Peptide deformylase is an essential metalloenzyme
required for the removal of the formyl group at the N terminus of
nascent polypeptide chains in eubacteria. The Escherichia
coli enzyme uses Fe2+ and nearly retains its activity
on substitution of the metal ion by Ni2+. We have solved
the structure of the Ni2+ enzyme at 1.9-Å resolution by
x-ray crystallography. Each of the three monomers in the asymmetric
unit contains one Ni2+ ion and, in close proximity, one
molecule of polyethylene glycol. Polyethylene glycol is shown to be a
competitive inhibitor with a KI value of 6 mM with respect to formylmethionine under conditions similar to those used for crystallization. We have also solved the
structure of the inhibitor-free enzyme at 2.5-Å resolution. The two
structures are identical within the estimated errors of the models. The
hydrogen bond network stabilizing the active site involves nearly all
conserved amino acid residues and well defined water molecules, one of
which ligates to the tetrahedrally coordinated Ni2+
ion.
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INTRODUCTION |
In eubacteria as well as in mitochondria and chloroplasts the
amino group of methionyl-tRNAfMet is
N-formylated by a formyltransferase during initiation of
protein synthesis (1). Consequently, all nascent polypeptides are
synthesized with N-formylmethionine at the N terminus.
During elongation of the polypeptide chain the formyl group is removed
hydrolytically by the enzyme peptide deformylase
(PDF,1 EC 3.5.1.27) (2, 3).
For Escherichia coli, deletion of the formyltransferase gene
leads to a strongly reduced cell growth rate (4), whereas deletion of
the PDF gene proves lethal (5). This formylation/deformylation cycle,
which appears to be a characteristic feature of eubacteria, does not
occur in the cytoplasm of eucaryotic cells. Therefore, PDF is an
attractive target for the design of new antibiotics.
PDF from E. coli, a monomeric protein of 168 residues,
shares the fingerprint motifs HEXXH (6), EGCLS, and
GXGXAAXQ (7) with PDF sequences of
other eubacteria, which suggests a common architecture of the catalytic
region in these proteins. PDF was reported to be a zinc enzyme (8) that
contains the motif HEXXH, known to be involved in zinc
binding in metalloproteases (9, 10). Meanwhile, it has been shown that
PDF utilizes Fe2+ as catalytic metal whereas the
Zn2+ form is nearly inactive (11, 12). Interestingly,
Fe2+ can be replaced by Ni2+ with a slight
reduction in PDF activity (11).
Recently, the structure of the core domain (residues 1-147) of PDF was
solved by NMR (8) and the structure of the full-length protein by x-ray
crystallography at 2.9-Å resolution (13). Both structures describe the
protein as isolated with a tightly bound zinc ion. We report the
structure of the catalytically active enzyme in the nickel-bound form
(PDF-Ni) at 2.5-Å resolution and at 1.9-Å resolution in complex with
a polyethylene glycol molecule (PDF-Ni/PEG), shown here to be a
competitive inhibitor of the enzyme.
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EXPERIMENTAL PROCEDURES |
PDF-Ni (specific activity, 900 units/mg; 0.6 Ni2+
ions per protein molecule) was isolated from overproducing E. coli cells and crystallized with 2 M
(NH4)2SO4 as precipitant in
the presence of 1% (w/v) PEG-1000 as
described.2 Crystals were
washed in 100 mM MOPS/NH3, 1% (w/v) PEG-1000,
2 M (NH4)2SO4 at pH
7.4. Three isomorphous derivatives were obtained by soaking crystals in
the above buffer with added heavy atom compounds. The mercury
derivative crystals were harvested after soaking 12 h in 0.1 mM ethylmercury phosphate (EMP), the platinum derivative
after soaking 24 h in 2 mM
K2PtCl4, and the double derivative after
soaking 24 h in 0.1 mM CH3HgCl and 2 mM K2PtCl4. PDF-Ni crystals without
PEG have been described.2
Diffraction data were collected at room temperature by the
rotation method and recorded by an electronic area detector (x-rays: CuK
, focused by Franks double-mirror optics; generator:
GX-18, Elliot/Enraf-Nonius, Delft, operated at 35 kV/50 mA; detector: X100, Siemens/Nicolet, Madison, WI; crystal to detector distance: 10 cm; rotation/image: 0.0417° or 0.0833°). Integrated intensities were extracted from the rotation images by the program XDS (15), which
includes routines for space group determination from the observed
diffraction pattern (16).
