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Anti-infectives
Giglione & Meinnel
Peptide deformylase as an emerging target for antiparasitic agents
Peptide deformylase as an emerging target
for antiparasitic agents
Carmela Giglione & Thierry Meinnel
Institut des Sciences Végétales, UPR2355, Centre National de la
Recherche Scientifique, Bâtiment 23, 1 avenue de la Terrasse, F-91198
Gif-sur-Yvette cedex, France.
http://www.ashley-pub.com
Review
1. Introduction
2. The PDF family: a new,
growing sub-family of the
HEXXH-containing
metalloprotease
super-family
3. The function of PDF
orthologues in eukaryotes
4. Many parasitic illnesses
and various PDFs to
inhibit
5. Parasite PDFs as drug
targets
6. Expert opinion: new
anti-parasitic drugs are
needed and PDF is a
target of choice
Acknowledgements
Bibliography
Websites
Peptide deformylases (PDFs) constitute a growing family of hydrolytic
enzymes previously believed to be unique to Eubacteria. Recent data from
our laboratory have demonstrated that PDF orthologues are present in
many eukaryotes, including several parasites. In this report we aim to
explain why PDF could be considered to be a potent target for human and
veterinary antiparasitic treatments.
Keywords: antibiotic, antiparasitic, Chagas’ disease, deformylase, malaria,
plasmodium, sequence homology, sleeping sickness, target, trypanosomatids
Emerging Therapeutic Targets (2001) 5(1):41-57
1. Introduction - Detecting and stabilising deformylase
activity in vitro
Polypeptide deformylase (PDF) was first detected in crude bacterial
extracts more than 3 decades ago [1,2]. This finding was the logical
extension of previous studies, which had shown that, although protein
synthesis started at a N-formylmethionine in bacteria, the N-formyl group
and often the methionine itself were absent from the N-termini of mature
proteins [3,4]. The deformylation step is part of the methionine cycle [5].
Deformylation plays a crucial role in this process as it is necessary for
subsequent removal of the unblocked methionine by methionine
aminopeptidase (MAP). MAP action is an essential part of the N-terminal
maturation process in all cells (for further details and references see also
[6,7]). The presence of an N-formyl group on the methionine residue at the
start of nascent polypeptides in bacteria seemed to contrast with the
situation in eukaryotes and Archae, in which nascent proteins synthesised
in the cytoplasm start with a free methionine. However, it was known that
proteins synthesised in eukaryote organelles also start with an N-formyl
methionine [8]. Most of the sequences of mitochondrial proteins obtained
from fungi and mammals indicate that the N-formyl group is retained. It was
therefore concluded that PDFs were unique to Eubacteria [9].
Data obtained at the end of 1960s indicated that PDF activity was very
unstable and was lost upon any attempt at molecular fractionation. This
lability prevented further characterisation of the protein for 25 years. The
PDF gene (def or fms) from Escherichia coli was finally cloned in 1993
[9,10]. PDF was then overproduced and the resulting protein characterised
in 1995 [11]. It was found that a metal cation was required for activity and
the nature of the three metal ligands was determined [12]. It is now
41
2001 © Ashley Publications Ltd. ISSN 1460-0412
42 Peptide deformylase as an emerging target for antiparasitic agents
generally accepted that bacterial PDF are linked to a
very unstable metal cation (Fe2+) and that the
atmospheric oxidation of this cation rapidly and
irreversibly inactivates the enzyme [13-15]. This
accounts perfectly for the instability of PDF activity.
The ferrous enzyme remains stable for less than one
minute in vitro.
The conditions required for the stabilisation of
deformylase activity were not determined until 1998.
They involve the use of either:
• reactive oxygen species scavengers such as TCEP
(Tris(2-carboxy- ethyl)-phosphine) and catalase, or
• the early replacement of the iron cation by metal
cations insensitive to oxygen such as nickel [13,16]
or cobalt [17,18], both of which preserve enzyme
activity, although cobalt much less so than nickel.
Several in vitro assays of PDF activity have recently
been developed and have proved very useful for
optimising conditions [19-21]. It should be noted that
determining the conditions required for stabilisation
of the metal cation and therefore of the activity of a
new PDF species remains a crucial and difficult step.
Since many new PDFs (see section 2) have recently
been discovered, before any in vitro data are
obtained, it cannot be excluded a priori that metals
other than iron and nickel may give them full
deformylase activity.
2. The PDF family: a new, growing
sub-family of the HEXXH-containing
metalloprotease super-family
This section of the review (and part of the next) will
take advantage of many in silico data [101-109]. Such
data are not yet regarded as ‘fact’ in the same way that
in vitro/vivo data are, but this may change as our data
sets enlarge and more supporting evidence accumulates. For instance, these in silico data [6] led most
recently to Pei’s work with the Plasmodium PDF (see
review by Pei, same issue) and our own most recent
work [7].
2.1 Three
conserved motifs and a conserved
arginine build the active site of eubacterial
PDFs
PDF was initially believed to occur in eubacteria only.
To date, more than 90 eubacterial PDF sequences
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have been determined [110]. Protein sequence
alignments have identified only 3 sets of
well-conserved residues, motif 1 {gφgφaapQ}, motif 2
{EgCφs} and motif 3 {HEφDHlxg} (where φ is any
hydrophobic aliphatic amino acid, with L > I > M > V;
see Figure 1 and [6,22]). Based on the 3D-structure
(see references quoted in [6]) and taking into account
site-directed mutagenesis data, it is now clear that the
three motifs build:
• the three sides of the active site, and
• part of the hydrophobic pocket in which the
methionine side chain of the substrate is buried.
More precisely, the two residues of motifs 1 and 3
shown in bold (see above) directly contribute to
catalysis, whereas the three underlined residues are
involved in binding to the metal cation. The acidic
side chains of the two residues shown in italics
hydrogen bond with the guanidium of a conserved,
buried arginine located between motifs 2 and 3. This
arginine is generally located in the vicinity of a
conserved valine (see VXR in Figure 1). Conservation
of the residues of the motif shown in lower case
appears, a priori, not to be strictly required although
the chemical nature of these residues (small,
hydrophobic and/or hydrophilic) is known to play a
role in generating the correct 3D structure of the active
site. Thus, a PDF may have activity, even in the
absence of several of the conserved residues of the 3
motifs, especially those of motif 1 [23]. Analysis of the
hydrophilic/hydrophobic nature of the side chains of
the residues of the various secondary structure
elements identified in the 3D-structure of the E. coli
enzyme strongly suggests that all PDFs have a
near-identical 3D folding pattern [22].
