Crystal structure of a complex between the actinomadura R39 dd -peptidase and a peptidoglycan-mimetic boronate inhibitor: Interpretation of a transition state analogue in terms of catalytic mechanism

Article abstract of DOI:10.1021/bi100757c

The Actinomadura R39 dd-peptidase is a bacterial low molecular weight class C penicillin-binding protein. It has previously been shown to catalyze hydrolysis and aminolysis of small d-alanyl-d-alanine terminating peptides, especially those with a side cha

Full text of DOI:10.1021/bi100757c

Biochemistry 2010, 49, 6411–6419 6411
DOI: 10.1021/bi100757c
Crystal Structure of a Complex between the Actinomadura R39 DD-Peptidase and a
Peptidoglycan-mimetic Boronate Inhibitor: Interpretation of a Transition State
Analogue in Terms of Catalytic Mechanism†
§
§
§
,‡
Liudmila Dzhekieva,‡, Mathieu Rocaboy,§, Frederic Kerff, Paulette Charlier, Eric Sauvage, and R. F. Pratt*
ꢀ ꢀ
‡
§
Department of Chemistry, Wesleyan University, Lawn Avenue, Middletown, Connecticut 06459, and Centre d’Ingeniere des
ꢀ
ꢀ
Proteines, Universite de Liege, B-4000 Sart Tilman, Liege, Belgium. Contributed equally to this work.
ꢁ
ꢁ
Received May 12, 2010; Revised Manuscript Received June 14, 2010
ABSTRACT: The Actinomadura R39 DD-peptidase is a bacterial low molecular weight class C penicillin-binding
protein. It has previously been shown to catalyze hydrolysis and aminolysis of small
terminating peptides, especially those with a side chain that mimics the amino terminus of the stem peptide
precursor to the bacterial cell wall. This paper describes the synthesis of ( -R-aminopimelylamino)- -1-
ethylboronic acid, designed to be a peptidoglycan-mimetic transition state analogue inhibitor of the R39 DD
D
-alanyl- -alanine
D
D
D
-
peptidase. The boronate was found to be a potent inhibitor of the peptidase with a Ki value of 32 ( 6 nM. Since
it binds some 30 times more strongly than the analogous peptide substrate, the boronate may well be a
transition state analogue. A crystal structure of the inhibitory complex shows the boronate covalently bound
to the nucleophilic active site Ser 49. The aminopimelyl side chain is bound into the site previously identified as
specific for this moiety. One boronate oxygen is held in the oxyanion hole; the other, occupying the leaving
group site of acylation or the nucleophile site of deacylation, appears to be hydrogen-bonded to the hydroxyl
group of Ser 298. The Ser 49 oxygen appears to be hydrogen bonded to Lys 52. If it is assumed that this
structure does resemble a high-energy tetrahedral intermediate in catalysis, it seems likely that Ser 298
participates as part of a proton transfer chain initiated by Lys 52 or Lys 410 as the primary proton donor/
acceptor. The structure, therefore, supports a particular class of mechanism that employs this proton transfer
device.
The bacterial DD-peptidases are a component of the meta-
bolic pathway leading to the bacterial cell wall. There they
catalyze the final steps of cell wall synthesis, incorporation of
the stem peptide monomer into polymeric peptidoglycan, and
sculpting of the final structure (1). From a human clinical
standpoint, these enzymes are important as a target for anti-
biotics, i.e., antibacterial drugs. They are inhibited, for exam-
ple, by the important β-lactam class of antibiotics and remain
as a well-validated option for further antibiotic design (2).
Scheme 1 shows the reactions catalyzed by these enzymes and
the reaction leading to their inhibition by β-lactams. The
reactions shown include transpeptidation, which extends the
peptidoglycan polymer, and carboxypeptidation and endo-
peptidation, both of which lead to reduced cross-linking of the
polymer.
low molecular weight (LMW)1 and high molecular weight
(HMW) classes (5). The latter is further subdivided into two
major groups, A and B, and the former into three groups, A, B,
and C. This classification was initially based on molecular
weight and amino acid sequence. Over the last 15 years, crystal
structures of members of all of these classes have been
obtained (6, 7). It is now clear that the classification relates
directly to protein structure and, most likely therefore, to some
degree at least, to function in vivo.
Although it seems clear that these enzymes must catalyze the
reactions of Scheme 1 in vivo, their activity as DD-peptidases
in vitro is less well characterized. To date, no strong affinity has been
demonstrated between any HMW or LMWA enzymes and any
specific elements of peptidoglycan structure, i.e., small peptides
of structure based on that of the relevant peptidoglycan (8, 9).
