Kinetic analyses and inhibition studies reveal novel features in peptide deformylase 1 from Trypanosoma cruzi

Article abstract of DOI:10.1016/j.molbiopara.2011.12.003

In eubacteria and eukaryotic organelles N-terminal methionine excision requires the sequential action of two activities, a peptide deformylase (PDF), which systematically removes the N-formyl group present on all nascent polypeptides and methionine aminop

Full text of DOI:10.1016/j.molbiopara.2011.12.003

Molecular & Biochemical Parasitology 182 (2012) 83–87
Contents lists available at SciVerse ScienceDirect
Molecular & Biochemical Parasitology
Short communication
Kinetic analyses and inhibition studies reveal novel features in peptide
deformylase 1 from Trypanosoma cruzi
a
a
a
b
Carlos A. Rodrígues-Poveda , Guiomar Pérez-Moreno , Antonio E. Vidal , Julio A. Urbina ,
a,∗
a
Dolores González-Pacanowska , Luis M. Ruiz-Pérez
Instituto de Parasitología y Biomedicina “López Neyra”, Parque Tecnológico Ciencias de la Salud, Av. del Conocimiento, s/n, Consejo Superior de Investigaciones Científicas, 18100
Armilla, Granada, Spain
a
b
Instituto Venezolano de Investigaciones Científicas, Centro de Bioquímica y Biofísica, Laboratorio de Química Biológica, Caracas 1020, Venezuela
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 3 November 2011
Received in revised form
In eubacteria and eukaryotic organelles N-terminal methionine excision requires the sequential action of
two activities, a peptide deformylase (PDF), which systematically removes the N-formyl group present on
all nascent polypeptides and methionine aminopeptidase (MAP), which exscinds methionine specifically
and depends on the previous removal of the N-formyl group. In Trypanosoma cruzi two genes encoding
bacterial PDF homologues have been identified and referred to as TcPDF-1 and TcPDF-2. Here we report
the biochemical characterization of a truncated soluble version of TcPDF-1 lacking the hydrophobic N-
terminal domain that is active with the bacterial PDF substrate formyl-methionyl-alanyl-serine but, in
1
2 December 2011
Accepted 13 December 2011
Available online 22 December 2011
Keywords:
Trypanosoma cruzi
Peptide deformylase
2
+
contrast to other PDFs, is not inhibited by actinonin. The enzyme is strongly activated by Cu and inhibited
2
+
by Ni . Our results show that T. cruzi PDF exhibits unique features thus providing a new avenue for the
design of potential inhibitors for use in the treatment of diseases caused by trypanosomatid parasites.
©
2012 Elsevier B.V. All rights reserved.
N-terminal methionine excision (NME) is an essential co-
translational process conserved from bacteria to eukaryotes that
determines the cellular fate of proteins [1]. First, the initiator
methionine is modified previously to translation initiation by the
that PDF orthologues are present and functional in eukaryotes
[3] including several parasites of biomedical importance such as
Plasmodium spp. [4] and species of the Trypanosomatidae family
[5] but they have not been found in Saccharomyces cerevisiae or
Caenorhabditis elegans. The enzyme is essential in procyclic forms
of Trypanosoma brucei [5].
incorporation of a formyl group in a reaction catalyzed by a spe-
fMet
cialized enzyme, methionyl-tRNA
transformylase (MTF). The
formyl group is necessary for optimal ribosomal translation [2].
Once the nascent protein emerges from the ribosomal tunnel, the
formyl group must be removed for further recognition by methi-
onine aminopeptidases. This enzymatic reaction plays a crucial
role in the posttranslational maturation of proteins and is cat-
alyzed by peptide deformylase (PDF), the enzyme responsible for
the hydrolytic excision of the formyl group (Fig. S1). PDF constitutes
a growing family of hydrolytic enzymes that contain, in addition
to the HEXXH signature sequence strictly conserved in the zinc
metalloprotease family, two short stretches of conserved amino
acids, EGCLS and GXGXAAXQ, that altogether build up the substrate
and metallic cofactor binding site [1]. PDF was previously believed
to be a unique feature to eubacteria, where the enzyme is essen-
tial for viability. More recently, several studies have demonstrated
Trypanosoma cruzi is a kinetoplastid parasite that causes Amer-
ican trypanosomiasis or Chagas’ disease which is one of the most
prevalent neglected parasitic diseases in the American continent. In
this organism, mitochondrial protein synthesis relies on imported
Met-e
tRNA
that becomes formylated by MTF after import and
is optimally used in translation initiation, while non-formylated
Met-e
tRNA
is used in elongation [6]. The formyl group is the main
determinant associated with the recognition of initiation factor 2
[7]. Recently it has been shown that mitochondrial translation is
essential in the clinically relevant bloodstream form of T. brucei [8],
which is an indication that formylation and probably deformylation
might be functional in this stage of the parasite. In trypanosomatids,
the mitochondrial DNA or kinetoplast encodes several proteins
involved in oxidative phosphorylation and the respiratory chain
[
9]. All these proteins are putative substrates of PDF which empha-
sizes the potential importance of formylation-deformylation cycles
in the mitochondrion of these parasites.