Demonstration of the importance of the HEXXH
sequence of motif 3 and its actual role in catalysis led
to the early assignment of PDF to the HEXXHcontaining metalloprotease super-family [24,25].
Indeed, the active site of bacterial PDF has a
secondary superstructure common to thermolysins
and matricins, the other two sub-families of the
super-family [26]. Extensive structural similarity
between PDF and matricins was also observed [23].
However, the nature and location of the third metal
ligand, the cysteine of motif 2, differs from those of
thermolysins and matricins. For this reason, PDFs
were classified as a third, novel, sub-family.
Emerging Therapeutic Targets (2001) 5(1)
Giglione & Meinnel 43
These structural data are important in definition of the
criteria for membership of the PDF family and for
deformylase activity.
2.2 A
2.2.1
phylogenetic tree for PDFs
Two classes of PDF in Eubacteria
From the sequence data obtained in bacteria and from
an analysis of the sets of deletions and insertions
(called I1 and I2 in Figure 1) located between identified secondary structures elements, it was concluded
that PDFs can be divided into at least two major
families [6]. Class I is typified by the E. coli enzyme and
the PDF of all Gram-negative bacteria fall systematically into this class (Figure 2). Class II PDF is typified
by Bacillus stearothermophilus PDF and contains PDF
from Mycoplasma and Gram-positive bacteria with
low G+C content (Figure 2). However, if two PDF
occur in such Gram-positive bacteria, the second is
often a Class I enzyme. PDF homologues significantly
dissimilar to both these classes were found, also in
Gram-positive bacteria with low G+C content, such as
Clostridium beijerinckii. Finally, the complete
genome sequence of the actinomycete Streptomyces
coelicolor revealed the presence of four def genes.
This corresponds to the largest number of PDF genes
detected in any eubacterium to date. Interestingly,
one of the four PDF of this actinomycete (S. coelicolor
1/4 in Figure 2) diverges from all other known PDFs.
It has been reported that actinomycetes naturally
produce a molecule with anti-PDF activity, actinonin
[27,28]. However, actinomycetes are themselves
resistant to actinonin, possibly due to the acquisition
of an actinonin-resistant PDF. This could explain the
presence of this divergent PDF in actinomycetes.
Alternatively, a function other than the classic
deformylation of nascent polypeptides may account
for this unusual PDF and the redundancy of def genes
in S. coelicolor.
2.2.2 The
PDFs from higher Eukaryotes
Analysis of the recently produced sequences of the
complete genomes of several higher eukaryotes
surprisingly revealed the presence of PDF
orthologues [6]. For instance, two Class I PDFs were
identified in the nuclear genome of both monocotyledonous and dicotyledonous flowering plants [7]. A
PDF sequence has also been identified in the genome
of the liverwort Marchantia polymorpha. This
indicates that PDFs are found in all higher plants
(Embryophyta). These enzymes differ from
© Ashley Publications Ltd. All rights reserved.
eubacterial PDFs in possessing N-terminal
pre-sequences that target the corresponding catalytic
domains to various compartments of the cell. Hence,
plant mitochondrial PDFs (mPDFs) are targeted to the
mitochondria only, whereas chloroplast cpPDFs are
the only PDFs found in the plastids (see Figure 2 and
[7]). The cpPDF are closely related to the PDFs of
cyanobacteria (Figure 2), which are believed to be
the ancestors of this photosynthetic organelle. mPDF
orthologues were also be identified in insects and the
tissues of various vertebrates and the corresponding
full-length cDNA has been cloned in humans [7]. All
animal PDF orthologues have sequences very similar
to those of mPDFs and are clearly derived from the
same branch of the PDF phylogenetic tree (Figure 2).
The bacterial sequence most similar to those of the
various mPDFs is the divergent PDF from S. coelicolor
(Figure 2).
2.2.3 The
PDF of lower Eukaryotes
PDF sequences have also been found in the genomes
of various eukaryotic protists. A cpPDF (i.e., a PDF
resembling the plastid PDFs of plants) was identified
in the malaria agent Plasmodium falciparum and is
thought to be targeted to the apicoplast [7,29]. A PDF
was also identified in the amoeba Dictyostelium
discoideum [7]. This PDF, which is probably targeted
to the mitochondria, more closely resembles a Class II
PDF (Figure 2). Finally, two different PDF
orthologues were found in the Kinetoplastids,
Trypanosoma spp. and Leishmania major (Figure 2;
[29]). These PDF species are clearly different from
Class I and Class II PDFs and we now propose that
they should be classified as a new class, Class III.