Such specificity, however, has been established for certain
members of the LMWB and LMWC classes. For example, it is
evident from peptide specificity studies and subsequent crystal
structures that the Streptomyces R61 DD-peptidase (LMWB) has
strong affinity for motif 1 (10-12) and that the Actinomadura
Largely because of their relevance to the β-lactam antibio-
tics, these enzymes have been closely studied for many
years (3-5). They have been classified into two groups, the
†This research was supported by National Institutes of Health Grant
AI-17986 (R.F.P.) and in part by the Belgian Program on Interuniver-
sity Poles of Attraction initiated by the Belgian State, Prime Minister’s
Office, Science Policy programming (IAP no. P6/19), the Fonds de la
Recherche Scientifique (IISN4.4505.00, IISN4.4509.09, FRFC2.4.508.01.
1Abbreviations: DIBAL, diisobutylaluminum hydride; DIPEA, dii-
sopropylethylamine; DMF, dimethylformamide; ESMS, electrospray
ionization mass spectroscopy; HATU, O-(7-azabenzotriazol-1-yl)-N,N,
N0,N0-tetramethyluronium hexafluorophosphate; HMW, high molecu-
lar weight; LMW, low molecular weight; MES, 4-morpholinoethane-
sulfonic acid; MOPS, 3-morpholinopropanesulfonic acid; NMR,
nuclear magnetic resonance; PBP, penicillin-binding protein; THF,
tetrahydofuran.
ꢁ
F, FRFC9.4.538.03.F, FRFC2.4.524.03), and the University of Liege
ꢀ
ꢀ
ꢀ
(Fonds speciaux, Credit classique, 2009). F.K. is Charge de Recherche of
the Fonds de la Recherche Scientifique (F.R.S.-FNRS, Brussels, Belgium).
*Corresponding author. Telephone: 860-685-2629. E-mail: rpratt@
wesleyan.edu. Fax: 860-685-2211.
r
2010 American Chemical Society
Published on Web 07/07/2010
pubs.acs.org/Biochemistry

6412 Biochemistry, Vol. 49, No. 30, 2010
Dzhekieva et al.
Scheme 1
R39 DD-peptidase (LMWC) strongly interacts with motif 2 (13).
These motifs are direct mimics of the stem peptides from the re-
spective Streptomyces and Actinomadura organisms.
recently, a variety of aryl boronates were found to inhibit the
LMWC DD-peptidase of Actinomadura R39 (39). A crystal
structure of a complex of the peptide boronate was interpreted
in terms of a transition state analogue (18), but this interpre-
tation is compromised somewhat by the nonspecific nature of
the peptide moiety (the molecule is not a close peptidoglycan-
mimetic and a large part of its peptide structure is not observed in
the crystal structure) and the (resulting?) lack of contact of Ser
110 (equivalent to Ser 298 of the R39 enzyme) with the boronate
moiety. In this paper, we describe 3, a specific peptidoglycan-
mimetic boronate inhibitor of the Actinomadura R39 DD-pepti-
dase, with a thermodynamic inhibition constant of 32 nM. We
suggest that this inhibitor generates a good transition state
analogue for this enzyme and interpret it in terms of mechanism
accordingly.
In each of these enzymes, therefore, it seems very likely that the
dominant substrates in vivo also contain these structural ele-
ments; i.e., the substrates have a non-cross-linked N-terminus.
These could be either monomers (carboxypeptidase activity) or
oligomers/polymers (endopeptidase activity). The low micromo-
lar Km values of these specific substrates (10-13) suggest that
structures such as 1 and 2 would be tightly trapped and processed
by these enzymes in vivo.
The detailed mechanism of action of these enzymes has also
been discussed at some length (6, 14-24). Much of the discussion
has been based on the more developed state of β-lactamase mec-
hanisms. This is rational since the active site structure of DD-pepti-
dases is closely analogous to that of the serine β-lactamases because
the latter enzymes are almost certainly evolutionary descendants of
the former (25, 26). The active sites of HMWA, HMWB, LMWA,
and LMWC DD-peptidases are closely analogous to that of a class
A β-lactamase, although Glu 166, recruited to catalyze β-lactam-
derived acyl-enzyme hydrolysis in the β-lactamase, is absent in
the DD-peptidases. Since Glu 166 probably also participates in
acylation (27-31), it is evident that both acylation and deacyla-
tion may be different in DD-peptidases than in β-lactamases.
One source of mechanistic information, and one that was rather
extensively employed with β-lactamases, is that of the structure of
transition state analogues. Typically, these are structures of the
β-lactamase complexed to inhibitors that form anionic tetrahedral
structures covalently bound to the nucleophilic active site serine
residue and which mimic the high-energy tetrahedral intermedi-
ates/transition states of acyl transfer turnover. Important exam-
ples are those generated from boronates (32-34) and phospho-
nates (35-37). To date, phosphonates have not yielded any
potent DD-peptidase inhibitors, although a weakly inhibitory
phosphonate was employed to obtain a transition state analogue
structure with the LMWB Streptomyces R61 DD-peptidase (38).
Boronates, however, have shown promise of stronger inhibition.
A peptide boronate has been shown to inhibit Escherichia coli
PBP5 with a dissociation constant of 13 μM (18), and more
MATERIALS AND METHODS
Synthesis. The synthesis of 3 is shown in outline in Scheme 2;
the details are provided below.