A comparison of the amino acid sequence of T. cruzi PDF-1
with other prokaryotic and eukaryotic PDFs reveals an overall low
degree of similarity (Fig. S1). The three typical motifs of this family
Abbreviations: PDF, peptide deformylase; EDTA, ethylenediaminetetraacetic
acid; f-MAS, formyl-methionyl-alanyl-serine.
∗ Corresponding author. Tel.: +34 958181621; fax: +34 95818 1632.
E-mail address: dgonzalez@ipb.csic.es (D. González-Pacanowska).
0
166-6851/$ – see front matter © 2012 Elsevier B.V. All rights reserved.
doi:10.1016/j.molbiopara.2011.12.003

8
4
C.A. Rodrígues-Poveda et al. / Molecular & Biochemical Parasitology 182 (2012) 83–87
of metalloproteases involved in catalysis are conserved in the T.
used in the characterization of E. coli, human and Plasmodium
peptide-deformylase activity. As shown in Fig. 1A, while the activity
increased with substrate concentration up to 35 mM, it drasti-
cally dropped thereafter, showing that TcPDF-1ꢀN37 is inhibited
by an excess of substrate. A similar substrate inhibition has been
described for bacterial [14] and human PDFs [12]. Consequently,
20 mM f-MAS was used in the enzymatic assay henceforth. Km and
Vmax values were obtained by hyperbolic regression fit of the data
to the Hill equation using Sigma Plot® software. The Km and Vmax
1
14
121
cruzi protein: motif 1
GVGLAAPQ
in Arabidopsis thaliana and
44
51
93
176
180
GIGLAATQ in Escherichia coli, motif 2
EGCLS
in A. thaliana
89
and EGCLS in E. coli and motif 3, HEXXH which is strictly con-
114
served in all PDFs studied to the date. The Gly of motif 1 (Gly
in
in A. thaliana,
Leu in E. coli) are crucial for catalysis and are substituted in H. sapi-
4
4
179
A. thaliana, Gly in E. coli) and Leu of motif 2 (Leu
92
1
08
173
108
115
ens for Cys
(
and Glu
in motif 1 ( CVGLSAPQ ) and motif 2
1
70
174
EGCES ) respectively. T. cruzi PDF-1 displays a similar substi-
8
3
83
90
−1
−1
,
tution in motif 1, Cys
(
CISFSAPK ) but maintains a hydrophobic
for f-MAS were 22.37 ± 0.137 mM and 200 ± 2.3 nmol min mg
Ile in motif 2 ( ENCIS138).
1
34
respectively. The calculated kcat value was 0.093 ± 0.065 s . While
we cannot fully discard that the His-tag may be detrimental to
enzyme activity, these values are of similar order of magnitude
to those described for other truncated recombinant eukaryotic
enzymes when f-MAS was used as substrate although the Vmax was
significantly lower in comparison to that of bacterial PDFs [4,5,12].
It is evident that the peptide f-MAS used in this study is not
the optimal substrate for TcPDF-1. It was designed to characterize
bacterial PDFs and is based on the fact that the complete bacterial
proteome is associated to formylation. In contrast, formylation in
trypanosomatids is limited to only 13 mitochondrial DNA-encoded
proteins [9]. Recent studies on human PDF have shown that the
enzyme is more efficient in deformylating substrates based on
the organelle-encoded proteome than other nonspecific substrates
including f-MAS [15]. We therefore designed a tripeptide based
on the N-terminal sequence of subunit 7 of the NADH dehydro-
genase complex (formyl-methionyl-leucyl-phenylalanine, f-MLP)
coded by mitochondrial DNA but its high hydrophobicity made it
inadequate for assay in aqueous media.
−1
All eukaryotic PDF orthologues have an extended N-terminal
domain which makes expression in bacterial systems difficult. This
extension plays a role in organellar targeting of the protein to
plastids and mitochondria in A. thaliana [1], to the apicoplast in api-
complexan parasites [4] and to the mitochondria in mammals [3].
