Peptide deformylase as an emerging target for antiparasitic agents

18 Pages·2001·0.33 MB·English

Giglione&Meinnel Anti-infectives
Peptidedeformylaseasanemergingtargetforantiparasiticagents
Peptide deformylase as an emerging target
for antiparasitic agents
CarmelaGiglione&ThierryMeinnel
http://www.ashley-pub.com InstitutdesSciencesVégétales,UPR2355,CentreNationaldela
RechercheScientifique,Bâtiment23,1avenuedelaTerrasse,F-91198
Gif-sur-Yvettecedex,France.
Review
1. Introduction Peptide deformylases (PDFs) constitute a growing family of hydrolytic
enzymespreviouslybelievedtobeuniquetoEubacteria.Recentdatafrom
2. The PDF family: a new,
our laboratory have demonstrated that PDF orthologues are present in
growing sub-family of the
many eukaryotes, including several parasites. In this report we aim to
HEXXH-containing
explainwhyPDFcouldbeconsideredtobeapotenttargetforhumanand
metalloprotease
veterinary antiparasitic treatments.
super-family
Keywords:antibiotic,antiparasitic,Chagas’disease,deformylase,malaria,
3. The function of PDF
plasmodium,sequencehomology,sleepingsickness,target,trypanosomatids
orthologues in eukaryotes
4. Many parasitic illnesses EmergingTherapeuticTargets(2001)5(1):41-57
and various PDFs to
inhibit
1.Introduction-Detectingandstabilisingdeformylase
5. Parasite PDFs as drug
activity invitro
targets
6. Expert opinion: new Polypeptide deformylase (PDF) was first detected in crude bacterial
anti-parasitic drugs are extracts more than 3 decades ago [1,2]. This finding was the logical
needed and PDF is a extension of previous studies, which had shown that, although protein
target of choice 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
Acknowledgements
proteins [3,4]. The deformylation step is part of the methionine cycle [5].
Bibliography Deformylation plays a crucial role in this process as it is necessary for
subsequent removal of the unblocked methionine by methionine
Websites
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]).ThepresenceofanN-formylgrouponthemethionineresidueatthe
start of nascent polypeptides in bacteria seemed to contrast with the
situationineukaryotesandArchae,inwhichnascentproteinssynthesised
inthecytoplasmstartwithafreemethionine.However,itwasknownthat
proteins synthesised in eukaryote organelles also start with an N-formyl
methionine [8]. Most of the sequences of mitochondrial proteins obtained
fromfungiandmammalsindicatethattheN-formylgroupisretained.Itwas
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].PDFwasthenoverproducedandtheresultingproteincharacterised
in1995[11].Itwasfoundthatametalcationwasrequiredforactivityand
the nature of the three metal ligands was determined [12]. It is now
41
2001©AshleyPublicationsLtd.ISSN1460-0412
42 Peptidedeformylaseasanemergingtargetforantiparasiticagents
generally accepted that bacterial PDF are linked to a have been determined [110]. Protein sequence
very unstable metal cation (Fe2+) and that the alignments have identified only 3 sets of
atmospheric oxidation of this cation rapidly and well-conservedresidues,motif1{gφgφaapQ},motif2
irreversibly inactivates the enzyme [13-15]. This {EgCφs} and motif 3 {HEφDHlxg} (where φ is any
accounts perfectly for the instability of PDF activity. hydrophobicaliphaticaminoacid,withL>I>M>V;
The ferrous enzyme remains stable for less than one see Figure 1 and [6,22]). Based on the 3D-structure
minute invitro. (seereferencesquotedin[6])andtakingintoaccount
site-directedmutagenesisdata,itisnowclearthatthe
The conditions required for the stabilisation of
three motifs build:
deformylase activity were not determined until 1998.
They involve the use of either:
(cid:127) the three sides of the active site, and
• reactive oxygen species scavengers such as TCEP
(Tris(2-carboxy-ethyl)-phosphine)andcatalase,or (cid:127) part of the hydrophobic pocket in which the
methionine side chain of the substrate is buried.
(cid:127) the early replacement of the iron cation by metal
More precisely, the two residues of motifs 1 and 3
cationsinsensitivetooxygensuchasnickel[13,16]
shown in bold (see above) directly contribute to
or cobalt [17,18], both of which preserve enzyme
catalysis, whereas the three underlined residues are
activity,althoughcobaltmuchlesssothannickel.
involved in binding to the metal cation. The acidic
Several in vitro assays of PDF activity have recently side chains of the two residues shown in italics
been developed and have proved very useful for hydrogen bond with the guanidium of a conserved,
optimisingconditions[19-21].Itshouldbenotedthat buriedargininelocatedbetweenmotifs2and3.This
determining the conditions required for stabilisation arginine is generally located in the vicinity of a
of the metal cation and therefore of the activity of a conservedvaline(seeVXRinFigure1).Conservation
newPDFspeciesremainsacrucialanddifficultstep. of the residues of the motif shown in lower case
Since many new PDFs (see section 2) have recently appears, a priori, not to be strictly required although
been discovered, before any in vitro data are
the chemical nature of these residues (small,
obtained, it cannot be excluded a priori that metals
hydrophobic and/or hydrophilic) is known to play a
other than iron and nickel may give them full
roleingeneratingthecorrect3Dstructureoftheactive
deformylase activity.