N,N-Dibenzyl-D-glutamic Acid Dibenzyl Ester (4) (40).
To a solution of D-glutamic acid (5.0 g, 34 mmol, 1 equiv) in water
(30 mL), together with potassium carbonate (18.8 g, 136 mmol,
4 equiv) and sodium hydroxide (2.75 g, 68 mmol, 2 equiv), heated
under reflux, was added benzyl bromide (25 mL, 136 mmol)
dropwise with stirring. The reaction was monitored by TLC
(hexane/ethyl acetate), and upon completion, the reaction mix-
ture was cooled to room temperature. The mixture was extracted
with diethyl ether (3 ꢀ 100 mL), and the combined organic layers
were washed with water (50 mL) and brine (50 mL) and dried
over magnesium sulfate. The ether was evaporated leaving a yel-
lowish oil (13 g). It was purified by chromatography on silica gel
(hexane:ethyl acetate, 7:1) yielding the product 4 as a colorless
oil (10.5 g, 60% yield). 1H NMR (CDCl3, 300 MHz) δ 2.1 (q, J =
7.5 Hz, 2H), 2.43, 2.56 (d quint, J = 9, 9 Hz, 2H,), 3.47 (t, J =
7.5 Hz, 1H), 3.55, 3.93 (AB q, J = 13.5, 4H), 5.26 (AB q, J =
3 Hz, 2H), 5.20, 5.32 (AB q, J = 12 Hz, 2H), 7.25-7.46 (m, 20H).
D-4-(N,N-Dibenzylamino)-4-(benzyloxycarbonyl)butanal
(5). To a stirred solution of 4 (3 g, 6 mmol, 1 equiv) in 30 mL of
anhydrous diethyl ether under nitrogen atmosphere was added

Article
Biochemistry, Vol. 49, No. 30, 2010 6413
Scheme 2
DIBAL (1 M in hexane, 6.5 mL, 1.1 equiv) slowly at -78 ꢀC. The
reaction was stirred for 15 min, and then water (0.3 mL) was
added. The reaction was allowed to warm to room temperature
and stirred additionally for 30 min after which it was dried over
magnesium sulfate, filtered, and evaporated leaving a colorless
gum, 5 (2.2 g), which was subjected to the next step without puri-
fication.1H NMR (CDCl3, 300 MHz) δ 1.21 (t, J = 7.8 Hz, 2H),
2.3-2.5 (m, 2H), 3.35 (t, J = 7.2 Hz, 1H), 3.52, 3.86 (AB q, J =
13.5 Hz, 4H), 5.20, 5.26 (AB q, J = 10.8 Hz, 2H), 7.2-7.5 (m,
15H), 9.58 (s, 1H).
(m, 1H), 3.34 (t, J = 7.5 Hz, 1H), 3.53, 3.87 (AB q, J = 13.5 Hz,
4H), 5.19, 5.25 (AB q, J = 10.8 Hz, 2H), 5.59 (d, J = 16 Hz, 1H),
6.7 (quint, J = 7.2 Hz, 1H), 7.2-7.4 (m, 15H). ES(þ)MS m/z
444.4 (M þ 1).
Methylboronic Acid Pinanediol Ester (8) (41). A mix-
ture of methylboronic acid (1.0 g, 16.7 mmol, 1 equiv) and (-)-
pinanediol (2.9 g, 16.7 mmol, 1 equiv) in diethyl ether (18 mL)
was allowed to stir at room temperature over sodium sulfate for
1 h. This mixture was partitioned between a 10% aqueous solu-
tion of sodium carbonate (25 mL) and dichloromethane (25 mL).
The organic solution was dried over sodium sulfate, evaporated,
and distilled under vacuum (bp 60 ꢀC, 1 Torr), yielding the pro-
D-6-(N,N-Dibenzylamino)-6-(benzyloxycarbonyl)-trans-
hex-2-enoic Acid tert-Butyl Ester (6). To a stirred solution of
5 (2.0 g, 5 mmol, 1 equiv) in dry THF (20 mL) was added (tert-
butoxycarbonylmethylene)triphenylphosphorane (2.6 g, 6.8 mmol,
1.2 equiv) at room temperature. The reaction was stirred for
1.5 h after which solvent was evaporated. The crude product
was purified by chromatography on silica gel (hexane:ethyl
acetate, 3:1), yielding the product 6 as a colorless oil (1.5 g,
1
duct 8 as a colorless oil (2.6 g, 80% yield). H NMR (CDCl3,
300 MHz) δ 0.26 (s, 3H), 0.82 (s, 3H), 1.10 (d, J = 10.9 Hz, 1H),
1.26 (s, 3H), 1.84-1.79 (m, 1H), 1.79 (s, 3H), 1.90-1.87 (m, 1H),
2.01 (t, J = 7 Hz, 1H), 2.29 (m, 1H), 4.23 (dd, J = 8.7, 1.9 Hz,
1H).
(R)-(Chloromethyl)boronic Acid Pinanediol Ester (9).