In this study, we explored the properties of a truncated version of
PDF-1 from T. cruzi lacking 37 amino acids from the amino terminus
using the formylated tripeptide formyl-methionyl-alanyl-serine (f-
MAS).
Initial attempts to produce a recombinant full-length soluble
T. cruzi enzyme were unsuccessful. Considering previous reports
on PDF from H. sapiens [3], and Plasmodium falciparum [4], we
generated two deletion mutants that lacked the N-terminal 33
and 37 amino acids. The full-length PDF-1 gene was amplified
ꢀ
by PCR from T. cruzi CL Brener genomic DNA with primers 5 -
ꢀ
ꢀ
GCC ATA TGC TGA GCC GTC TGT CAC GGA-3 and 5 -GGA TCC CAT
TGG TTC AGA CGA GCA TCC-3 , which were specifically designed
ꢀ
according to the information obtained from the genome sequence
database (GenBank accession number TcCLB.506871.100). Addi-
The influence of the ionic strength on deformylase activity was
assessed by supplementation of the reaction buffer with increasing
concentrations of KCl. An increase in ionic strength had a significant
impact on TcPDF-1ꢀN37, giving rise to a stimulation of the activity
which reached a maximum at 0.5 M KCl (Fig. 1B).
ꢀ
ꢀ
tional primers, 5 -GCC ATA TGG CGG AGG CGC AGG TGA AGT 3 and
5
ꢀ
ꢀ
-GCC ATA TGG TGA AGT CCC GAG TGG CCT-3 , were designed to
generate the truncated proteins where 33 (ꢀN33) and 37 (ꢀN37)
amino acids were eliminated from the N-terminus respectively. The
primers contain NdeI and BamHI restriction sites (underlined) for
cloning into the pET-22b vector. The cloning procedure resulted
in the addition of a six-histidine tag to the C-terminus of PDF. Of
the two constructs tested, major amounts of soluble enzyme were
obtained with TcPDF-1ꢀN37, a PDF lacking 37 amino acids from
the amino terminus.
Next, we studied the effect of the nature and concentration of
the metallic cofactor on TcPDF-1 deformylation capacity. To this
end, several oxidation insensitive divalent cations were added to
the reaction buffer at different concentrations ranging from 10 to
100 ␮M. The enzyme was preincubated with the metallic cofactor
2
+
2+
2+
for 10 min before initiating the reaction. While Co , Mg , Mn
The purification and biochemical characterization of bacterial
PDFs have largely remained a major challenge because the enzyme
did not affect significantly the activity, Ni2+ had a pronounced
inhibitory effect on peptide deformylation at all concentrations
tested. On the other hand, the addition of Cu induced a 50%
2+
2+
is extraordinarily labile due to oxidation of its catalytic Fe . It has
been shown that substitution of the ferrous ion for Co2 or Ni2+ dur-
ing the purification procedure of bacterial [10,11], P. falciparum [4]
and human PDFs [12] gives highly active and stable enzyme prepa-
rations. Here TcPDF-1ꢀN37 protein was overexpressed in E. coli
cells that were grown in medium enriched with Co2 (100 ␮M).
Under these conditions, TcPDF-1ꢀN37 was purified to over 99%
purity by metal-affinity chromatography and exhibits an estimated
molecular mass of ∼28–30 kDa (Fig. S2).
+
increase in the measured activity at 50 ␮M (Fig. 1C). To our knowl-
edge, our study is the first to report the activation of a PDF enzyme
by this metal ion. Both P. falciparum and E. coli PDFs differ from
TcPDF-1 and are highly active with both Co2+ and Ni , retaining
the same catalytic activity as the iron-native enzyme with the lat-
2+
+
2+
ter [4,10,11]. In contrast, human PDF is only active with Co [12]
while A. thaliana PDF is highly active when associated to Zn2+
.
The effect of the chelating agents EDTA and 1,10-phenantroline
on deformylase activity was studied at concentrations ranging from
1 to 20 mM, and 1 to 5 mM respectively. E. coli PDF was included as
a control assay. For this purpose, a segment of the E. coli def gene
Truncation of the first 37 N-terminal amino acids gave a pro-
tein that proved to be catalytically active and exhibited kinetic
parameters similar to other eukaryotic PDFs. A similar strategy was
employed by Schneiders and coworkers who obtained an active
version of T. brucei PDF-1 by eliminating the first 30 amino acids,
thus suggesting the presence of a structural core responsible for
deformylase activity [5].