site. Thus, a PDF may have activity, even in the
absenceofseveraloftheconservedresiduesofthe3
motifs,especiallythoseofmotif1[23].Analysisofthe
2.ThePDFfamily:anew,growing
hydrophilic/hydrophobicnatureofthesidechainsof
sub-familyoftheHEXXH-containing
the residues of the various secondary structure
metalloproteasesuper-family elements identified in the 3D-structure of the E. coli
enzyme strongly suggests that all PDFs have a
This section of the review (and part of the next) will near-identical 3D folding pattern [22].
takeadvantageofmanyinsilicodata[101-109].Such
dataarenotyetregardedas‘fact’inthesamewaythat
Demonstration of the importance of the HEXXH
invitro/vivodataare,butthismaychangeasourdata
sequenceofmotif3anditsactualroleincatalysisled
sets enlarge and more supporting evidence accumu-
to the early assignment of PDF to the HEXXH-
lates. For instance, these in silico data [6] led most
containing metalloprotease super-family [24,25].
recentlytoPei’sworkwiththePlasmodiumPDF(see
Indeed, the active site of bacterial PDF has a
review by Pei, same issue) and our own most recent
secondary superstructure common to thermolysins
work [7].
and matricins, the other two sub-families of the
super-family [26]. Extensive structural similarity
2.1Threeconservedmotifsandaconserved
between PDF and matricins was also observed [23].
argininebuildtheactivesiteofeubacterial
However, the nature and location of the third metal
PDFs
ligand, the cysteine of motif 2, differs from those of
PDFwasinitiallybelievedtooccurineubacteriaonly. thermolysins and matricins. For this reason, PDFs
To date, more than 90 eubacterial PDF sequences were classified as a third, novel, sub-family.
©AshleyPublicationsLtd.Allrightsreserved. EmergingTherapeuticTargets(2001)5(1)
Giglione&Meinnel 43
Thesestructuraldataareimportantindefinitionofthe eubacterial PDFs in possessing N-terminal
criteria for membership of the PDF family and for pre-sequencesthattargetthecorrespondingcatalytic
deformylase activity. domainstovariouscompartmentsofthecell.Hence,
plantmitochondrialPDFs(mPDFs)aretargetedtothe
2.2AphylogenetictreeforPDFs mitochondria only, whereas chloroplast cpPDFs are
theonlyPDFsfoundintheplastids(seeFigure2and
2.2.1TwoclassesofPDFinEubacteria [7]). The cpPDF are closely related to the PDFs of
cyanobacteria (Figure 2), which are believed to be
Fromthesequencedataobtainedinbacteriaandfrom
theancestorsofthisphotosyntheticorganelle.mPDF
an analysis of the sets of deletions and insertions
orthologueswerealsobeidentifiedininsectsandthe
(calledI1andI2inFigure1)locatedbetweenidenti-
tissues of various vertebrates and the corresponding
fied secondary structures elements, it was concluded
full-length cDNA has been cloned in humans [7]. All
that PDFs can be divided into at least two major
animalPDForthologueshavesequencesverysimilar
families[6].ClassIistypifiedbytheE.colienzymeand
to those of mPDFs and are clearly derived from the
the PDF of all Gram-negative bacteria fall systemati-
samebranchofthePDFphylogenetictree(Figure2).
callyintothisclass(Figure2).ClassIIPDFistypified
The bacterial sequence most similar to those of the
byBacillusstearothermophilusPDFandcontainsPDF
variousmPDFsisthedivergentPDFfromS.coelicolor
from Mycoplasma and Gram-positive bacteria with
(Figure2).
low G+C content (Figure 2). However, if two PDF
occur in such Gram-positive bacteria, the second is
2.2.3ThePDFoflowerEukaryotes
oftenaClassIenzyme.PDFhomologuessignificantly
dissimilar to both these classes were found, also in PDFsequenceshavealsobeenfoundinthegenomes
Gram-positivebacteriawithlowG+Ccontent,suchas of various eukaryotic protists. A cpPDF (i.e., a PDF
Clostridium beijerinckii. Finally, the complete resemblingtheplastidPDFsofplants)wasidentified
genome sequence of the actinomycete Streptomyces in the malaria agent Plasmodium falciparum and is
coelicolor revealed the presence of four def genes. thoughttobetargetedtotheapicoplast[7,29].APDF
ThiscorrespondstothelargestnumberofPDFgenes was also identified in the amoeba Dictyostelium
detected in any eubacterium to date. Interestingly, discoideum[7].ThisPDF,whichisprobablytargeted
oneofthefourPDFofthisactinomycete(S.coelicolor tothemitochondria,morecloselyresemblesaClassII
1/4inFigure2)divergesfromallotherknownPDFs. PDF (Figure 2). Finally, two different PDF
It has been reported that actinomycetes naturally orthologues were found in the Kinetoplastids,
produce a molecule with anti-PDF activity, actinonin Trypanosomaspp.andLeishmaniamajor(Figure2;
[27,28]. However, actinomycetes are themselves [29]). These PDF species are clearly different from
resistant to actinonin, possibly due to the acquisition Class I and Class II PDFs and we now propose that
of an actinonin-resistant PDF. This could explain the they should be classified as a new class, Class III.
presence of this divergent PDF in actinomycetes.