This compound was prepared as described in ref 42. The crude
material was purified by chromatography on silica gel (hexane:
ethyl acetate, 24:1) and the product 9 obtained as a colorless oil
(2.0 g, 80% yield). 1H NMR (CDCl3, 300 MHz) δ 0.85 (s, 3H),
1.17 (d, J = 10.9 Hz, 1H), 1.3 (s, 3H), 1.42 (s, 3H), 1.58 (d, J =
7.2 Hz, 3H), 1.85-2.43 (m, 5H), 3.6 (q, J = 7.5 Hz, 1H), 4.37 (dd,
J = 1.9, 8.7 Hz, 1H).
D-1-(Aminoethyl)boronic Acid Pinanediol Ester Hydro-
chloride (10). To a stirred solution of (R)-(chloromethyl)boro-
nic acid pinanediol ester (2.0 g, 8.2 mmol, 1 equiv), in dry THF
(20 mL) under an argon atmosphere at -100 ꢀC, was added a 1 M
solution in THF of lithium hexamethyldisilazane (9.3 mL, 1.13
equiv) dropwise down the cold wall. The reaction mixture was
1
60% yield). H NMR (CDCl3, 300 MHz) δ 1.47 (s, 9H), 1.86
(m, 2H), 2.07 (m, 1H), 2.32 (m, 1H), 3.34 (t, J = 7.5 Hz, 1H),
3.53, 3.87 (AB q, J = 13.5 Hz, 4H), 5.19, 5.25 (AB q, J = 10.8
Hz, 2H), 5.59 (d, J = 16 Hz, 1H), 6.70 (quint, J = 7.2 Hz, 1H),
7.2-7.4 (m, 15H).
D-6-(N,N-Dibenzylamino)-6-(benzyloxycarbonyl)-trans-
hex-2-enoic Acid (7). The ester 6 (0.5 g, 1 mmol, 1 equiv) was
dissolved in 5 mL of dichloromethane. To this solution, stirred in
an ice bath, was added trifluoroacetic acid (6 mL) slowly. The
reaction mixture was stirred for 1 h at room temperature after
which solvent was evaporated. After the residue was dried under
vacuum, the gummy product, 7, was obtained (0.4 g, 90% yield).
1H NMR (CDCl3, 300 MHz) δ 1.86 (m, 2H), 2.07 (m, 1H), 2.32

6414 Biochemistry, Vol. 49, No. 30, 2010
Dzhekieva et al.
allowed to warm to room temperature and stand for 24 h. The
next reaction was carried out in situ. The reaction solution was
cooled to -78 ꢀC, and 2 M hydrochloric acid in diethyl ether
(12.3 mL, 3 equiv) was added dropwise down cold wall. The re-
action mixture was allowed to warm to room temperature and
then concentrated under reduced pressure to give a brown solid.
This material was washed with ethyl acetate and dissolved in ace-
tonitrile and the solution filtered. The filtrate was dried under
reduced pressure to give a solid material, which was recrystalli-
zed from acetonitrile, affording the product 10 as a white solid
(0.85 g, 40% yield). 1H NMR (CDCl3, 300 MHz) δ 0.83 (s, 3H),
1.15 (d, J= 10.9 Hz, 1H), 1.3 (s, 3H), 1.4 (s, 3H), 1.52 (d, J=
7.2 Hz, 3H), 1.83-2.43 (m, 5H), 3.07 (br s, 1H), 4.39 (d, J = 8.5 Hz,
1H), 8.23 (br s, 3H).
D-1-[6-(N,N-Dibenzylamino)-6-(benzyloxycarbonyl)-trans-
hex-2-enoylamino]ethylboronic Acid Pinanediol Ester (11).
To a stirred solution of the acid 6 (300 mg, 0. 68 mmol, 1 equiv)
were added HATU (260 mg, 0.68 mmol, 1 equiv), diisopropylet-
hylamine (230 mL, 1.36 mmol, 2 equiv) in dry DMF (10 mL), and
the boronate ester 10 (180 mg, 0.75 mmol, 1.1 equiv) at 0 ꢀC. The
reaction mixture was then allowed to warm to room temperature
and stirred overnight. Subsequently, it was concentrated under
reduced pressure and the residue dissolved in ethyl acetate (20 mL).
The ethyl acetate solution was washed with 0.1 N hydrochloric acid,
saturated sodium bicarbonate, and brine and dried over magnesium
sulfate. After evaporation of the organic solvent, the residual
gum was further purified by chromatography on silica gel (3%
MeOH/97% CHCl3), yielding the product 11 as a colorless foam
(300 mg, 70% yield). 1H NMR (CDCl3, 300 MHz) δ 0.86 (s, 3H),
1.2 (d, J = 7.5 Hz, 3H), 1.28 (s, 3H), 1.44 (s, 3H), 1.75-2.4 (m,
13H), 2.93 (m, 1H), 3.35 (m, 1H), 3.54, 3.92 (AB q, J = 13.5 Hz,
4H), 4.22 (d, J = 9 Hz, 1H), 5.22 (AB q, J = 20, 10.8 Hz, 2H),
5.35 (d, J = 16 Hz, 1H), 6.74 (quint, J = 7.2 Hz, 1H), 7.24 (s,
5H), 7.26 (s, 5H), 7.28 (s, 5H).