2.2.4 The
PDF orthologues of Archae
PSI-BLAST [30] is now recognised to be a powerful
tool for identifying biologically relevant sequence
similarities. Using this program with several PDF
sequences, we recently identified for the first time two
new sequences from Archaea displaying strong
similarity to PDF (Figure 1 and Figure 2). These
archaeal PDFs were found in the euryarchaeota
Methanothermobacter thermoautotrophicus
(Genbank accession number AE000809) and
Methanothermus fervidus (Genbank accession
number CAA70987). These two sequences are most
closely related to the sequences found in kinetoplastids (i.e., Class III PDFs). A specific feature of archaeal
PDFs is the occurrence of a distal insertion between
motifs 2 and 3 (Figure 1). Given the alternation of
Emerging Therapeutic Targets (2001) 5(1)
I2
Motif 1
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TREQRLKERVAARMELEAQVKSR-VACYPHRSLTRPA-LRLERHQVNTP-LFHSQLLNLNKMATDLQ—----———-CISFSAPKGHWDAAI
T. cruzi1/2
T. brucei
FTLFD—————-------NSVFINPVNLDEEVWRAEAARQGMSWVAFEEEKMREL-RAEGLTGFAWEPCASSGF-LLHYIERPLTVRMRALDE
EGCφS
VXR
-VTRFDEDEMIGRTLEIVYLFLNPRIISEEG——---------------——————————— TVYRFEKCGSRNE-—RELVSRPYRVVVDGDYI
Methanothermobacter
NP
-VLIKGHPNEA—--—-NFEVWVNPTVPGYDDRHSIAP——-----------—————————-MYGMWENCISCGA-CTAWVIRPQSITCSGLDE
T. brucei
T. cruzi1/2
-ILIKSNPDET-----EYEVWVNPSVPGYDDRNAVAP----—————---------————-MYGMWENCISCGT-ATAWVVRPQRITCSGYDE
-VLIKSHPDEE—----VFEVWVSPSVPDYDARTSIAP--—-----------————————-MYGMWENCISCGA-TAAWVIRPQSVTCSGWDE
L. major2/2
L. major1/2
FTLFD—————-------GSVFINPVNLDLLEVEAAGSRSGM-PIAEAEAQWVASCRREGKTCFAWEPCASCCF-LMHYIERPATVRIRAIGA
T. cruzi2/2
—IAVHVTDENGT—-LYSYALFNPKIVSHSVQQC-------------—————————————YLTTGEGCLSVDRDVPGYVLRYARITVTGTTL
FLLKLPSQEGLNCPNFPLTAFFNPKIKLIDQDNN-------------—————————————TITMLESCLSVPN-IFAHVQRSKRCIITFLDI
B. stearothermophilus
--IVWNALYEKRKEE-NERIFINPSIVEQSLV--------------——————————————KLKLIEGCLSFPG-IEGKVERPSIVSISYYDI
D. discoideum
Motif2
P. falciparum
Central insertion
--IVIDVSENRDE—--—RLVLINPELLEKSG--------------——————————————-ETGIEEGCLSIPE-QRALVPRAEKVKIRALDR
gφgφaapQ
E. coli
Lr
MDDADLKKLLRFTITEKRVIEKLQIPPDAFLPLL-FSIRFGGDW—----———-SLRKNSSRFMAIKEK
AREKRLKDRVAARLELEAQVKSR-VACYPHRSLTRPA-LRLDRTQVNTP-LFQSQLLSLKKMASDLR—----———-CISFSAPKGHWDATV
L. major1/2
Methanothermobacter
SSNSSGSFADSSRPYVPGQAVQE—-TYPIIQLPARSLWCRQYALDARRVAQGEYAGLISQVREARHYYQ—---——YPSMSAPQTGWNVQM
SSSPSFKETVEGKLEKEAEALRR-VACYPHRSMTRPV-MPVPTSQILSP-VFMSSLMDLNQLATGLH—----———-CLSFSAPKAHWDAAV
L. major2/2
????LCAPQIGWNVQM
T. cruzi2/2
MITMKDIIKEGHPTLRKVAEPVPLPP-SEEDKRILQSLLDYVKMSQDPELAAKYGLRPGIGLAAPQINVSKRM
FILFYFFYNILIKFKMEIVNISKNGVKVGNRVLREKALPWS-——KEKLNDVRRVEKLLEKMYKEMKDCT----———GTGIAAPQIGVNKQL
D. discoideum
B. stearothermophilus
SNIKQKRKGSLYLLKNEKDEIK—IVKYPDPILRRRSEEVT-——NFDDNLKRVVRKMFDIMYESK------—————GIGLSAPQVNISKRI
P. falciparum
SVLQVLHIPDERLRKVAKPVE-——EVNAEIQRIVDDMFETMYAEE------—————GIGLAATQVDIHQRI
I1
MLRHLFRCT——AAWAPKRSA
T. brucei
N-terminal pre-sequence
MLSRLSRTVPLLG——PRRSA
T. cruzi1/2
E. coli
MRRCVPWRRLVCGGTSLLRGGVDAGGAPL
L. major1/2
???????????????????????????
MRTSAAAAVAAAAHAGVRALHTATGVAGNSCGASPRATVALPPHSGLTLPSFTRCGASSAAFITRAHSVG—CCGCAAAVKQHRLYSSHGC
L. major2/2
MLMYYSLSLFNLIICCNVTSIYGYIHNVRSLEPYIKNDQIKNYS
D. discoideum
N-terminal pre-sequence
P. falciparum
Figure.1: Alignment of eukaryotic protist PDFs
44 Peptide deformylase as an emerging target for antiparasitic agents
Emerging Therapeutic Targets (2001) 5(1)
HGN———-------————HKVQVLDGMRARCLMHELDHLMGKTIFHQAVGPEFVVSSVAMAQRYLWPANFPSAEAYVTTPGQFFDYVQNETV
L. major1/2
© Ashley Publications Ltd. All rights reserved.
HEφDHLXG
IPPGMEWWYAQNVREEFSNEQIGQ
??PGMEWWYAQNMQQHFQDARLNQ
IPPGMEWFYAQSMNQQFEDARLSH
L. major1/2
T. cruzi1/2
T. brucei
PDF amino acid sequences from eukaryotic protists were aligned using ClustalX software and by hand. The sequence indicated as Methanothermobacter corresponds to the
protein from Methanothermobacter thermoautotrophicus, Genbank accession number AE000809. The three previously defined motifs and the other conserved residues are
shown in bold-typeface. A series of question marks indicates unknown parts of the sequence. Special features are indicated above the sequence.
LDRKFEDGIYPGCEQDRQQRIELTAMEEIQRNVWRKEKAKRKEGGQQCGRGDVTAVEDDDGSGAAGAR
C-terminal extension
L. major2/2
A
LRAVLDPLDLKIRLKRLEKPLRFTGSGAYGVAHEMEHLEGEESEGTPFWEFEYEIEE
HGK——————-------—PFTVTLDKMRARMALHELDHLQGVLFTRRVVDTDHVVPMEGFVTMSDWSDDYRGCPARTPTRSFVSDAVPDGNL
L. major2/2
Methanothermobacter
DGH—————-------——PFEVTLEKMRARMALHELDHLSGVLFTRRIPDSNHVVPLEGFSTLSGWSDDFPSLEAPQTFLYTTLTSPYTF
T. cruzi2/2
YGN——————-------—EKTELLDGMRARCLMHELDHLTGKTILHQALGPEFIGSGIAMGQ??????????????????????????????
DGE————-------———EVTLRLKGLPAIVFQHEIDHLNGIMFYDRINPADPFQVPDGAIPIGR
B. stearothermophilus
YGN—————-------——EKTEVLDGMRARCLMHELDHLSGKTILDQAQGPEFIVSGIAMGQRDLWPPNFPSAEAYMTSPHQFFDYVKNGPI
TGK—————-------——ERIIEADGILAACFQHEYDHLLGKIFIDRIDKSELSNKLIYTTELTEDNLREIFKLHGDFQIIK
T. brucei
NGY————-------———KHLKILKGIHSRIFQHEFDHLNGTLFIDKMTQVDKKKVRPKLNELIRDYKATHSEEPAL
D. discoideum
T. cruzi1/2
DGK——————-------—PFELEADGLLAICIQHEMDHLVGKLFMDYLSPLKQQRIRQKVEKLDRLKARA
dispensible C-terminus domain
P. falciparum
Motif3
E. coli
Distal insertion
Figure.1: Alignment of eukaryotic protist PDFs (continued)
Giglione & Meinnel 45
Emerging Therapeutic Targets (2001) 5(1)
46 Peptide deformylase as an emerging target for antiparasitic agents
hydrophobic and hydrophilic residues in this
insertion, its only effect is likely to be the slight
extension of an antiparallel β-sheet far from the active
site. However, it should be stressed that motif 1 is only
weakly conserved in this species.