Alternatively, a function other than the classic 2.2.4ThePDForthologuesofArchae
deformylation of nascent polypeptides may account
PSI-BLAST [30] is now recognised to be a powerful
forthisunusualPDFandtheredundancyofdefgenes
tool for identifying biologically relevant sequence
in S.coelicolor.
similarities. Using this program with several PDF
sequences,werecentlyidentifiedforthefirsttimetwo
2.2.2ThePDFsfromhigherEukaryotes
new sequences from Archaea displaying strong
Analysis of the recently produced sequences of the similarity to PDF (Figure 1 and Figure 2). These
complete genomes of several higher eukaryotes archaeal PDFs were found in the euryarchaeota
surprisingly revealed the presence of PDF Methanothermobacter thermoautotrophicus
orthologues [6]. For instance, two Class I PDFs were (Genbank accession number AE000809) and
identifiedinthenucleargenomeofbothmonocotyle- Methanothermus fervidus (Genbank accession
donous and dicotyledonous flowering plants [7]. A number CAA70987). These two sequences are most
PDFsequencehasalsobeenidentifiedinthegenome closelyrelatedtothesequencesfoundinkinetoplas-
of the liverwort Marchantia polymorpha. This tids(i.e.,ClassIIIPDFs).Aspecificfeatureofarchaeal
indicates that PDFs are found in all higher plants PDFs is the occurrence of a distal insertion between
(Embryophyta). These enzymes differ from motifs 2 and 3 (Figure 1). Given the alternation of
©AshleyPublicationsLtd.Allrightsreserved. EmergingTherapeuticTargets(2001)5(1)
44 Peptidedeformylaseasanemergingtargetforantiparasiticagents
S ? C L A A I I L M M M V V I K R I I L A E E E E I
Y ? G P S S R R Q R Q Q A T A E D D D T G D D D D Y
N ? H A R R Q K K K V V A A A K L Y L T I L Y W L D
K ? S G R K H S N S N N D D D I A Y F G A A G G G G
I ? S G P P I I V V W W W W W A R S T T R R S S S D
Q ? Y A — A D N G N G G H H H M I I I V I M C C C V
D ? L D — W V V I I I T A G G F K S I T R R T T T V
N ? R V G A Q Q Q Q Q Q K K K R Q V V C I V V I V I V
K ? H G L A 1 T P P P P P P P P S p K I R R T T R S S R
EPYI ???? AVKQ LLRG TVPL CT—— otif GLAA GLSA GIAA GLAA ?LCA SMSA SFSA SFSA SFSA RKNS gφaa PRAE ERPS QRSK LRYA ERPA ERPL VRPQ IRPQ IRPQ SRPY XR
L ? A S R R M I I T I ? P L I I L φ V V V V I I V V V V V
S ? A T S F G G G G ? Y C C C S g L K H Y Y Y W W W L
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V ? G G R H — — — R — — — — — R E F P M L T A T R
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G ? V R - - - A Q - - - - I F V V C S C C C R
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I S T I K V S N K N S E K T Y T T M M M T
L S E D E Y I L L L F - — — — K L - - -
N A F F L D L D S N - — — — — G G — — — —
F G M M L L G M L L L — — — — E E — — — —
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S R D R E S Y S Q Q P — — — — R R — — — —
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YYS PSF QRI KRV VRR RIL AQG VFM LFQ LFH DAF tio ——— ——— ——— ——— VAS REL --- ——— ——— ———
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M GL NA DD KL EE AR LS NT NT QI ns —— —— —— —— EA EE -- -- -- -—
S V F E S D I V V L i — — - - A E - - - -
LPPH I1-——E -——N -——K LPP- QYAL PTSQ DRTQ ERHQ VIEK ntral ---— ---- ---- ---- PIAE WVAF ———— ---- ---- ----
A E T S P R V L L R e - - - - - S — - - -
V V V W V C P R R K C - - - - M M - - - -
T P E P P W M L L E - - - - G G - — - -
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R A S A A S V A A I - - - - R R - - — -
P V R K V R P P P T - - - - S A P P P -
S K R E K A R R R F - - - - G A A A A -
A R R R R P T T T R r - - - - A E V I I -
G L L L L L M L L L L - - N C A A A S S -
C R I V T Q S S S L - - N Q E R N T H —
S E P R P I R R R K - V D Q V W R R R —
N D D N H I H H H K G L Q V E V D A D G
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Figure.1:Alig P.falciparum D.discoideum L.major2/2 L.major1/2 T.cruzi1/2 T.brucei E.coli P.falciparum D.discoideum B.stearothermo T.cruzi2/2 L.major2/2 L.major1/2 T.cruzi1/2 T.brucei Methanothermo E.coli P.falciparum D.discoideum B.stearothermo T.cruzi2/2 L.major2/2 L.major1/2 T.cruzi1/2 T.brucei Methanothermo
©AshleyPublicationsLtd.Allrightsreserved. EmergingTherapeuticTargets(2001)5(1)
Giglione&Meinnel 45
ee
thar
os
te
su
dd
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pr
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ctercons
F GNL ETV ??? GPI mobaotherce.