Inhibition of PDF by PEG-1000 was determined by the following
procedure. PDF activity was measured at 30 °C with formyl-Met (1-32
mM) at pH 7.2 (100 mM MOPS/NaOH, 2 M
Li2SO4, 1 mM TCEP) in a total
volume of 50 µl. The reaction was started by 420 ng of PDF-Ni (5 µl) and stopped after 5 min by 4% HClO4 (50 µl). The
amount of hydrolyzed substrate was determined according to the method
of Fields (17) using 2,4,6-trinitrobenzolsulfonic acid
(
420 = 22 mM
1
cm
1). For inhibition studies, up to 10 mM
PEG-1000, pretreated with TCEP (350 mM PEG/35
mM TCEP, pH 7.2) for 1 h, was included in the assay
mixture. Vmax and apparent KM
values were estimated from double-reciprocal plots (1/v
versus 1/[S]), and the KI value was
calculated from secondary replots of the slopes versus
PEG-1000 concentrations.
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RESULTS |
Structure Determination--
Crystallographic data used for
structure determination are summarized in Table
I. The structure of PDF-Ni/PEG crystals
was solved by the multiple isomorphous replacement method and
exploitation of the 3-fold redundancy of the electron density in the
asymmetric unit. The atomic model was obtained by several rounds of
model building (18) and correction followed by refinement (19) and map
calculation. Atomic coordinates for about 50% of the residues were
restrained to obey non-crystallographic symmetry. Unexpected density
near the metal ion occurred in all three independent monomers and
improved to a clear feature during the refinement process. It was then
assumed to be an ordered part of a PEG molecule, consistent with
subsequent biochemical studies showing that PEG is a competitive inhibitor of PDF. Only parts of the PEG-1000 molecules
(HO(C2H4O)nH,
= 22) are found in the map at a density above 5% of the map
maximum. The visible part is modeled with n = 10 for
the molecule length. The final model consists of 4122 (3 × 1346 + 84 alternate locations) non-hydrogen protein atoms, 3 Ni2+
ions, 2 SO42
ions, 3 PEG, and 205 water molecules. The estimated coordinate error is 0.21 Å, and r.m.s.
deviations from ideal geometry are 0.01 Å and 1.2° for bond length
and bond angles, respectively.
Structure determination of PDF-Ni crystals was based on the above
atomic model. The refined structure consists of 4038 (3 × 1346)
non-hydrogen protein atoms, 3 Ni2+ ions, 2 SO42
ions, and 100 water molecules.
The estimated coordinate error is 0.30 Å, and r.m.s. deviations from
ideal geometry are 0.015 Å and 1.4° for bond length and bond angles,
respectively. In both structures, coordinates for residues Arg-167 and
Ala-168 are ill defined, and Pro-9 is found in a
cis-conformation.
Overall Structure--
Basically confirming the results of Chan
et al. (13), PDF-Ni is an
+
protein consisting of a
five-stranded antiparallel
-sheet (
1,
2,
3,
6,
7), a two-stranded
-ribbon (
4,
5), three regular
-helices, and three short
310 helices (Figs. 1 and
2). It has been noticed (8) that this
overall arrangement of secondary and tertiary structure is quite
different from other metalloproteases such as thermolysin (20) or
stromelysin-1 (21), which also contain parallel
-strands.

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Fig. 1.
Stereo view of superimposed C- traces of
the three crystallographically independent copies of peptide
deformylase. The numbers refer to amino acid residues. The
transformations for optimal superposition were determined from
equivalent C- atoms using molecule A as a reference and applied to
the Ni2+ ion (marked as Ni) and the PEG molecule
as well.
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Fig. 2.
Schematic representation of secondary
structure as analyzed by the program DSSP (14). First and last
amino acid residues in the helices and sheet strands are
specified.
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The structures of the three molecules in the asymmetric unit are rather
similar except for the more flexible regions 62-68 and 164-168 (Fig.
1). Moreover, a comparison of the monomer structures with and without
bound PEG shows no significant differences. In fact, the r.m.s.
deviation between corresponding C
atoms is 0.25 Å (residues 66-68 and 161-168 are omitted from the comparison), which
is within the expected coordinate errors of the models. The monomer
structure is apparently rigid and structurally insensitive to the
binding of PEG and to different environments in the crystal.
