 Ni
2+
-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 experi-
ment with lyase subunits, the chromophore was prop-
erly attached; here the presence of DOHH either had
no influence (PecE ⁄ F; Fig. 3D) or was somewhat inhib-
itory (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 pro-
tein 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 gen-
erates a small background absorption at 645 nm. Crude
extracts with expressed (green lane) and nonexpressed
(red lane) DOHH protein showed a reduced back-
ground reaction, and also, in small yield, the addition
of PecA at the central methine bridge of the chromo-
phore 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). More-
over, a 10–100-fold higher absorption would be gener-
ated 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 nonradio-
active system was established. First, we modified the
D. Frommholz et al. Deoxyhypusine hydroxylase from Plasmodium

FEBS Journal 276 (2009) 5881–5891 ª 2009 The Authors Journal compilation ª 2009 FEBS 5883
eIF-5A protein from P. vivax to the deoxyhypusinylat-
ed 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. Micro-
con-YM 100 and 30 kDa. In Fig. 4, lane 2 the first
size-exclusion chromatography step with the Micro-
con-YM100 is presented showing that both proteins
are recovered in the eluate. Subsequent size-exclusion
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 anal-
ysis 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
Fig. 1. Multiple amino acid alignment of
DOHH proteins from four different eukary-
otes (Saccharomyces cerevisiae, Schizosac-
charomyces 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. falci-
parum 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 pro-
teins involved in energy metabolism and
conversion. The most significant amino acid
identity to HEAT repeats in CpcE from Syn-
echococcus elongates is found in E-Z-type
HEAT repeat domains 1 and 2 (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.
Deoxyhypusine hydroxylase from Plasmodium D. Frommholz et al.
5884 FEBS Journal 276 (2009) 5881–5891 ª 2009 The Authors Journal compilation ª 2009 FEBS
in the complete DHS assay (Fig. 5, eIF-5A, lane A)
and in the eluate (Fig. 5, eIF-5A, lane E) after Micro-
con-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 chro-
matography 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 derivatiza-
tion with methyl chloroformate [18] which esterifies
reactive side chains and carboxyl groups. In addition
to the molecular ion [M]
+•
at m ⁄ z 347, several promi-
nent fragments of deoxyhypusine were detected, i.e.
[M-NH-C(O)OCH
3
] at [M-74]
+
, [M-C(O)OCH
3
] with
[M-59]
+
, [M-2(NH-C(OO)CH
3
)
+
] with [M-2Æ74]
+
and

[M-2ÆNHC(O)OCH
3
-C(O)OCH
3
-OCH
3
] with [M-2Æ74-
59-31]
+
.
In order to assay the activity of recombinant DOHH
derived from P. falciparum, the nonradioactively modi-
fied eIF-5A (Dhp) was incubated with purified recom-
binant 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-OCH
3
] with [M-31]
+
.
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 deoxyhypus-
inylated eIF-5A. The data demonstrate that a com-
plete 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
123 4 56 M
42 kDa
55 kDa
43 kDa
29 kDa
20 kD
a
Fig. 2. Expression and purification of DOHH. (A) Purification of
histidine-tagged recombinant DOHH by nickel-chelate affinity chro-
matography under native conditions. M, Roti standard protein mar-
ker. Lane 1, lyzed crude cell extract; 2, flow-through; 3 and 4, wash
fractions; 5, eluate fraction containing recombinant DOHH 6) sec-
ond eluate faction.
0.05
0.10
0.15
0.4
0.8
1.2

B
Absorption
A
0.06
0.12
0.18
400 500 600 700 800
0.7
1.4
2.1
D
Absorption
C
λ (nm)
Fig. 3. Assay of DOHH for phycocyanin C-a84 lyase (A,B) and phy-
coerythrocyanin C-a84 lyase ⁄ isomerase (C,D) activities. Absorption
spectra of acceptor proteins, CpcA and PecA, after treatment with
PCB, and purification by Ni
2+
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 isomeri-
zation 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).
D. Frommholz et al. Deoxyhypusine hydroxylase from Plasmodium
FEBS Journal 276 (2009) 5881–5891 ª 2009 The Authors Journal compilation ª 2009 FEBS 5885
curved layers of a helices arranged in a regular right-

handed superhelix with the repeats arranged about a
common axis [19]. These superhelical structures pres-
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 resem-
ble those found originally in phycocyanin lyase subun-
its 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 isomeri-
zation [3]; the binding can be followed chromatograph-
ically 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 phyco-
biliprotein-type light-harvesting complexes, but subse-
quently adapted to a new role as a hydroxylase during
evolution. Because the dohh gene is not part of the api-
coplast genome, this would imply a gene transfer to

the nucleus.
A structural annotation in MADIBA [20] for selec-
tion 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 pro-
moter regions which are found in chlorophyll a ⁄ b
binding proteins [21].
dohh is present as a single-copy gene on chromo-
some 13 in P. falciparum and is transcribed in asexual
blood stages ( 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 devel-
opment.
One interesting feature, according to the prediction
of the PlasmoAP bioinformatic tool [23] is the
123
212 kDa
118 kDa
66 kDa
43 kDa