IK YTTLTSPYT SFVSDAVPD GQFFDYVQN ????????? HQFFDYVKN AR Methanotherotifsandtheethesequen
FQI TFL PTR TTP ??? TSP AAG asdmbov
L D Q T V ? M G dea
n EEPA KLHG LEAP CPAR AEAY ???? AEAY DDGS dicatedefincated
sibleC-terminusdomai KQQRIRQKVEKLDRLKARA DKKKVRPKLNELIRDYKATHS ELSNKLIYTTELTEDNLREIF DPFQVPDGAIPIGR NHVVPLEGFSTLSGWSDDFPS DHVVPMEGFVTMSDWSDDYRG EFVVSSVAMAQRYLWPANFPS EFIGSGIAMGQ?????????? EFIVSGIAMGQRDLWPPNFPS FEYEIEE KAKRKEGGQQCGRGDVTAVED ndbyhand.Thesequencein00809.Thethreepreviouslyce.Specialfeaturesareindi
n L V S A S T P P P E E a0n
Figure.1:AlignmentofeukaryoticprotistPDFs(continued) DistalinsertionMotif3dispe DGK——————-------—PFELEADGLLAICIQHEMDHLVGKLFMDYLSPE.coli NGY————-------———KHLKILKGIHSRIFQHEFDHLNGTLFIDKMTQP.falciparum TGK—————-------——ERIIEADGILAACFQHEYDHLLGKIFIDRIDKD.discoideum DGE————-------———EVTLRLKGLPAIVFQHEIDHLNGIMFYDRINPB.stearothermophilus DGH—————-------——PFEVTLEKMRARMALHELDHLSGVLFTRRIPDT.cruzi2/2 HGK——————-------—PFTVTLDKMRARMALHELDHLQGVLFTRRVVDL.major2/2 HGN———-------————HKVQVLDGMRARCLMHELDHLMGKTIFHQAVGL.major1/2 YGN——————-------—EKTELLDGMRARCLMHELDHLTGKTILHQALGT.cruzi1/2 YGN—————-------——EKTEVLDGMRARCLMHELDHLSGKTILDQAQGbruceiT. LRAVLDPLDLKIRLKRLEKPLRFTGSGAYGVAHEMEHLEGEESEGTPFWMethanothermobacter AHEφDHLXG C-terminalextension LDRKFEDGIYPGCEQDRQQRIELTAMEEIQRNVWRKL.major2/2 IPPGMEWWYAQNVREEFSNEQIGQL.major1/2 ??PGMEWWYAQNMQQHFQDARLNQT.cruzi1/2 IPPGMEWFYAQSMNQQFEDARLSHT.brucei PDFaminoacidsequencesfromeukaryoticprotistswerealignedusingClustalXsoftwareMethanothermobacterthermoautotrophicusproteinfrom,GenbankaccessionnumberAEshowninbold-typeface.Aseriesofquestionmarksindicatesunknownpartsoftheseque
©AshleyPublicationsLtd.Allrightsreserved. EmergingTherapeuticTargets(2001)5(1)
46 Peptidedeformylaseasanemergingtargetforantiparasiticagents
hydrophobic and hydrophilic residues in this 3.1.2AnimalPDForthologues
insertion, its only effect is likely to be the slight
extensionofanantiparallelβ-sheetfarfromtheactive Ourgrouphasrecentlyobtainedconvincingevidence
that mRNAs encoding proteins homologous to
site.However,itshouldbestressedthatmotif1isonly
mitochondrial PDF are expressed by the nuclear
weakly conserved in this species.
genomesofinsects,fishandhumans[6,7,29].Animal
PDFs are derived from the same common ancestor
Thus,itisclearthat,incontrasttowhatwasbelieved
and from the same original function as the
untilveryrecently,PDForthologuesarenotrestricted
homologues identified in the mitochondria of higher
to Eubacteria, but instead occur in most of the
plants (mPDF; Figure2). Thus, they are presumably
branchesofthephylogenetictreeoflivingorganisms
involved in the removal of the N-formylmethionine
(i.e., also in the Archaea, lower and higher
fromnewlysynthesisedproteinsinanimalmitochon-
eukaryotes). However, some organisms, such as
dria, which would make it more difficult to use
nematodes,fungiandmostArchaea,clearlylackPDF
inhibitors of these enzymes in human therapeutics.
