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

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 a

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 Marciniak1and Annette E Kaiser1

1 Hochschule Bonn-Rhein-Sieg, Rheinbach, Germany

2 Department of Biologie I-Botanik, Universita¨t Mu¨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: kaiser@microbiology-bonn.de

(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 ) by enzymatic studies ( MI:0415 )

l MINT-7255326 : DOHH (uniprotkb: Q8I701 ) enzymaticly reacts ( MI:0414 ) with eIF-5A (uni-protkb: Q710D1 ) by enzymatic 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.

host) [1] Completion of the parasitic life-cycle requires

rapid changes in its environment such as the

stimula-tion and inhibistimula-tion 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 D3,31

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 A S 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

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 (http://plasmodb.org/plasmo/)

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 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 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

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.

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

(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)OCH3] at [M-74]+, [M-C(O)OCH3] with

[M-59]+, [M-2(NH-C(OO)CH3)+] with [M-2Æ74]+ and

[M-2ÆNHC(O)OCH3-C(O)OCH3-OCH3] 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 purimodi-fied

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-OCH3] 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

1 2 3 4 5 6 M

42 kDa

55 kDa

43 kDa

29 kDa

20 kDa

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

A

0.06 0.12 0.18

400 500 600 700 800 0.7

1.4

2.1

D

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).

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 solvpres-ent-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 (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 devel-opment

One interesting feature, according to the prediction

of the PlasmoAP bioinformatic tool [23] is the

212 kDa

118 kDa

66 kDa

43 kDa

29 kDa

20 kDa

E 30

E 100

FT 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

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

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.

HN

O

O

A

– 74

– 74 – 59 – 59

– 59

O

B

32 000 79.1

253.0 52.1

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

40 000

36 000

32 000

28 000

24 000

20 000

16 000

12 000 8000 4000

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-OCH3] 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.

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

CGACAAC-3¢ 1 lm, downstream primer DOHH reverse

MgSO4, 1 mm, AMV reverse transcriptase 0.1 UÆlL)1and a proof-reading ReproFast Taq polymerase (Genaxxon, Ulm,

P falciparum strain NF54 First-strand synthesis was

94C for 2 min The following program was applied for sec-ond-strand synthesis and PCR amplification: 94C for 30 s,

60C for 1 min, 68 C for 2 min (40 cycles) The final exten-sion was performed for 7 min at 68C 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,

2 mm EDTA), centrifuged, resuspended in lysis buffer and sonicated twice at 4C for 30 s (tip 1 at 50% using a Bran-son Bran-sonifier) After centrifugation for 10 min at 16 000 rpm

glycerol, 0.3% bromphenol blue) heated at 100C 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 lL of

Benzon-aseNuclease were added The suspension was incubated

on ice for 15–30 min Centrifugation was applied at

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

Ni-NTA spin column and centrifuged for 5 min at 270 g.

The Ni-NTA spin column was washed twice with 600 lL

NaCl, 5 mm imidazole, pH 8.0 and centrifuged for 2 min at

890 g DOHH was eluted from the column with 300 lL

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)

GATCCCTAGTGAACCTCTATAGATAT-3¢ The

result-ing fragment of 1236 bp was digested with NotI and

vector and re-sequenced For subcloning into pET-15b,

which was digested with NdeI and BamHI, the

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

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

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 120C 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 falciparumNF54 strain (7.5 lg), 50 mm NaCl⁄ PipH 7.4,

(Dhp) in a reaction volume of 600 lL Incubation was

recovered by size-exclusion chromatography and hydro-lyzed in 6 m HCl at 120C for 24 h Hypusine was isolated

as deoxyhypusine and derivatized by methylchloroformate and determined by GC⁄ MS [19]

GC/MS

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

flow of 0.928 cm3Æmin)1 was used The temperature of the split⁄ splitless injector was 250 C The electron impact ion

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 mixtempera-ture was centri-fuged for 15 min at 15 000 g to remove any particulate

absorption and light-induced absorption changes [4]

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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