29 kDa
20 kDa
E 30E 100FT 30
elF-5A
DHS
Fig. 4. Separation of modified eIF-5A on SDS ⁄ PAGE after size-
exclusion chromatography with a Microcon-YM 100 kDa and a
Microcon-YM-30 kDa column. 1, Flow-through after the Microcon-
YM-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
FT E E A
M
YM 30
YM 30
YM 100
69 kDa
29 kDa
eIF-5A
20 kDa
Fig. 5. Western blot experiment after size-exclusion chromatogra-
phy of modified eIF-5A (Dhp); 1 : 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.
Deoxyhypusine hydroxylase from Plasmodium D. Frommholz et al.
5886 FEBS Journal 276 (2009) 5881–5891 ª 2009 The Authors Journal compilation ª 2009 FEBS
[M]
+
HN

NH NH
O
OCH
3
OCH
3
H
3
CO
O
A
– 74
– 74
– 59
– 59
– 59
O
B
32

000
79.1
253.0
52.1
AbundanceAbundanceAbundanceAbundance
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
0
20
7000
6000
5000
4000
3000
2000
1000
0
342 343 344 345 346 347 348 349 350 351
40 60 80 100 120 140 160 180 200 220 240 260 280 300 320 340 360 380 400
119.0
158.1
m/z >
m/z >
m/z >
m/z >
346.0
347.0
348.1
349.1
405.1
375.1
195.0
222.9
281.0
331.1

346
401.1
327.1
297.0
253.0
195.0
156.1
119.1
79.1
36.0
339.0
341.1
342.1
345.1
344.1
346.1
347.0
348.0
223.0
343.1
120

000
110

000
100

000
90

000
80

000
70

000
60

000
50

000
40

000
30

000
20

000
10

000
0
20 40 60 80 100 120 140 160 180 200 220 240 260 280 300 320 340 360 380 400 420
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
0
337 338 339 340 341 342 343 344 345 346
[M-31]
+

347 348 349
Fig. 6. (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 deoxyhypu-
sine. (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-OCH
3
] with [M-31]
+
repre-
senting the hydroxyl group were identified.
The most characteristic fragment ions are
presented in the structure of the hypusine
derivative. Deoxyhypusine is also present.
D. Frommholz et al. Deoxyhypusine hydroxylase from Plasmodium
FEBS Journal 276 (2009) 5881–5891 ª 2009 The Authors Journal compilation ª 2009 FEBS 5887
occurrence of a signal peptide with a cleavage site at
the N-terminal position 26 in the dohh gene. In photo-
synthetic plants, and in Plasmodia which contain the

nonphotosynthetic apicoplasts, transit peptides [23,24],
direct proteins to the apicoplast that has certain plant-
like-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 target-
ing [24]; however, the N-terminus of the DOHH pro-
tein 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 [
14
C]-
labeled spermidine [25]. We combined eIF-5A from
P. vivax and human DHS for the synthesis of deoxy-
hypusine 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 deriv-
atives [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 deoxy-
hypusine in the DOHH activity assay.
To investigate how the additional E-Z-type HEAT

repeat present in DOHH from Plasmodium may influ-
ence hydroxylase activity, a quantitative assay with
nonradioactively labeled eIF-5A [26] and purified
DOHH enzyme from human and the parasite is cur-
rently 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
(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¢ 1 lm, downstream primer DOHH reverse 5¢-
CTAGTGAACCTCTATAGATATAA-3¢ 1 lm, 25 mm
MgSO
4
,1mm, AMV reverse transcriptase 0.1 UÆlL
)1
and a
proof-reading ReproFast Taq polymerase (Genaxxon, Ulm,
Germany) 0.1 UÆlL