Thus, it is clear that, in contrast to what was believed
until very recently, PDF orthologues are not restricted
to Eubacteria, but instead occur in most of the
branches of the phylogenetic tree of living organisms
(i.e., also in the Archaea, lower and higher
eukaryotes). However, some organisms, such as
nematodes, fungi and most Archaea, clearly lack PDF
orthologues. Of course, it may be possible that these
organisms have a PDF with homology below the
BLAST threshold, but in our opinion this is unlikely.
Indeed, searches in various databases were achieved
unsuccessfully by various and different means. Since
new complete genome sequences will become
available in the next few years, it is therefore of value
to identify those organisms that do not contain a PDF.
3. The function of PDF orthologues in
Eukaryotes.
The detection of so many PDF orthologues in
organisms other than eubacteria was unexpected.
However, the detection of these orthologues does not
necessarily imply that their role is the same and that
their activity in eukaryotic cells is the cleavage of
N-formyl groups from nascent polypeptides.
3.1 Do
all PDF orthologues display deformylase
activity?
3.1.1 Plant
PDF orthologues
Two plant PDFs have recently been shown to complement a def Ts bacterial strain and deformylation
activity was measured in vitro [7]. Analysis of the
N-terminal sequences of many chloroplast-encoded
proteins indeed reveals systematic deformylation and
the subsequent removal of the first methionine in
some cases, depending on the nature of the second
residue (see data quoted in [7]). Although less
complete, a similar analysis in plant mitochondria led
to the same conclusion [31-35]. These two sets of data
indicate that deformylation of nascent polypeptides is
performed efficiently in plant organelles.
© Ashley Publications Ltd. All rights reserved.
3.1.2
Animal PDF orthologues
Our group has recently obtained convincing evidence
that mRNAs encoding proteins homologous to
mitochondrial PDF are expressed by the nuclear
genomes of insects, fish and humans [6,7,29]. Animal
PDFs are derived from the same common ancestor
and from the same original function as the
homologues identified in the mitochondria of higher
plants (mPDF; Figure 2). Thus, they are presumably
involved in the removal of the N-formylmethionine
from newly synthesised proteins in animal mitochondria, which would make it more difficult to use
inhibitors of these enzymes in human therapeutics.
H o w ever, an al ys i s o f th e s eq u en ces of
mitochondrially-encoded proteins in animals has
shown that virtually all retain their N-formyl group, as
if PDF was not present or active in this organelle (see
data in [6,7]). N-terminal sequence data are available
for 6 such proteins in cattle. None of these proteins
were found to undergo N-deformylation [36-41].
Additional unpublished data strongly suggest that all
mitochondrial proteins retain their N-formyl group in
bovine systems (see discussion in [7]). The human
PDF sequence was studied with Target P software
[42,104], for prediction of the subcellular location of
proteins. It was predicted that this protein would be
targeted to the secretory pathway, whereas all plant
mPDFs were predicted to be located in the mitochondria. In contrast, the mouse PDF amino acid sequence,
derived from its full-length cDNA (T. Meinnel,
unpublished results), was studied with the same
software and was predicted with a high probability to
be routed to the mitochondria. Clearly, animal PDFs
require experimental studies before any definitive
conclusions can be drawn about their actual subcellular location.
Even if animal PDFs were present in the mitochondria, their structure might account for their intrinsic
absence of deformylase activity. We have observed
that the conserved hydrophobic residue of motif 2
(generally a leucine; see 2.1 and Figure 1) is systematically replaced by a hydrophilic residue (i.e., a
glutamate) in vertebrate PDFs. Given the importance
of this residue in bacterial PDF [22,25], this change is
likely to have profound consequences for the
hydrophilic activity of PDF. Indeed, the side chain of
this leucine makes hydrophobic contact with that of
one of the conserved hydrophobic residues of motif 1.
This contact has two major effects: (i) the closing of
one end of the active site and (ii) on the location of the
Emerging Therapeutic Targets (2001) 5(1)
Giglione & Meinnel 47
Figure 2: A phylogenetic tree for PDF reveals three distinct classes.
Class 1 PDF
cpPDF
Arabidopsis thaliana
Plasmodium falciparum
cyanobacterial PDF
Tomato
Calothrix
Rice
Alfalfa
Barley
Synechocystis
Aquifex aeolicus
Prochlorococcus marinus
Heliobacter pylori
Rickettsia prowazekii
Borrelia burgdorferi
Vibrio cholerae1/2
Treponema pallidum
Escherichia coli
Deinococcus radiodurans
Haemophilus influenzae
Thermus thermophilus
Neisseria gonorrhoeae
Myobacterium tuberculosis
Pseudomonas aeruginosa1/2
Pseudomonas aeruginosa2/2
Clostridium acetobutylicum
Thermotoga maritima
Chlamydia trachomatis
Bacillus subtilis1/2
Legionella pneumophila
Dictyostelium discoideum
Mouse
Mycoplasma pneunomiae
Rat
Animal
PDF
Human
Staphylococcus aureus
Bacillus stearothermophilus
Fish
Fruit-fly2/2
Bacillus subtilis2/2
Fruit-fly1/2
Streptococcus pyogenes1/2
Enterococcus faecalis
Mosquito
Streptomyces coelicolor1/4
mPDF
Class 2 PDF
Corn
Wheat
Tomato
Arabidopsis thaliana
Alfalfa
Streptococcus pneumoniae2/2
Clostridium beijerinckii
Mycoplasma thermoautotrophicum
Trypanosoma
cruzi2/2
Methanothermus fervidus
Leishmania major1/2
Trypanosoma brucei
Trypanosoma cruzi1/2
Leishmania major2/2
archaeal PDF
Class 3 PDF
From the > 120 sequences of PDF orthologues available in databases, 56 PDF sequences were selected as representative of the sequence diversity of this protein. The sequences were aligned with ClustalX software and a phylogenetic tree constructed with TreeView1.6. A number (1, 2 or 4) indicates that the sequence is one of the two (or four) PDF species of this organism. cpPDF = plastid
PDF, mPDF = mitochondrial PDF.
backbone NH which makes a contact with the oxygen
of the formyl moiety of the substrate. We have
constructed a model of the active site of human PDF
(Figure 3). Although it is only a working model, it
suggests that the result of replacing the leucine by a
glutamate alone is considerable movement (5 Å) of
this side chain. This leads to the active site becoming
larger and open to the outside, unlike that of E. coli
PDF. Thus, vertebrates PDFs may well have acquired
a new substrate specificity that is currently unknown.