orthologues.Ofcourse,itmaybepossiblethatthese
organisms have a PDF with homology below the
However, analysis of the sequences of
BLAST threshold, but in our opinion this is unlikely.
mitochondrially-encoded proteins in animals has
Indeed,searchesinvariousdatabaseswereachieved
shownthatvirtuallyallretaintheirN-formylgroup,as
unsuccessfully by various and different means. Since
ifPDFwasnotpresentoractiveinthisorganelle(see
new complete genome sequences will become
data in [6,7]). N-terminal sequence data are available
availableinthenextfewyears,itisthereforeofvalue
for 6 such proteins in cattle. None of these proteins
toidentifythoseorganismsthatdonotcontainaPDF.
were found to undergo N-deformylation [36-41].
Additional unpublished data strongly suggest that all
mitochondrialproteinsretaintheirN-formylgroupin
bovine systems (see discussion in [7]). The human
3.ThefunctionofPDForthologuesin
PDF sequence was studied with Target P software
Eukaryotes.
[42,104], for prediction of the subcellular location of
proteins. It was predicted that this protein would be
The detection of so many PDF orthologues in targeted to the secretory pathway, whereas all plant
organisms other than eubacteria was unexpected. mPDFswerepredictedtobelocatedinthemitochon-
However,thedetectionoftheseorthologuesdoesnot dria.Incontrast,themousePDFaminoacidsequence,
necessarily imply that their role is the same and that derived from its full-length cDNA (T. Meinnel,
their activity in eukaryotic cells is the cleavage of unpublished results), was studied with the same
N-formyl groups from nascent polypeptides. softwareandwaspredictedwithahighprobabilityto
be routed to the mitochondria. Clearly, animal PDFs
require experimental studies before any definitive
3.1DoallPDForthologuesdisplaydeformylase
conclusions can be drawn about their actual subcel-
activity?
lular location.
Even if animal PDFs were present in the mitochon-
3.1.1PlantPDForthologues
dria, their structure might account for their intrinsic
TwoplantPDFshaverecentlybeenshowntocomple- absence of deformylase activity. We have observed
ment a def Ts bacterial strain and deformylation that the conserved hydrophobic residue of motif 2
activity was measured in vitro [7]. Analysis of the (generallyaleucine;see2.1andFigure1)issystem-
N-terminal sequences of many chloroplast-encoded atically replaced by a hydrophilic residue (i.e., a
proteinsindeedrevealssystematicdeformylationand glutamate) in vertebrate PDFs. Given the importance
the subsequent removal of the first methionine in ofthisresidueinbacterialPDF[22,25],thischangeis
some cases, depending on the nature of the second likely to have profound consequences for the
residue (see data quoted in [7]). Although less hydrophilicactivityofPDF.Indeed,thesidechainof
complete,asimilaranalysisinplantmitochondrialed this leucine makes hydrophobic contact with that of
tothesameconclusion[31-35].Thesetwosetsofdata oneoftheconservedhydrophobicresiduesofmotif1.
indicatethatdeformylationofnascentpolypeptidesis This contact has two major effects: (i) the closing of
performed efficiently in plant organelles. oneendoftheactivesiteand(ii)onthelocationofthe
©AshleyPublicationsLtd.Allrightsreserved. EmergingTherapeuticTargets(2001)5(1)
Giglione&Meinnel 47
Figure2:AphylogenetictreeforPDFrevealsthreedistinctclasses.
Class 1 PDF cpPDF
Arabidopsisthaliana
Plasmodiumfalciparum Tomato Rice
cyanobacterialPDF Calothrix Alfalfa Barley
Synechocystis Aquifexaeolicus
Prochlorococcusmarinus Heliobacterpylori
Borreliaburgdorferi Rickettsiaprowazekii
Vibriocholerae1/2
Treponemapallidum
Escherichiacoli
Deinococcusradiodurans Haemophilusinfluenzae
Thermusthermophilus
Neisseriagonorrhoeae
Myobacteriumtuberculosis
Pseudomonasaeruginosa1/2
Pseudomonasaeruginosa2/2 Clostridiumacetobutylicum
Chlamydiatrachomatis Thermotogamaritima
Bacillussubtilis1/2
Legionellapneumophila
Dictyosteliumdiscoideum
Mouse
Rat Mycoplasmapneunomiae
Animal Human Staphylococcusaureus
Bacillusstearothermophilus
PDF Fish
Fruit-fly2/2 Bacillussubtilis2/2
Fruit-fly1/2 Streptococcuspyogenes1/2
Enterococcusfaecalis
Mosquito
Class 2 PDF
Streptomycescoelicolor1/4 Corn
Wheat
mPDF Tomato
Arabidopsisthaliana
Alfalfa
Mycoplasmathermoautotrophicum
Streptococcuspneumoniae2/2 Methanothermusfervidus
Trypanosoma
Clostridiumbeijerinckii cruzi2/2 Leishmaniamajor1/2 archaealPDF
Trypanosomabrucei
Trypanosomacruzi1/2 Class 3 PDF
Leishmaniamajor2/2
Fromthe>120sequencesofPDForthologuesavailableindatabases,56PDFsequenceswereselectedasrepresentativeofthese-
quencediversityofthisprotein.ThesequenceswerealignedwithClustalXsoftwareandaphylogenetictreeconstructedwithTree-
View1.6.Anumber(1,2or4)indicatesthatthesequenceisoneofthetwo(orfour)PDFspeciesofthisorganism.cpPDF=plastid
PDF,mPDF=mitochondrialPDF.