)1
and 4.5 lg total cellular RNA from
P. falciparum strain NF54. First-strand synthesis was per-
formed at 45 °C for 45 min. AMV ⁄ RT was inactivated at
94 °C for 2 min. The following program was applied for sec-
ond-strand synthesis and PCR amplification: 94 °C for 30 s,
60 °C for 1 min, 68 °C for 2 min (40 cycles). The final exten-
sion was performed for 7 min 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 lgÆmL
)1
) and kanamycin (15 lgÆmL
)1
) was
used for expression in pET-28a.
One milliliter samples from the expressing strain was
taken and centrifuged at 13 000 rpm for 2 min. Cells were
lysed with 400 lL lysis buffer (50 mm Tris ⁄ HCl, pH 8.0,
2mm EDTA), centrifuged, resuspended in lysis buffer and
sonicated twice at 4 °C for 30 s (tip 1 at 50% using a Bran-

son sonifier). After centrifugation for 10 min at 16 000 rpm
at 4 °C, samples were diluted 1 : 1 in loading buffer
(20 mm Tris, pH 6.8, 2% w ⁄ v SDS, 2 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 5 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 lLof
10 mgÆmL
)1
) and 3 UÆmL
)1
culture volume Benzon-
aseÒNuclease were added. The suspension was incubated
on ice for 15–30 min. Centrifugation was applied at
12 000 g for 30 min at 4 °C. A Ni-NTA spin column was
equilibrated with 600 lL lysis buffer containing 10 mm
imidazole. Centrifugation for 2 min at 890 g followed. Six
hundred microliters of the cleared lysate containing the 6·
His-tagged protein was loaded onto the pre-equilibrated
Deoxyhypusine hydroxylase from Plasmodium D. Frommholz et al.
5888 FEBS Journal 276 (2009) 5881–5891 ª 2009 The Authors Journal compilation ª 2009 FEBS
Ni-NTA spin column and centrifuged for 5 min at 270 g.
The Ni-NTA spin column was washed twice with 600 lL
washing buffer containing 5.0 mm NaH
2
PO

4
, 300 mm
NaCl, 5 mm imidazole, pH 8.0 and centrifuged for 2 min at
890 g. DOHH was eluted from the column with 300 lL
elution buffer 50 mm NaH
2
PO
4
, 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 geno-
mic DNA of P. falciparum strain NF54 using primers with
restriction enzymes for NotI (recognition site is underlined)
DOHH express
forw
5¢-TTAAGCGGCCGCATGGGAGAA
AATAACGA-3¢ and BamHI DOHH express
rev
5¢-AAAAG
GATCCCTAGTGAACCTCTATAGATAT-3¢. The result-
ing 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 amplifica-
tion was performed with primers DOHH express
for
5¢-
AAAAAA

CATATGGAGAAAATAAC-3¢ and DOHH
express
rev
5¢-AAAAGGATCCCTAGTGAACCTCTATAG
ATATT-3¢. The restriction sites for NdeI and Bam HI 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 respec-
tive 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 con-
taining the respective plasmids. After induction of the cells,
extraction and purification of the acceptor protein by che-
lating 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 chroma-
tography under native conditions. Buffer exchange was per-
formed with a Sephadex-G25 column before the incubation
of DHS activity. A reaction mixture of 1 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 2 h. The un-modified and modified eIF-5A
precursor protein was recovered by a Microcon-YM
100 kDa column (Amicon, Millipore, Schwalbach, Ger-
many), retaining DHS. A subsequent application of a Micro-
con-YM 30 kDa column enriched both forms of eIF-5A.
Protein hydrolysis was performed under nitrogen in 6 m HCl
at 120 °C for 24 h. The eluate was evaporated to dryness,
derivatized with methyl chloroformate according to the pro-
tocol 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 ⁄ P
i
pH 7.4,
1mm NAD, 1 mm dithiothreitol and  20 lg eIF-5A
(Dhp) in a reaction volume of 600 lL. Incubation was per-
formed for 3 h at 37 °C. The modified eIF-5A protein was
recovered by size-exclusion chromatography and hydro-
lyzed in 6 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 chro-
matograph 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 thick-
ness 0.25 lm. The temperature of the column was pro-
grammed from 60 °C (1 min hold) and increased by
5 °CÆmin
)1
to 280 °C (50 min hold). A constant helium
flow of 0.928 cm
3
Æmin
)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 tempera-
ture (details presented in the Result), the mixture was centri-
fuged for 15 min at 15 000 g to remove any particulate
matter and the supernatant was investigated by UV ⁄ Vis
absorption and light-induced absorption changes [4].
D. Frommholz et al. Deoxyhypusine hydroxylase from Plasmodium
FEBS Journal 276 (2009) 5881–5891 ª 2009 The Authors Journal compilation ª 2009 FEBS 5889

Acknowledgements
We thank Professor Dr J. Hauber (Heinrich-Pette-
Institut, 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 Forschungsgemeins-
chaft (SFB 553) and KHZ support from the National
Natural Science Foundation of China (grants30670489
and 30870541).
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Supporting information
The following supplementary material is available:
Fig. S1. RT-PCR of the dohh gene from Plasmo-
dium 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
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should be addressed to the authors.
D. Frommholz et al. Deoxyhypusine hydroxylase from Plasmodium
FEBS Journal 276 (2009) 5881–5891 ª 2009 The Authors Journal compilation ª 2009 FEBS 5891