Further study of this issue is required before the use of
anti-PDF drugs is extended.
As no in vitro analysis has yet been reported, the
presence of PDF orthologues in vertebrates remains a
mystery. One possible reason for the presence of PDF
© Ashley Publications Ltd. All rights reserved.
homologues in animals may be that these proteins are
remnants of ancient PDFs that no longer function in
deformylation in the mitochondria. It is not known
whether these homologues have substrates outside of
the mitochondria or whether the product of the PDF
open reading frame has acquired another function or
location in animal cells.
3.1.3
cpPDF from Apicomplexa and green algae
No protein sequences from the apicoplast of Plasmodium falciparum are currently available, but the
strong resemblance of this organelle to the chloroplast
of the green alga Chlamydomonas reinhardtii makes
it possible to make certain predictions. In this green
alga, protein sequences for plastid-encoded proteins
Emerging Therapeutic Targets (2001) 5(1)
48 Peptide deformylase as an emerging target for antiparasitic agents
Figure 3: A 3D model of human PDF suggests an opening of its active site
Glu
Motif 2
5Å
3Å
Leu
Motif 3
Motif 1
The amino acid sequence of the human PDF sequence was superimposed over that of E. coli PDF and its 3D structure reconstructed by
homology modelling with the known crystal structure [96], using SwissPdbViewer [104]. Minimisation was carried out with the Insight
II package (MSI). The catalytic metal cation is shown as a light blue sphere.
are available and provide clear evidence of deformylase activity (see data quoted in [29]). This would be
consistent with the existence of PDF activity in the
apicoplast of Apicomplexa. However, only three
proteins of the apicoplast of P. falciparum, namely
ribosomal proteins S3 and L14 and ORF105, would be
predicted to undergo removal of the first methionine.
The removal of the first methionine would be
catalysed by the apicoplast MAP recently identified on
chromosome 5 of P. falciparum, which resembles its
plant counterpart [7]. More generally, it should be
borne in mind that Apicomplexa display significant
molecular similarity to plants, indicative of a common
evolutionary origin [43,44].
3.1.4
Class III PDFs
Class III PDFs remain difficult to analyse. First, no
sequence data are available for proteins synthesised
in the mitochondria of kinetoplastids. Second, we
have cloned and overexpressed the PDF from
Trypanosoma brucei in E. coli (C. Lazennec and T.
Meinnel, unpublished results). The full-length protein
sequence and three N-terminally truncated variants,
unlike plant PDFs, were unable to complement a defTs
bacterial strain. Nevertheless, using the same strategy
© Ashley Publications Ltd. All rights reserved.
with plant PDFs, we found that it was not easy to
achieve complementation with the full-length
mitochondrial form [7]. We therefore cannot exclude
that we did not express the appropriate, most soluble
form of T. brucei PDF in the bacterium. Third, we
found that the purified protein from T. brucei had no
significant deformylase activity in vitro, with a kcat/KM
greater than 1 M-1 s-1 (C. Lazennec and T. Meinnel,
unpublished results). Given the difficulties in
stabilising the metal cation in the enzyme, no definitive conclusion can be drawn from experiments with
negative results such as this, as previously indicated
(see section 1). Finally, we have found that the PDF
orthologue of T. brucei has a lysine instead of the
crucial glutamine in motif 1 (Figure 1). A
post-translational modification that does not occur in
E. coli has already been reported for urease involving
carbamylation of the side chain of the lysine of the
active site [45]. We therefore wondered whether such
a modification could occur also in kinetoplastids.
Unfortunately, replacement by site-directed
mutagenesis of the lysine by a glutamine, which
mimics a carbamyl-lysine did not improve PDF
activity in vitro or in vivo.
Emerging Therapeutic Targets (2001) 5(1)
Giglione & Meinnel 49
No information is yet available concerning the second
type of PDF from kinetoplastids. The sequence of this
PDF is more closely related to other PDFs than to that
from T. brucei. In particular, it contains the important
glutamine of motif 1. It is not yet clear whether this
PDF has deformylase activity, although we believe
that this may well be the case.
Last but not least, the presence of PDF orthologues in
Archaea remains a true mystery. In this organism, it
has been known for twenty years that there is no
N-formylation of nascent polypeptides [8]. We believe
that, as in the case of human PDF (see 3.1.1), this
protein may have a slightly different proteolytic
activity.
PDF activity is essential in the organelles of
some eukaryotes but has probably disappeared
from others, including humans
3.2
Given current knowledge and the data available on
plant mPDFs, animal PDFs appear to be inactive in
mitochondria. This raises the questions as to why the
protein deformylation is not required in animal cells
but does occur in other organisms, such as plants and
many eukaryotic protists.
Correlation between gene number and
deformylase activity in organelles
3.2.1
To date, although many PDF genes have been
sequenced, none has been identified in an organelle
genome. The same is true for other crucial enzymes
such as MAP and aminoacyl-tRNA synthetases (aaRS)
(for a review on aaRS see [46]). The mitochondrial
genome of the protozoon Reclinomonas americana
contains the largest (97) collection of genes identified
to date in an organelle [47]. It has been suggested that
this genomes resembles the ancestral genome of
mitochondria, but it contains no aaRS, PDF or MAP
gene. Interestingly, the number of genes in R.
americana is similar to that of the smallest bacterial
genome described so far, Mycoplasma genitalium. It
contains 470 ORFs, of which only 250-350 are considered to be strictly required for cell survival [48]. Like
aaRS and MAP, its PDF genes are absolutely required
for survival. Thus, like aaRS and MAP, PDF genes
were among the first genes corresponding to essential
functions to be transferred from the organelle to the
nucleus.