backboneNHwhichmakesacontactwiththeoxygen homologuesinanimalsmaybethattheseproteinsare
of the formyl moiety of the substrate. We have remnants of ancient PDFs that no longer function in
constructed a model of the active site of human PDF deformylation in the mitochondria. It is not known
(Figure 3). Although it is only a working model, it whetherthesehomologueshavesubstratesoutsideof
suggests that the result of replacing the leucine by a the mitochondria or whether the product of the PDF
glutamate alone is considerable movement (5 Å) of openreadingframehasacquiredanotherfunctionor
thissidechain.Thisleadstotheactivesitebecoming location in animal cells.
larger and open to the outside, unlike that of E. coli
PDF.Thus,vertebratesPDFsmaywellhaveacquired
3.1.3cpPDFfromApicomplexaandgreenalgae
anewsubstratespecificitythatiscurrentlyunknown.
Furtherstudyofthisissueisrequiredbeforetheuseof NoproteinsequencesfromtheapicoplastofPlasmo-
anti-PDF drugs is extended. dium falciparum are currently available, but the
strongresemblanceofthisorganelletothechloroplast
As no in vitro analysis has yet been reported, the ofthegreenalgaChlamydomonasreinhardtiimakes
presenceofPDForthologuesinvertebratesremainsa it possible to make certain predictions. In this green
mystery.OnepossiblereasonforthepresenceofPDF alga, protein sequences for plastid-encoded proteins
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48 Peptidedeformylaseasanemergingtargetforantiparasiticagents
Figure3:A3DmodelofhumanPDFsuggestsanopeningofitsactivesite
Glu
Motif 2
5¯
Leu
3¯
Motif 3
Motif 1
TheaminoacidsequenceofthehumanPDFsequencewassuperimposedoverthatofE.coliPDFandits3Dstructurereconstructedby
homologymodellingwiththeknowncrystalstructure[96],usingSwissPdbViewer[104].MinimisationwascarriedoutwiththeInsight
IIpackage(MSI).Thecatalyticmetalcationisshownasalightbluesphere.
areavailableandprovideclearevidenceofdeformy- with plant PDFs, we found that it was not easy to
lase activity (see data quoted in [29]). This would be achieve complementation with the full-length
consistent with the existence of PDF activity in the mitochondrial form [7]. We therefore cannot exclude
apicoplast of Apicomplexa. However, only three thatwedidnotexpresstheappropriate,mostsoluble
proteins of the apicoplast of P. falciparum, namely form of T. brucei PDF in the bacterium. Third, we
ribosomalproteinsS3andL14andORF105,wouldbe foundthatthepurifiedproteinfromT.bruceihadno
predictedtoundergoremovalofthefirstmethionine. significantdeformylaseactivityinvitro,withak /K
cat M
The removal of the first methionine would be greater than 1 M-1 s-1 (C. Lazennec and T. Meinnel,
catalysedbytheapicoplastMAPrecentlyidentifiedon unpublished results). Given the difficulties in
chromosome5ofP.falciparum,whichresemblesits stabilising the metal cation in the enzyme, no defini-
plant counterpart [7]. More generally, it should be tiveconclusioncanbedrawnfromexperimentswith
borne in mind that Apicomplexa display significant negative results such as this, as previously indicated
molecularsimilaritytoplants,indicativeofacommon (see section 1). Finally, we have found that the PDF
evolutionary origin [43,44]. orthologue of T. brucei has a lysine instead of the
crucial glutamine in motif 1 (Figure 1). A
3.1.4ClassIIIPDFs post-translationalmodificationthatdoesnotoccurin
E.colihasalreadybeenreportedforureaseinvolving
Class III PDFs remain difficult to analyse. First, no
carbamylation of the side chain of the lysine of the
sequence data are available for proteins synthesised
activesite[45].Wethereforewonderedwhethersuch
in the mitochondria of kinetoplastids. Second, we
a modification could occur also in kinetoplastids.
have cloned and overexpressed the PDF from
Unfortunately, replacement by site-directed
Trypanosoma brucei in E. coli (C. Lazennec and T.