Scheme 3
enzyme concentration of 94.5 nM. Under these conditions, the
Km value of the substrate was 38.4 μM (S. A. Adediran and R. F.
Pratt, unpublished). Measurements of initial velocity vs boronate
concentration were fitted to Scheme 3 by the Dynafit pro-
gram (44) to obtain the Ki value directly.
R39 DD-Peptidase Crystallography. Protein Purifica-
tion and Crystallization. The R39 DD-peptidase was expressed
and purified as described previously (45). Crystals were grown at
20 ꢀC by hanging drop vapor diffusion. Crystals were obtained by
mixing 4 μL of a 25 mg mL-1 protein solution (also containing
5 mM MgCl2 and 20 mM Tris, pH 8), 2 μL of well solution (2.0 M
ammonium sulfate and 0.1 M MES, pH 6), and 0.5 μL of 0.1 M
CoCl2 solution. Crystals were soaked in 9 μL of a solution
containing 3.0 M ammonium sulfate and 0.1 M MES, pH 6, and
0.1 μL of 3 (0.1 M).
Data Collection, Structure Determination, and Refine-
ment. Data were collected at 100 K on an ADSC Q315r CCD
detector at a wavelength of 0.9797 A on beamline BM30A at the
European Synchrotron Radiation Facility (ESRF, Grenoble,
France). X-ray diffraction experiments were carried out under
cryogenic conditions (100 K) after transferring the crystals into
45% glycerol and 1.8 M ammonium sulfate. Intensities were
indexed and integrated using Mosflm (46). Data were scaled with
SCALA of the CCP4 program suite (47). Refinement was carried
out using REFMAC5 (48), TLS (49), and Coot (50). The
structure of the R39 DD-peptidase bound to 3 was refined to
2.4 A with Rcryst and Rfree values of 20.0% and 26.2%, respec-
tively. The ligand occupancy was refined as 1.0. Data statistics
and refinement are summarized in Table 1.
(D-R-Aminopimelylamino)-D-1-ethylboronic Acid Pina-
nediol Ester (12). The boronate ester 11 (200 mg, 0.30 mmol,
1 equiv) was dissolved in 10 mL of methanol, and 10% Pd on
activated carbon (20 mg) was added. The hydrogenation reaction
was carried out at 40 psi and at room temperature overnight. Pd/
C was then removed by filtration, and methanol was evaporated,
leaving a sticky gum. The residue was dissolved in anhydrous eth-
anol, and a white solid was precipitated by THF. This material,
the product 12, was washed with THF and hexane and dried under
high vacuum (90 mg, 80% yield). 1H NMR (CD3OD, 300 MHz)
δ 0.88 (s, 3H), 1.11 (d, J = 7.2 Hz, 3H), 1.3 (s, 3H), 1.35 (s, 3H),
1.59-2.43 (m, 14H), 2.58 (q, J = 7 Hz, 1H), 3. 74 (t, J = 6.5 Hz,
1H), 4.14 (d, J = 8.5 Hz, 1H). High-resolution ES(þ)MS m/z
381.2557 (M þ 1), calcd for C19H34N2O5B 381.2561.
RESULTS AND DISCUSSION
Synthesis of the boronate inhibitor, 3, was achieved according
to Scheme 2. This molecule incorporates the peptidoglycan-
mimetic -R-aminopimelyl side chain, a specific binding motif
D
for this enzyme (13, 51), and a boronate reaction center. It was
found to be a very powerful inhibitor of the R39 DD-peptidase,
with an inhibition constant (assumed competitive in view of the
structure of the inhibitor and supported subsequently by the
structure of the complex, described below) of 32 ( 6 nM. The
inhibition appeared to be fast and reversible under manual
mixing conditions since no time-dependent phenomena were
observed. The Km value of 16 as a substrate is 1.3 μM (51). Since
deacylation is rate-determining at saturation (S. A. Adediran and
R. F. Pratt, unpublished), this Km value probably encompasses
formation of the covalent acyl-enzyme intermediate, and thus the
noncovalent dissociation constant must be greater than 1 μM.
The considerably tighter binding of 3 suggests that it may form a
specific transition state analogue structure. This conclusion is
supported by the crystal structure itself, discussed below. The
boronate complexes of serine proteases (52) and serine β-
lactamases (32-34) have generally been interpreted in this
fashion.
(D-R-Aminopimelylamino)-D-1-ethylboronic Acid (3). The
boronate ester 12 (20 mg, 0.05 mmol) was dissolved in water
(10 mL) and extracted five times with dichloromethane (2 mL)
over 24 h. The aqueous layer was freeze-dried, and the product 3
1
was obtained as a colorless glassy solid (7 mg, 57% yield). H
NMR (D2O, 300 MHz) δ 0.91 (d, J = 7 Hz, 3H), 1.15-1.35 (m,
2H), 1.49-1.62 (m, 2H), 1.67-1.79 (m, 2H), 2.27 (t, J = 7.2 Hz,
2H), 2.47 (q, J = 8.1 Hz, 1H), 3.6 (t, J = 7.2 Hz, 1H).