This raises questions concerning (i) the actual role of
N-terminal protein processing and (ii) the reasons
why it has been retained in the organelles of some
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organisms (i.e., all plastids and the mitochondria of
higher plants and of some protists) but has
disappeared from the mitochondria of animals and
fungi (humans, C. elegans and S. cerevisiae for
instance). It should be borne in mind that the
genomes of plants and the genomes of protist’s
organelles encode 30-100 proteins versus only nine in
yeast and 13 in animal mitochondria. Moreover, in the
mitochondria of all animal including C. elegans, which
contains no PDF, a common set of 13 proteins is
synthesised: 7 subunits of NADH:ubiquinone
oxidoreductase, the three subunits of cytochrome
oxidase, cytochrome b and subunits 8 and 9 of ATP
synthase. It seems most likely that the genes encoding
several proteins strictly requiring deformylation to
achieve their final function were retained in the
organelles genomes of some organisms, but not in
those of others such as yeast. The loss of proteins
requiring deformylation would thus have led to the
loss of the deformylase function. Clearly, none of the
13 proteins encoded by animal mitochondria require
deformylation for full activity.
Determination of the smallest set of
physiological substrates requiring the
deformylation function
3.2.2
The set of additional proteins encoded by plant
organelles includes ribosomal proteins, subunits of
RNA polymerase, translation factors and the proteins
involved in the specific functions of the organelle,
such as the large subunit of Rubisco in plastids [49-52].
Rubisco is one of the most abundant proteins on earth
and is the motor of photosynthetic function. This
protein is known to undergo further processing in the
plastids. Once PDF and MAP have removed the
N-formylmethionine of the large subunit of Rubisco,
the serine in position 2 is removed and this is followed
by N-acetylation [53] or even further N-methylation
[54]. Given the crucial role of Rubisco, we cannot
exclude the possibility that PDF and MAP have been
retained to ensure the correct processing at least of
this protein. However, the impact of these modifications on Rubisco activity and stability is unknown and
the 3D structure of this molecule provides no further
insight into this issue [55]. No Rubisco is present in the
apicoplast of P. falciparum, in which deformylation is
clearly required [56]. The reason for the necessity of
N-formyl removal probably lies in the function of one
or several of the ribosomal proteins (i.e., translation
machinery) encoded by all organelle genomes
(mitochondrial and plastid).
Emerging Therapeutic Targets (2001) 5(1)
50 Peptide deformylase as an emerging target for antiparasitic agents
In conclusion, we believe that the requirement for
PDF has disappeared only in organisms in which the
number of genes in the organelle genome has been
reduced to a very small number. This provides strong
evidence that the pathway for the processing of the
first methionine plays a crucial, general role (for a
further discussion see [5]). Thus, it seems probable
that deformylation is essential for the function of the
organelle, in those organelles in which it occurs.
Blocking PDF function should lead to the death of the
corresponding organism. The essentiality of PDF will
deserve however to be experimentally addressed in
each biological system where it is present.
4. Many parasitic illnesses and various
PDFs to inhibit
Six major tropical diseases, malaria, filariasis, schistosomiasis, African trypanosomiasis, Chagas’ disease
and leishmaniasis, together account for the deaths of
more than one million people each year and cause
enormous suffering in hundreds of millions more
[111-113]. Resistance to the drugs currently used to
cure most of these diseases is clearly on the increase
and new medicines are required in the short-term.
PDF function is required in eubacteria but absent in
humans. Given that most of the organisms responsible
for these major diseases have PDFs, this raises
possibilities for the use of inhibitors of PDF as potent
medicines.
4.1
Apicomplexa
The phylum Apicomplexa includes a large family of
unicellular parasites that cause various diseases. This
family of protists includes the causative agent of
malaria (Plasmodium spp.), opportunistic pathogens
associated with immunodeficiency (e.g., the
AIDS-associated pathogen Toxoplasma gondii,
Cryptosporidium and Sarcocystis) and pathogens of
poultry, livestock and shellfish (e.g., Eimeria,
Theileria, Babesia). The members of the Apicomplexa
have recently been shown to contain a small plastid
called the apicoplast [57,58]. The 35 kb sequence of
the apicoplast genomes of two Apicomplexa species
(i.e., T. gondii and P. falciparum) have been
determined [59,102]. These data reveal a high level of
conservation of the 25 proteins encoded by these
genomes including 17 ribosomal proteins, elongation
factor Tu (EFTu), one subunit of the clp protease and
the three subunits of RNA polymerase. The apicoplast
gen o m e
strongly
res embles
that
of
© Ashley Publications Ltd. All rights reserved.
non-photosynthetic plants, with no genes encoding
the proteins of the photosynthetic machinery.
The discovery of a plastid in Apicomplexa has rapidly
led to suggestions that this organelle would be a good
target for antiparasitic drugs. Indeed, apicoplast
function has been shown to be required for parasite
survival [60,61]. The apicoplast has been shown to be
the pharmacological target of many antibiotics known
to be specific for eubacteria and organelles. These
drugs (e.g., chloramphenicol, clindamycin,
thiostrepton, rifamycin, azithromycin, fluoroquinolones) specifically block apicoplast protein
synthesis, transcription or DNA replication [62,63].
The blocking of the activities of the plastids with such
drugs results in a characteristic pattern of delayed
death that contrasts with the more immediate effect
seen with drugs such as chloroquine. However,
inhibition of the apicoplast EFTu by various drugs,
such as kirromycin or enacyloxin IIa, results in the
rapid onset of inhibition [64]. Interestingly, plastid
function can be blocked by these drugs and the
parasite killed at various stages of its sexual development [65]. Although the apicoplast is clearly the site of
many crucial metabolic processes, such as branched
and aromatic amino acid biosynthesis, it is unclear
why the expression of its genome is important for cell
survival. No apicoplast gene product appears to play a
specific role in processes other than genome expression. However, the functions of several open reading
frames of the apicoplast genome are unknown. The
key to understanding the essential function of the
apicoplast probably lies in the identification of the
functions of these genes.
To date, P. falciparum PDF is the only species of this
group for which a PDF has been described [29]. Given
the high level of conservation of apicoplast
sequences, there is probably a PDF sequence in all
Apicomplexa. No direct proof has yet been provided
that the P. falciparum PDF is located in the apicoplast.