ꢀ
ꢀ
was amplified by PCR using forward primer 5 -CGC ATA TGT CAG
ꢀ
TTT TGC AAG TGT TAC-3 and reverse primer 5 -GGA TCC TTC AGC
ꢀ
GGT GAC AGA TAA TCC AT-3 and cloned in pET22b. The result-
ing expression construct codes for a bacterial PDF (EcPDFꢀC18)
that lacks 18 C-terminal residues and contains a C-terminal His-
tag. The conditions for expression and purification were similar
to those used for TcPDF-1ꢀN37 (see supplementary information).
Purified EcPDFꢀC18 was active and more stable than the full-
length enzyme [16]. Before measuring the activity, the enzyme
was preincubated with the chelating agent for 10 min. Strikingly,
both EDTA and 1,10-phenantroline strongly activated deformy-
lase activity while 1,10-phenantroline was a potent inhibitor of
The PDF activity assay was based on the quantification of for-
mate using formate dehydrogenase [13]. The reaction mixture
+
contained 50 mM KH PO pH 7.5, 12 mM NAD , 1.2 U/ml FDH, and
2
4,
TcPDF-1ꢀN37 enzyme as indicated. The reaction was performed at
◦
3
7 C for 300 s and the resulting increase in absorbance of NADH at
45 nm was measured with a UV-VIS spectrophotometer.
The catalytic activity of TcPDF-1ꢀN37 was assessed with f-
3
MAS as substrate. f-MAS is a bacterial PDF specific substrate

C.A. Rodrígues-Poveda et al. / Molecular & Biochemical Parasitology 182 (2012) 83–87
85
the E. coli enzyme. In the case of EDTA, TcPDF-1ꢀN37 activity
increased up to 70% at 10 mM (Fig. 2A) while a stunning 6-fold
increase was observed in the presence of 1 mM 1,10-phenantroline
(Fig. 2B). This unusual observation suggests that an unknown diva-
lent cation metal with inhibitory properties is strongly associated
to the enzyme in our preparation. We can hypothesize that other
active divalent cations present in the bacterial expression system
during cell growth or in the chemicals used for enzyme purifica-
tion such as inhibitory Ni2+ ions could have been incorporated to
the enzyme active site during the purification process.
The occurrence of the HEXXH motif involved in metal-binding
characteristic of zinc metalloproteases and present in all PDFs,
prompted us to explore whether the parasite enzyme would be
inhibited by a series of previously characterized metalloprotease
inhibitors. Bestatin is a compound originally isolated from Strep-
tomyces olivoreticulli and was one of the first potent inhibitors of
metallo-aminopeptidases with broad spectrum of action and has
been extensively investigated in biological systems both in vitro
and in vivo. Other compounds tested included CP-101537, a matrix
metalloprotease (MMP) inhibitor and CL-82198 which is a selec-
tive inhibitor of MMP-13. However, none of the compounds had
significant effect on PDF-1 activity at concentrations up to 100 ␮M.
Actinonin is a naturally occurring inhibitor of bacterial pep-
tide deformylase with an IC50 value of 0.8–90 nM, depending on
the metal cation of the bacterial enzyme [17]. The inhibitor has
been shown to be also active on eukaryotic PDFs such as human,
Plasmodium and plants both in vitro and in vivo [4,12,18]. Wies-
ner et al. have reported that actinonin inhibits the growth of both
wild-type P. falciparum (IC50 = 3.0 ␮M) and a multidrug-resistant
strain (IC50 = 3.6 ␮M) [18]. In addition, actinonin inhibits human
deformylase activity with an IC50 of 43 nM [12].
Here, we examined the inhibitory effect of actinonin at concen-
trations up to 100 ␮M which completely inhibit the activity of E. coli
PDF, yet this compound had no impact on the T. cruzi deformylase
activity (Fig. 2C). The antiproliferative effects of actinonin were also
evaluated on the epimastigote form of T. cruzi in culture (Fig. 2D).
The drug induced a dose-dependent inhibition of growth with an
IC50 of 500 ␮M. These observations suggest that the structural ele-
ments that determine actinonin binding in other PDF enzymes may
be absent in this parasite. Several crystallographic studies avail-
able describe the binding of actinonin to representative members
of type I and type II PDFs [19]. In E. coli PDF, the actinonin bind-
ing site consists in a deep invagination that involves 18 residues.