Kinetics Studies. An equilibrium constant for inhibition of
the Actinomadura R39 DD-peptidase by the boronate 3 (0.05-
1.0 μM) was obtained from steady-state competition experiments
where N-phenylacetylglycyl-D-thiolactate (43) was employed as a
chromophoric (245 nm) substrate (0.5 mM). The reaction con-
ditions were 20 mM MOPS buffer, pH 7.50, 25 ꢀC, and an

Article
Biochemistry, Vol. 49, No. 30, 2010 6415
410 Nζ (3.1 A), as is Lys 52 Nζ to Ser 49 Oβ (3.1 A). A re-
presentation of this situation is shown as 13.
Table 1: Data Collection and Refinement Statistics
data collection
space group
P21
cell dimensions (A, deg)
a = 103.3, b = 91.6,
c = 106.9, β = 94.37
33.1-2.4 (2.53-2.4)
77588
resolution range (A)a
no. of unique reflections
R
merge (%)a,b
9.5 (47.5)
redundancy a
3.6 (3.6)
completeness (%)a
ÆIæ/ÆσIæa
99.7 (99.4)
9.7 (2.6)
refinement
resolution range
32.4-2.4
14096
303
The most likely incorporation of hydrogens into the putative
hydrogen bonds of 13 yields 14. If structure 14 is a direct analogue
of a tetrahedral intermediate generated during the acyl transfer
reaction of normal catalysis, then that intermediate would have
structure 15. In structures 13-15 and generally below, Ser 1 cor-
responds to the nucleophilic active site serine (Ser 49 in the R39
DD-peptidase), Lys 1, the lysine of the KT(S)G motif (Lys 410 in
R39), Ser 2, the serine of the SXN motif (Ser 298), and Lys 2, the
lysine of the SXXK motif (Lys 52). Although boronates (and
phosphonates) have been interpreted in terms of the tetrahedral
intermediate/transition states of both enzyme acylation and
deacylation steps (18, 32-38, 57), because of the two hydroxyl
groups on boron, boronates appear to most directly represent de-
acylation intermediates, generated by enzyme-catalyzed attack of
water on the acyl-enzyme.
The presence of a sulfate ion, from the crystallization medium,
in the crystal structure directly adjacent to the boronate moiety of
the inhibitor is, however, interesting, in particular since one of its
oxygen atoms is apparently hydrogen-bonded to the Thr 411
hydroxyl group. A sulfate at this position was also observed in the
apoenzyme structure but was displaced by the β-lactam nitroce-
fin (53). This position approximates to the site where the carboxy-
no. of non-hydrogen protein atoms
no. of water molecules
Rcryst (%)
20.0
Rfree (%)
26.2
rms deviations from ideal stereochemistry
bond lengths (A)
0.009
1.24
26.5
28.1
bond angles (deg)
mean B factor (all atoms) (A2)
mean B factor (ligand) (A2)
Ramachandran plot
most favored region (%)
additionally allowed regions (%)
generously allowed regions (%)
disallowed regions (%)
rmsd of CR atom with native structure (A)
PDB code
91.7
7.7
0.5
0.1
0.4c
2XDM
aStatistics for the highest resolution shell are given in parentheses.
P
P
bRmerge
=
|Ii - Im|/ Ii, where Ii is the intensity of the measured reflection
and Im is the mean intensity of all symmetry-related reflections. Numbers
within parentheses are for the outer resolution shell. cMonomer B.
The overall fold of the R39 DD-peptidase complex with 3 is
similar to that of the native enzyme structure (53) (rms deviation
0.4 A). The asymmetric unit of the R39/3 complex contains four
protein molecules. The active sites of monomers B and C are each
occupied by a molecule of 3, whereas the active sites of monomers
A and D are partly blocked by a symmetric molecule and are
observed free of ligand. The rms difference between monomers B
and C is 0.4 A. The position of 3 in the active site of monomers B
and C is similar, and the following description refers to monomer
B. Electron density around the tetrahedral boronate moiety is
well-defined.
late of the terminal D-alanine of a substrate might bind, probably
also hydrogen-bonded to Thr 411 (11-13, 38, 53). Figure 2 shows
a model of a tetrahedral intermediate derived from the excellent
substrate 16 and directly built onto the boronate of the crystal
structure. The substrate carboxylate can indeed occupy the same
general space as the sulfate ion. In this way, to some degree at
least, the boronate structure may also be seen as an acylation
tetrahedral intermediate analogue.