Nevertheless, the amino acid sequence of this PDF
resembles that of cpPDF and it is rooted on the same
branch of the PDF phylogenic tree as plant cpPDF
(Figure 2). The recent demonstration that cpPDFs are
systematically targeted to the plastids of plants
strongly suggests that P. falciparum PDF is targeted to
the apicoplast [7]. Moreover, like other nuclearencoded apicoplast proteins, this PDF has a bipartite
N-terminal pre-sequence consisting of a signal
peptide for entry into the secretory pathway and a
transit peptide similar to those found in plant proteins
Emerging Therapeutic Targets (2001) 5(1)
Giglione & Meinnel 51
targeted to the chloroplast, for subsequent import into
the apicoplast [66,67].
4.2
Trypanosomatids
possible that other closely related human parasites
like Entamoeba spp. (responsible for several
intestinal diseases) also contain a PDF that would be
sensitive to anti-PDF drugs.
Trypanosomatids cause many serious parasitic
diseases. The most famous is African trypanosomiasis
or sleeping sickness, which is transmitted by the
Tsetse fly (Glossina spp.). This disease has
re-emerged as a major health problem in sub-Saharan
Africa. Sleeping sickness actually covers two distinct
diseases with different clinical signs in humans
depending on the epidemic area. This reflects the two
very different causative trypanosomes that cause
them: Trypanosoma brucei gambiense (north west of
the African rift) and T. brucei rhodesiense (south east
of the African rift). Chagas’ disease is another major
disease caused by a trypanosome, Trypanosoma
cruzi, in South America. Leishmaniasis is caused by
Leishmania major and transmitted by phlebotomine
sand-fly vectors.
No PDF sequence has been detected in the genome of
the nematode C. elegans. This suggests that the
nematodes responsible for filariasis such as Brugia
malayi are unlikely to be sensitive to anti-PDF drugs.
Similarly, although little is known about the number
of genes in the mitochondria of Shistosoma spp.
[70,71], the mitochondrial genome of another related
member of the Platyhelminthes, the turbellarian
Echinococcus multilocularis [102], contains a similar
number of genes to that of C. elegans. Therefore,
these parasites are also unlikely to be sensitive to
anti-PDF drugs.
The origin, location and function of the two PDFs in
trypanosomatids is unknown. Their N-terminal
pre-sequence resemble mitochondrial (e.g., L. major
and Trypanosoma sequences 1/2 in Figure 1) or
plastid import sequences (e.g., Leishmania 2/2 in
Figure 1). These flagellates are very closely related to
euglenids which, unlike trypanosomatids, display a
plastid. Some of these protists are photosynthetic
(Euglena gracilis) whereas others are not (Astasia
longa). The N-formylation of proteins has been
shown to occur and to be strictly required in the two
organelles of these organisms [68]. We therefore
cannot exclude the possibility that the second PDF is a
remnant of the occurrence of an ancient plastid,
which has now disappeared. Alternatively, given the
high level of similarity between the two PDFs, the
second PDF may result from an early duplication of
the original gene.
There is increasing evidences that PDF would be a
good target for new antibacterial agents and many
efficient inhibitors of PDF have been described (see
accompanying review in this issue and reference [6]).
The most efficient agents appear to be hydroxamate
[28,72] and thiol derivatives [73-75]. Structure-based
drug design could be the approach of choice to
improve further their specificity and affinity [76].
Hence, powerful automated NMR methods, aimed at
discovering and producing high-affinity ligands, have
been described [77] and are currently being used by
Hoffmann-Laroche to screen for inhibitors of PDF [78].
It is now clear that the availability of large amounts of
purified parasitic PDF may become a limiting step.
Little is yet known about the potency of parasite PDFs
as drug targets, but promising results have been
obtained for P. falciparum PDF.
5. Parasite PDFs as drug targets
5.1 Plasmodium
4.3
Other parasites
The amoeba D. discoideum possesses a PDF that is
very likely to be routed to its mitochondria [7]. The
mitochondrial genome of this species encodes a large
number of proteins (> 40). In this respect, it closely
resembles the genome of plant mitochondria, in
which PDF is known to be functional. The mitochondrial genome of Acanthamoeba castellanii [69]
encodes a similar number of genes and based on the
reasoning described above (see 3.1) is likely to
possess a PDF. This amoeba is responsible for several
opportunistic infections in humans. It is therefore
© Ashley Publications Ltd. All rights reserved.
PDF is naturally inhibited by
actinonin
Studies with P. falciparum PDF have not yet been
reported in vitro, but the resemblance of this protein
to tomato cpPDF led us to study this tomato PDF as a
model for PDFs from the Apicomplexa. We showed
that this protein was as sensitive as bacterial PDF to
the antibiotic actinonin [79]. The antibacterial properties of actinonin were discovered in 1962 [27] but the
specific intracellular molecular target of this molecule
in bacteria was only recently shown to be PDF [28].
Actinonin has also proved to be a potent inhibitor of
Plasmodium growth with a half-maximal inhibitory
Emerging Therapeutic Targets (2001) 5(1)
52 Peptide deformylase as an emerging target for antiparasitic agents
concentration (3 µM) lower than that of the most
sensitive bacteria [56,79]. However, actinonin has no
effect against malaria in vivo in a rodent model of
malaria. Although actinonin is known to inhibit a
variety of other enzymes (e.g., matricins; see our
conclusion in [6]), the effect of actinonin on Plasmodium growth strongly suggests that PDF is a good
target for new antimalarial agents, particularly for
many of the diseases caused by members of the
phylum Apicomplexa. As recently discussed however
[79], the essentiality of PDF in P. falciparum deserves
to be proved.
5.2 How
can we improve the in vivo potency of
anti-PDF drugs?
PDF is a peptidase that recognises three contiguous
peptide bonds, including the amide of the formyl
group [23,74]. It is therefore not surprising that the
most potent inhibitors belong to peptide derivative
series (see discussion and references in [6]) and that
non-peptide inhibitors of PDF, such as those
described by Pfizer and Merck, lack potency [80,81].
The rational design of peptide derivatives has led to
the discovery of several potent drugs (see the review
by Pei, same issue). However, peptides have limitations in terms of bioavailability and activity when
given by the oral route. Actinonin has low potency in
vivo in humans both as an antibacterial drug [82] and
as an antimalarial agent [56]. It has been suggested
that this is due to metabolic inactivation. Peptide
mimicry (for a recent review read [83]) is therefore one
way in which the bioavailability of actinonin and of
other thiol derivatives could be improved.
Fortunately, the active site of PDF has been reported
to be very similar to that of matricins (see 2.1), a class
of proteases of broad therapeutic interest. As a result,
most of the strategies to improve the in vivo stability of
anti-PDF drugs take advantage of the lessons learned
with anti-matricin drugs.