Four of these amino acids are strictly conserved in eukaryotic PDFs,
including A. thaliana, Plasmodium spp. and human enzymes; three
of them, Gly46, Leu and Gln are involved in the interaction with
47
51
90
the hydroxamate moiety of actinonin and the fourth, Gly , contacts
its valine group. In T. cruzi, the actinonin-binding residues described
8
5
86
90
for E. coli and H. sapiens are substituted by Ser , Phe , Lys and
Asn
135
respectively, thus providing a reasonable explanation for the
apparent insensitivity of the parasite PDF to actinonin (Fig. S1). To
elucidate whether the absence of one or more of these residues, and
in particular of Gly90, is responsible for lack of inhibition of TcPDF-1,
a systematic mutation analysis is currently underway. These stud-
ies together with the structural characterization of Tc-PDF1 would
help to identify the residues that shape the inhibitor binding site in
PDFs. This information would be especially relevant in the design
of compounds with improved activity and in devising strategies to
overcome potential drug resistance resulting from point mutations.
In summary, we have purified and characterized biochemically
one of the PDF orthologues identified in the T. cruzi genome and
demonstrated that the enzyme possesses deformylating activity
in vitro. The enzyme is strongly modulated by the nature of the
metallic cofactor and the ionic strength suggesting that the activ-
ity might be physiologically relevant under the adequate reaction
conditions. The unique features regarding metal ion requirements
Fig. 1. Substrate inhibition, divalent ion requirements and influence of ionic
strength on ꢀ37TcPDF-1. (A) Substrate inhibition. The concentration of f-MAS
ranged between 10 and 40 mM. The activity was measured as a function of sub-
strate f-MAS using the formate dehydrogenase-coupled PDF assay. The experiments
are representative of at least 2 independent determinations. (B) Influence of ionic
strength on the relative rate of deformylation of ꢀ37TcPDF-1. Relative activity of
1
00% was assigned to the rate value obtained under standard assay conditions.
(
C) Graphical representation of the effect of several divalent metallic cations on
+
deformylase activity using standard assay conditions (50 mM KH2PO4, 12 mM NAD ,
0
.1 mg/ml ꢀ37TcPDF-1 and 20 mM f-MAS). The error bars correspond to standard
deviations. The asterisk indicates significant differences with control data (p < 0.05;
Student’s t test).

8
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C.A. Rodrígues-Poveda et al. / Molecular & Biochemical Parasitology 182 (2012) 83–87
Fig. 2. Effect of chelating agents and inhibitors on the deformylase activity of ꢀ37TcPDF-1 and EcPDFꢀC18 and on the growth of T. cruzi epimastigotes. (A) EDTA. (B)
◦
1
,10-phenantroline. The enzyme was incubated for 5 min at 37 C with the indicated concentration of the chelating agent. The asterisk indicates significant differences with
◦
control data (p < 0.05; Student’s t test). (C) Effect of actinonin on the deformylase activity of ꢀ37TcPDF-1 and EcPDFꢀC18. The enzyme was incubated for 5 min at 37 C in
the presence of increasing concentrations of compound. Relative activity of 100% was assigned to the rate value obtained in the standard assay (50 mM KH2PO4, 12 mM
+
+
NAD , 0.1 mg/ml ꢀ37TcPDF-1 and 20 mM f-MAS). Assay conditions for EcPDFꢀC18 were 50 mM KH2PO4, 12 mM NAD , 0.005 mg/ml enzyme and 2 mM f-MAS. The error
bars correspond to standard deviations. (D) Effect of actinonin on the proliferation of T. cruzi epimastigotes. The (Y) strain of the epimastigote form of T. cruzi was cultured in
◦
liver infusion tryptose (LIT) medium, supplemented with 10% newborn calf serum (Invitrogen) at 28 C with agitation. For determination of inhibitor sensitivity, the cultures
were initiated at 2 × 10 epimastigotes/ml and cell densities were measured with an electronic particle counter (model Z1 Beckmam CoulterTM). Time zero corresponds to
6
the moment of addition of the drug.
and inhibition profile exhibited by T. cruzi PDF and the observation
that PDF-1 is essential in procyclic forms of T. brucei [5], support
the study of trypanosomal peptide deformylase as a biochemical
target for antiprotozoan drug design.
[3] Serero A, Giglione C, Sardini A, Martinez-Sanz J, Meinnel T. An unusual pep-
tide deformylase features in the human mitochondrial N-terminal methionine
excision pathway. J Biol Chem 2003;278:52953–63.
[
[
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4] Bracchi-Ricard V, Nguyen KT, Zhou Y, Rajagopalan PT, Chakrabarti D, Pei D.
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Acknowledgments
Financial support for this research came from the Plan Nacional
(
SAF2010-20059), the RICET FIS Network (RD06/0021), the Junta
de Andalucía (BIO-199) and the Howard Hughes Medical Institute
grant number 5500062).
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Appendix A. Supplementary data
[
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2011;27(10):429–33.
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crystallization of functionally competent Escherichia coli peptide deformylase
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