mutagenesis of the lysine by a glutamine, which
Meinnel,unpublishedresults).Thefull-lengthprotein
mimics a carbamyl-lysine did not improve PDF
sequence and three N-terminally truncated variants,
activity invitro or invivo.
unlikeplantPDFs,wereunabletocomplementadefTs
bacterialstrain.Nevertheless,usingthesamestrategy
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Giglione&Meinnel 49
Noinformationisyetavailableconcerningthesecond organisms (i.e., all plastids and the mitochondria of
typeofPDFfromkinetoplastids.Thesequenceofthis higher plants and of some protists) but has
PDFismorecloselyrelatedtootherPDFsthantothat disappeared from the mitochondria of animals and
fromT.brucei.Inparticular,itcontainstheimportant fungi (humans, C. elegans and S. cerevisiae for
glutamine of motif 1. It is not yet clear whether this instance). It should be borne in mind that the
PDF has deformylase activity, although we believe genomes of plants and the genomes of protist’s
that this may well be the case. organellesencode30-100proteinsversusonlyninein
yeastand13inanimalmitochondria.Moreover,inthe
Lastbutnotleast,thepresenceofPDForthologuesin
mitochondriaofallanimalincludingC.elegans,which
Archaea remains a true mystery. In this organism, it
contains no PDF, a common set of 13 proteins is
has been known for twenty years that there is no
synthesised: 7 subunits of NADH:ubiquinone
N-formylationofnascentpolypeptides[8].Webelieve
oxidoreductase, the three subunits of cytochrome
that, as in the case of human PDF (see 3.1.1), this
oxidase, cytochrome b and subunits 8 and 9 of ATP
protein may have a slightly different proteolytic
synthase.Itseemsmostlikelythatthegenesencoding
activity.
several proteins strictly requiring deformylation to
achieve their final function were retained in the
3.2PDFactivityisessentialintheorganellesof
organelles genomes of some organisms, but not in
someeukaryotesbuthasprobablydisappeared
those of others such as yeast. The loss of proteins
fromothers,includinghumans
requiring deformylation would thus have led to the
Given current knowledge and the data available on lossofthedeformylasefunction.Clearly,noneofthe
plant mPDFs, animal PDFs appear to be inactive in 13 proteins encoded by animal mitochondria require
mitochondria.Thisraisesthequestionsastowhythe deformylation for full activity.
protein deformylation is not required in animal cells
butdoesoccurinotherorganisms,suchasplantsand
3.2.2Determinationofthesmallestsetof
many eukaryotic protists.
physiologicalsubstratesrequiringthe
deformylationfunction
3.2.1Correlationbetweengenenumberand
deformylaseactivityinorganelles The set of additional proteins encoded by plant
organelles includes ribosomal proteins, subunits of
To date, although many PDF genes have been
RNA polymerase, translation factors and the proteins
sequenced, none has been identified in an organelle
involved in the specific functions of the organelle,
genome. The same is true for other crucial enzymes
suchasthelargesubunitofRubiscoinplastids[49-52].
suchasMAPandaminoacyl-tRNAsynthetases(aaRS)
Rubiscoisoneofthemostabundantproteinsonearth
(for a review on aaRS see [46]). The mitochondrial
and is the motor of photosynthetic function. This
genome of the protozoon Reclinomonas americana
proteinisknowntoundergofurtherprocessinginthe
containsthelargest(97)collectionofgenesidentified
plastids. Once PDF and MAP have removed the
todateinanorganelle[47].Ithasbeensuggestedthat
N-formylmethionine of the large subunit of Rubisco,
this genomes resembles the ancestral genome of
theserineinposition2isremovedandthisisfollowed
mitochondria, but it contains no aaRS, PDF or MAP
by N-acetylation [53] or even further N-methylation
gene. Interestingly, the number of genes in R.
[54]. Given the crucial role of Rubisco, we cannot
americana is similar to that of the smallest bacterial
exclude the possibility that PDF and MAP have been
genome described so far, Mycoplasmagenitalium. It
retained to ensure the correct processing at least of
contains470ORFs,ofwhichonly250-350areconsid-
this protein. However, the impact of these modifica-
ered to be strictly required for cell survival [48]. Like
tionsonRubiscoactivityandstabilityisunknownand
aaRSandMAP,itsPDFgenesareabsolutelyrequired
the3Dstructureofthismoleculeprovidesnofurther
for survival. Thus, like aaRS and MAP, PDF genes
insightintothisissue[55].NoRubiscoispresentinthe
wereamongthefirstgenescorrespondingtoessential
apicoplastofP.falciparum,inwhichdeformylationis
functions to be transferred from the organelle to the
clearly required [56]. The reason for the necessity of
nucleus.
N-formylremovalprobablyliesinthefunctionofone
Thisraisesquestionsconcerning(i)theactualroleof or several of the ribosomal proteins (i.e., translation
N-terminal protein processing and (ii) the reasons machinery) encoded by all organelle genomes
why it has been retained in the organelles of some (mitochondrial and plastid).