It was found early on that the presence of a sterically
demanding group at the P2’ position (such as
C(CH3)3) increases the activity of drugs given orally
by preventing hydrolysis at adjacent hydrolysable
centres. This led to the synthesis of the earlier
‘peptoid’ matricin inhibitor BB-2516 by British
Biotech, this drug also being known as marimastat
[84]. The synthesis of one inhibitor of this type,
BB-3497, with an n-butyl instead of the branched
butyl at P1’ in BB-2516 to confer specificity for PDF,
was recently undertaken [85]. Another company
(Versicor) used a similar approach and an hydroxamic
© Ashley Publications Ltd. All rights reserved.
inhibitor VRC3324 was synthesised [86]. In both cases,
the inhibitors effectively cured septicaemia in mice
when administered via the various possible injection
routes.
Another way to bypass metabolic hydrolysis of the
compound is to synthesise β-sulfonyl- and β-sulfinylhydroxamic acids, as described for CGS27023A, a
broad-spectrum matricin inhibitor [87]. In the case of
PDF, such an approach was successfully developed
by Hoffmann-Laroche after they re-discovered
actinonin by screening for inhibitors of E. coli PDF
[72]. Although drug resistance is frequently observed
with such compounds in bacteria, this need not
necessarily to be the case in parasites, against which
these drugs may be effective.
6. Expert opinion: new anti-parasitic drugs
are needed and PDF is a target of choice
Parasitic diseases such as malaria are clearly
increasing in importance world-wide due to the
acquisition of resistance. Hence, P. falciparum
malaria caused more than 1 million deaths worldwide among 273 million cases in 1998 [112-113].
Unfortunately, malaria and most of the parasitic
diseases caused by PDF-containing protists are not
economically attractive targets for the pharmaceutical
industry [88]. However, a recent analysis by Jeffrey
Sachs, a Harvard health economist, of the economic
and social costs of malaria concluded that controlling
this disease is highly cost-effective [89]. This led the
World Health Organisation (WHO) to instigate the
WHO Roll Back Malaria campaign, which seeks to
halve the incidence of malaria by 2010. There are also
frequent predictions and warnings that global climate
changes, induced by greenhouse gases, will result in
the world being warmer in the future. If this process of
global warming occurs rapidly during the 21st century
[90], then the vectors of major parasitic diseases,
Anopheles mosquito, Tsetse fly or sand fly, may reach
the central or northern regions of Europe and large
parts of America. Although some climate change
predictions foresee little change in the distribution of
malaria for instance [91], the opposite is also predicted
in other studies [92-95]. Such difficulties in predicting
their own near future should provide northern
countries with a major incentive to commit
themselves to the search for new drugs to fight
parasitic illnesses.
Emerging Therapeutic Targets (2001) 5(1)
Giglione & Meinnel 53
PDF is clearly one of several promising targets for the
treatment of parasitic diseases. Many tools are already
available to study the effects of PDF inhibition in vivo
and in vitro. Actinonin and other thiopeptide derivatives are already available and have been shown to
inhibit Plasmodium growth in a rodent model. PDF is
already considered a promising target for new
antibacterial agents [6] and many major pharmaceutical companies (e.g., Aventis, British-Biotech, Glaxo,
Hoffmann-Laroche, Merck, Novartis, Pfizer,
Sm ith Klin e-Beecham)
have
deve l o p ed
high-throughput screening protocols to search for
possible anti-PDF drugs. We believe that discovering
drugs that, like actinonin, could have both antibacterial and antiparasitic effects should be very easy. This
should result in much lower initial investment costs
for pharmaceutical companies. A study of the
biochemical properties of the various PDFs as well as
the differences between them should rapidly help the
search for very specific, efficient drugs.
This should encourage and stimulate the search by
private companies for new drugs aimed at blocking
the PDF activity of parasites. This will, as for all new
medicines today, require the means and resources of a
large pharmaceutical company.
4.
MARCKER K, SANGER F: N-formyl-methionyl-S-RNA. J.
Mol. Biol. (1964) 8:835-840.
5.
MEINNEL T, MECHULAM Y, BLANQUET S: Methionine as
translation start signal: a review of the enzymes of the
pathway in Escherichia coli. Biochimie (1993)
75:1061-1075.
6.
GIGLIONE C, PIERRE M, MEINNEL T: Peptide deformylase as a target for new generation, broad spectrum
a n ti m i cr o b i a l a g e n ts . M o l . M i c r o b i o l . (2 0 0 0 )
36:1197-1205.
A recent review of PDF and of its use as a target for antimicrobial drugs.
••
7.
••
8.
KOZAK M: Comparison of initiation of protein
synthesis in procaryotes, eucaryotes and organelles.
Microbiol. Reviews (1983) 47:1-45.
9.
MAZEL D, POCHET S, MARLIERE P: Genetic characterization of polypeptide deformylase, a distinctive enzyme
of eubacterial translation. EMBO J. (1994) 13:914-923.
Reports the cloning of E. coli PDF using an elegant genetic
screen.
•
10.
•
Acknowledgements
The authors would like to thank all of their collaborators in Gif/Yvette who have collectively contributed to
this work. This work was supported by ATIPE, PCV
and MCT grants from the C.N.R.S. to TM and by the
Fondation pour la Recherche Médicale. CG holds a
post-doctoral fellowship from the Association pour la
Recherche sur le Cancer (ARC, Villejuif, France).
MEINNEL T, BLANQUET S: Enzymatic properties of
Escherichia coli peptide deformylase. J. Bacteriol.
(1995) 177:1883-1887.
12.
MEINNEL T, LAZENNEC C, BLANQUET S: Mapping of the
active site zinc ligands of peptide deformylase. J. Mol.
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13.
GROCHE D, BECKER A, SCHLICHTING I et al.: Isolation
and crystallization of functionally competent Escherichia coli peptide deformylase forms containing either
iron or nickel in the active site. Biochem. Biophys. Res.
Commun. (1998) 246:342-346.
An excellent work on the various means to stabilise PDF
activity.
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Malaria foundation international
Carmela Giglione & Thierry Meinnel†
Institut des Sciences Végétales, UPR2355, Centre National de la
Recherche Scientifique, Bâtiment 23, 1 avenue de la Terrasse,
F-91198 Gif-sur-Yvette cedex, France.
Tel.: 33+1 69 82 36 12; Fax: 33+1 69 82 36 07;
E-mail: [email protected]
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