Active Site--
Sequence alignment of deformylases from other
eubacteria reveals three conserved regions,
G43XGXAAXQ,
E88GCLS (7), and H132EXXH, which are believed
to form the catalytic site of the enzyme involved in metal and
substrate binding. Our electron density map at 1.9-Å resolution allows
an unambiguous assignment of all non-hydrogen atoms forming the active
site (Fig. 3). As reported for the zinc
structures (8, 13), the Ni2+ ion is found to be
tetrahedrally ligated (for review of metal liganding see Ref. 22) to
the N-
2 atoms of His-132 and His-136 in the HEXXH motif,
to the S-
atom of Cys-90, and to an oxygen atom of the group W1
modeled as a water molecule (Figs. 4 and 5). All four ligands are precisely
aligned by an intricate network of hydrogen bonds involving conserved
residues of the enzyme family. The side chain of His-132 forms a
hydrogen bond with the side chain of Glu-88, which itself is fixed by
Arg-102 and indirectly by Asp-135. His-136 is held in place by Leu-13
mediated by a water molecule W3. The fourth ligand W1 is fixed by
hydrogen bonds with the side chain oxygens of Glu-133 and a water
molecule W2. The side chain of Gln-50, although it comes close to W1,
is oriented in such a way that it cannot form a reasonable hydrogen
bond with W1. In fact, there is no accepting group for the second
proton of W1 in our structure, which leads us to speculate that it
could well be a hydroxyl anion instead of a water molecule. The side chain of the conserved Gln-50 forms hydrogen bonds donating protons to
water W2 and to the hydroxyl group of Ser-92. Interestingly, water W2,
the side chain amide of Gln-50, and the main chain amide of Leu-91 are
found in the arrangement required for the tetrahedral transition state,
if W2 were replaced by the carbonyl oxygen of the formyl group. The
amides of Gln-50 and Leu-91 could well serve as an anion trap to
compensate the negative charge developing at the formyl oxygen.

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Fig. 3.
Stereo pair of the final
A-weighted 2F0 - Fc map at
1.9-Å resolution covering the active site of peptide deformylase with
the bound Ni2+ ion and the ordered part of a PEG
molecule. Density is contoured at 12% of the map maximum.
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Fig. 4.
Stereo pair of the active site of peptide
deformylase showing all non-hydrogen atoms. Side chain atoms of
unlabeled amino acids have been omitted.
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Fig. 5.
Interaction scheme between the catalytic
Ni2+ ion, water molecules W1, W2, W3, and amino acid
residues in the active site of peptide deformylase. With the
exception of Leu-6, all residues shown in the figure are well
conserved. Bonds and bond angles between the Ni2+ ion and
its ligands are shown. Dashed lines indicate hydrogen bonds
with distances between donor and acceptor atom given in Å. The second
proton of W1 and the distance to the side chain of Gln-50 are in
brackets to indicate the absence of a hydrogen bond.
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The PEG molecule fits into a pocket formed by residues Gly-43,
Ile-44, Ile-86, Glu-88-Leu-91, side chain of Arg-97, Cys-129, His-132,
Glu-133, and the Ni2+ ion. The interactions are
predominantly hydrophobic except for the non-conserved Arg-97, which
forms a hydrogen bond with the PEG molecule. The close proximity of the
PEG molecule to the Ni2+ ion and to the conserved residues
suggests that it might overlap with the enzyme's substrate/product
binding site. For clarification, inhibition studies were carried out as
specified under "Experimental Procedures." For PEG-1000
concentrations up to 10 mM, identical kcat values of 4.7 ± 0.2 s
1
were found, whereas the apparent KM values increased linearly from 4.1 mM (without PEG-1000) to 12.5 mM
(with 10 mM PEG-1000), yielding a calculated
KI value of 6 mM. The observed pattern shows that inhibition by PEG-1000 is competitive with respect
to formyl-Met.
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DISCUSSION |
Since its discovery about 30 years ago PDF is known to rapidly
lose its activity, which makes its purification difficult (2, 23). Even
in recent work with the recombinant enzyme, specific activities have
been reported that are off by orders of magnitude (6, 24). Meanwhile,
it has been shown that PDF is a Fe2+ enzyme (11, 12)
instead of a zinc metalloprotease as previously believed (8). Whereas
the Zn2+ form is nearly inactive (11, 12), we routinely
observe 1200 units/mg specific activity for PDF-Fe2+ and
900 units/mg for PDF-Ni2+ (11).
The three structures now known appear to be generally similar although
significant differences remain that could well be of importance for
understanding the enzyme mechanism. The two zinc structures (8, 13)
have been compared previously (13), and the following discussion
focuses on the differences between the zinc and nickel structures
determined by x-ray analysis. These differences include Pro-9, which is
in a cis-conformation as well as two of the three
310 helices, Glu-11-Arg-14 and Phe-142-Tyr-145, which
have been modeled as
-helices
1 and
4
in the zinc structure (13). Other structural differences are found in
the region Glu-87-Leu-99, which includes the
-ribbon. In the active
site region the fourth metal ligand corresponding to W1 seems to be
displaced, and the important water molecule W2 appears to be absent
from the zinc structure. Also, the side chain of Gln-50 is more
involved in stabilizing W2 rather than W1, and the amide hydrogens of
Leu-91 and Ala-47 are found at distances 5 and 4.6 Å from W1,
respectively, which is too far for a hydrogen bond as reported
previously (13).
We thank Karin Fritz-Wolf, Arnon Lavie, and
Klaus Scheffzek for critical discussions and help at various stages of
the project, Hans Wagner for excellent maintenance of the x-ray
facilities at the MPI Heidelberg, and Ken Holmes and Joachim Knappe for
continuous support.