The structure shows the boronate strongly bound to the
enzyme active site (Figure 1). The aminopimelyl side chain is snu-
gly accommodated in the specific binding pocket identified in the
previous peptide and β-lactam structures (13). In particular, the
amido group is held between its hydrogen bond partners, Asn 300
(NH2) and Thr 413 (CdO), the tetramethylene side chain abuts
the hydrophobic side chains of Met 414 and Tyr 147, and the
polar (ionic) carboxylate and ammonium termini are hydrogen
bonded to Ser 415, Arg 351, and Asp 412. These interactions
obviously define the specificity of the enzyme for motif 2.
The deacylation tetrahedral intermediate 15 might then be
generated by mechanism a, b, or c (Scheme 4) and break down to
products by either d, e, or f. In mechanism a, nucleophilic attack
of water on the acyl-enzyme is assisted by Lys 2 acting as a gen-
eral base although proton transfer is indirect via Ser 2. In the
alternative mechanism b, water attack is facilitated by Lys 1 as a
general base, again indirectly via Ser 2. Direct action of Lys 2 as a
general base is the centerpiece of mechanism c, where Ser 2 and
Lys 1 appear to have only spectator or electrostatic roles.
Breakdown of the tetrahedral intermediate also has three
alternative paths, d, e, and f, which are symmetrical analogues
of a, b, and c, respectively. In d, the general acid catalyst facilita-
ting departure of Ser 1 is Lys 2, whereas in e, it is the protonated
Closer to the reaction center, the
into a hydrophobic pocket in the surface (surrounded by Gly 148,
Leu 349, and Met 414), as now seems likely to be typical of DD
D-methyl group is directed
-
peptidases (26, 54-56); a larger side chain would not fit into this
pocket. The boron is found in a tetrahedral, presumably anionic,
configuration, covalently bound to the active site Ser 49 hydroxyl
oxygen atom (bond length 1.44 A). One boronate oxygen is
firmly (2.9, 3.0 A) hydrogen-bonded into the oxyanion hole (Ser
49 NH, Thr 413 NH) while the other is closely attended (2.7 A)
by the hydroxyl group of Ser 298. The latter is also close to Lys

6416 Biochemistry, Vol. 49, No. 30, 2010
Dzhekieva et al.
F
IGURE 1: Crystal structure of the R39 DD-peptidase in complex with the specific boronate 3. In this stereoview, the electron density is a |Fo| - |Fc|
difference map calculated from the final coordinates of the model refined in the absence of ligand. The resulting positive density is shown with
brown hatching and is contoured at 2.5σ. The protein backbone and secondary structure are in green, and the boronate is in yellow. Heteroatoms
are red (oxygen), blue (nitrogen), orange (sulfur), and pink (boron). Also shown is a sulfate of crystallization bound at the active site. This figure
was generated using PYMOL (www.pymol.sourceforge.net).
F
IGURE 2: A model of the tetrahedral intermediate generated on reaction of the enzyme with the substrate 16. The sulfate of crystallization is also
shown. The model was constructed by replacement of the appropriate boronate oxygen in the structure of Figure 1 with a D-alanine leaving group
(salmon) and shows the overlap between the leaving group carboxylate and the sulfate anion. The other colors are as for Figure 1. This figure was
generated using PYMOL (www.pymol.sourceforge.net).
Lys 1, both acting by way of Ser 2. In mechanism f, Lys 2 acts
alone as a general acid. With the assumption of mechanistic sym-
metry, the simplest possibility, these mechanisms can then be
extrapolated to analogous mechanisms of acylation (Scheme 5,
of a (a0) and e (e0) has also been suggested by Nicola et al. (18) and
Diaz et al. (21). To some extent at least, the distinction between
mechanisms a (a0) and b (b0) centers around the relative pKa
values of Lys 1 and Lys 2 and thus relates to the pH-rate profiles
of various DD-peptidases. This issue has been much discussed
(see ref 8, for example, for a review of the situation). In the
present authors’ opinion, the currently available evidence prob-
ably favors a lower pKa for Lys 2, but the issue is still open,
particularly with respect to the lysine pKas in the various inter-
mediate complexes.
where L is the leaving group,
transpeptidase reaction).
D-Ala, in a carboxypeptidase or
Of these mechanisms, almost all have been previously propo-
sed at one time or another for one reason or another. In partic-
ular, the combination of a0 and d0 in acylation, and thus a and d in
deacylation, has been seriously discussed (15). The combination

Article
Biochemistry, Vol. 49, No. 30, 2010 6417
Scheme 4
Scheme 5
One mechanism, specific to acylation, and involving the
This employs Lys 2 as the sole general acid/base catalyst and is
supported by computational results from Zhang et al. (22). The
downside of this proposal has always been the Ser 2, Lys 1 com-
bination, which in this mechanism appears to play no obvious
role. This dyad has previously been observed in many crystal
structures of covalent DD-peptidase/β-lactam complexes, in close
association with the ligand. Some examples are found in refs
(58-61). In the current structure, we find Ser 2 apparently tightly
hydrogen-bonded to the boronate “leaving group” oxygen. If the
boronate is indeed an analogue of a high-energy intermediate, it
seems unlikely that Ser 2 is not part of the mechanism. An issue
that clouds the computational study referred to above (22) is that
the system studied was E. coli PBP5. This DD-peptidase is unusual
substrate carboxyl group as a member of the proton donation
path to the leaving group, has also been considered (20, 21).