©AshleyPublicationsLtd.Allrightsreserved. EmergingTherapeuticTargets(2001)5(1)
50 Peptidedeformylaseasanemergingtargetforantiparasiticagents
In conclusion, we believe that the requirement for non-photosynthetic plants, with no genes encoding
PDFhasdisappearedonlyinorganismsinwhichthe the proteins of the photosynthetic machinery.
number of genes in the organelle genome has been
reducedtoaverysmallnumber.Thisprovidesstrong ThediscoveryofaplastidinApicomplexahasrapidly
evidence that the pathway for the processing of the ledtosuggestionsthatthisorganellewouldbeagood
first methionine plays a crucial, general role (for a target for antiparasitic drugs. Indeed, apicoplast
further discussion see [5]). Thus, it seems probable function has been shown to be required for parasite
that deformylation is essential for the function of the survival[60,61].Theapicoplasthasbeenshowntobe
organelle, in those organelles in which it occurs. thepharmacologicaltargetofmanyantibioticsknown
BlockingPDFfunctionshouldleadtothedeathofthe to be specific for eubacteria and organelles. These
correspondingorganism.TheessentialityofPDFwill drugs (e.g., chloramphenicol, clindamycin,
deserve however to be experimentally addressed in thiostrepton, rifamycin, azithromycin, fluoroqui-
each biological system where it is present. nolones) specifically block apicoplast protein
synthesis, transcription or DNA replication [62,63].
Theblockingoftheactivitiesoftheplastidswithsuch
4. Manyparasiticillnessesandvarious drugs results in a characteristic pattern of delayed
PDFstoinhibit death that contrasts with the more immediate effect
seen with drugs such as chloroquine. However,
Sixmajortropicaldiseases,malaria,filariasis,schisto- inhibition of the apicoplast EFTu by various drugs,
somiasis, African trypanosomiasis, Chagas’ disease such as kirromycin or enacyloxin IIa, results in the
andleishmaniasis,togetheraccountforthedeathsof rapid onset of inhibition [64]. Interestingly, plastid
more than one million people each year and cause function can be blocked by these drugs and the
enormous suffering in hundreds of millions more parasitekilledatvariousstagesofitssexualdevelop-
[111-113]. Resistance to the drugs currently used to ment[65].Althoughtheapicoplastisclearlythesiteof
cure most of these diseases is clearly on the increase many crucial metabolic processes, such as branched
and new medicines are required in the short-term. and aromatic amino acid biosynthesis, it is unclear
PDF function is required in eubacteria but absent in whytheexpressionofitsgenomeisimportantforcell
humans.Giventhatmostoftheorganismsresponsible survival.Noapicoplastgeneproductappearstoplaya
for these major diseases have PDFs, this raises specificroleinprocessesotherthangenomeexpres-
possibilitiesfortheuseofinhibitorsofPDFaspotent sion.However,thefunctionsofseveralopenreading
medicines. frames of the apicoplast genome are unknown. The
key to understanding the essential function of the
4.1Apicomplexa apicoplast probably lies in the identification of the
The phylum Apicomplexa includes a large family of functions of these genes.
unicellularparasitesthatcausevariousdiseases.This
family of protists includes the causative agent of Todate,P.falciparumPDFistheonlyspeciesofthis
malaria (Plasmodium spp.), opportunistic pathogens groupforwhichaPDFhasbeendescribed[29].Given
associated with immunodeficiency (e.g., the the high level of conservation of apicoplast
AIDS-associated pathogen Toxoplasma gondii, sequences, there is probably a PDF sequence in all
Cryptosporidium and Sarcocystis) and pathogens of Apicomplexa.Nodirectproofhasyetbeenprovided
poultry, livestock and shellfish (e.g., Eimeria, thattheP.falciparumPDFislocatedintheapicoplast.
Theileria,Babesia).ThemembersoftheApicomplexa Nevertheless, the amino acid sequence of this PDF
have recently been shown to contain a small plastid resemblesthatofcpPDFanditisrootedonthesame
called the apicoplast [57,58]. The 35 kb sequence of branch of the PDF phylogenic tree as plant cpPDF
the apicoplast genomes of two Apicomplexa species (Figure2).TherecentdemonstrationthatcpPDFsare
(i.e., T. gondii and P. falciparum) have been systematically targeted to the plastids of plants
determined[59,102].Thesedatarevealahighlevelof stronglysuggeststhatP.falciparumPDFistargetedto
conservation of the 25 proteins encoded by these the apicoplast [7]. Moreover, like other nuclear-
genomesincluding17ribosomalproteins,elongation encoded apicoplast proteins, this PDF has a bipartite
factorTu(EFTu),onesubunitoftheclpproteaseand N-terminal pre-sequence consisting of a signal
thethreesubunitsofRNApolymerase.Theapicoplast peptide for entry into the secretory pathway and a
genome strongly resembles that of transitpeptidesimilartothosefoundinplantproteins
©AshleyPublicationsLtd.Allrightsreserved. EmergingTherapeuticTargets(2001)5(1)