Apart from the clear, although not decisive, defect that this
mechanism could not apply to deacylation, is the observation that
molecules such as 17, where the carboxylate is very differently
placed with respect to the scissile bond, can be excellent substrates
of the R39 DD-peptidase (54). This observation suggests that such
participation by the substrate carboxylate is not essential to facile
acylation.
One pathway, distinguished by its simplicity and its similarity to
many protease mechanisms, is the combination of c and f in dea-
cylation (Scheme 4) along with c0 and f0 in acylation (Scheme 5).

6418 Biochemistry, Vol. 49, No. 30, 2010
Dzhekieva et al.
and unique (except for its close homologue E. coli PBP6 (62)) in
having a broadened active site, at least in the crystal structure.
This property is illustrated, for example, by the larger than usual
distance between CR of Ser 2 (Ser 298) and CR of Gly 413 (of the
KS(T)G motif) (13) which leads to a considerable separation
between the Ser 1 and Ser 2 side chains. In the crystal structure of
a nonspecific boronate complex with PBP5 (18), the active site
breadth has decreased to around the average value (13), suggest-
ing that the PBP5 active site may undergo a significant con-
formational charge on reaction with a substrate. Although Zhang
et al. (22) were aware of this problem and apparently attempted
to address it, there remains uncertainty as to whether they suc-
ceeded. Certainly, their computed intermediates appear to retain
the original separation of Ser 1 and Ser 2.
The present structure of Figure 1 therefore is probably best
interpreted, taking into account all currently available evidence,
in terms of a catalytic mechanism of deacylation represented by
the sequences a and d, and thus, assuming symmetry between
acylation and deacylation, a0 followed by d0 in acylation. Only the
uncertainty of the actual pKas of Lys 1 and Lys 2 seems to remain
as an issue that may affect this conclusion. It is interesting that a
popular mechanism of acylation of class A β-lactamases follows a
similar path involving the homologue of Ser 2 in proton transfer
to the leaving group (27, 63, 64).
11. McDonough, M. A., Anderson, J. W., Silvaggi, N. R., Pratt, R. F.,
Knox, J. R., and Kelly, J. A. (2002) Structures of two kinetic
intermediates reveal species specificity of penicillin-binding proteins.
J. Mol. Biol. 322, 111–122.
12. Silvaggi, N. R., Josephine, H. R., Kuzin, A. P., Nagarajan, R., Pratt,
R. F., and Kelly, J. A. (2005) Crystal structures of complexes between
the R61 DD-peptidase and peptidoglycan-mimetic β-lactams: a non-
covalent complex with a “perfect penicillin”. J. Mol. Biol. 345, 521–
533.
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P., Davies, C., and Pratt, R. F. (2008) Crystal structures of complexes
of bacterial DD-peptidases with peptidoglycan-mimetic ligands: the
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14. Morlot, C., Pernot, C., LeGouellec, A., DiGiulmi, A. M., Vernet, T.,
Dideberg, O., and Dessen, A. (2005) Crystal structure of a peptido-
glycan synthesis regulatory factor (PBP 3) from Streptococcus pneu-
moniae. J. Biol. Chem. 280, 15984–15991.
15. Sauvage, E., Duez, C., Herman, R., Kerff, F., Petrella, S., Anderson,
ꢁ
J. W., Adediran, S. A., Pratt, R. F., Frere, J.-M., and Charlier, P.
(2007) Crystal structure of the Bacillus subtilis penicillin-binding
protein 4a and its complex with a peptidoglycan-mimetic peptide.
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Gutheil, W. G. (2006) Overexpression and enzymatic characterization
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271, 23–32.
17. Rhazi, N., Charlier, P., Dehareng, D., Engher, D., Vermeire, M.,
ꢁ
ꢁ
ꢀ
Frere, J.-M., Nguyen-Disteche, M., and Fonze, E. (2003) Catalytic
mechanism of the Streptomyces K15 DD-transpeptidase/penicillin-
binding protein probed by site-directed mutagenesis and structural
analysis. Biochemistry 42, 2895–2906.
The strong affinity of 3 for the R39 DD-peptidase suggests that
specific boronates may generally be very powerful DD-peptidase
inhibitors and thus, possibly, antibiotics. Gutheil has previously
raised this possibility (65). For it to be achieved, however, the
specificity puzzle of high molecular weight PBPs (7, 8, 13) will
have to be solved.
18. Nicola, G., Peddi, S., Stefanova, M., Nicholas, R. A., Gutheil, W. G.,
and Davies, C. (2005) Crystal structure of Escherichia coli penicillin-
binding protein 5 bound to a tripeptide boronic acid inhibitor: a role
for Ser 110 in deacylation. Biochemistry 44, 8207–8217.
19. Thomas, B., Wang, Y., and Stein, R. (2001) Kinetic and mechanistic
studies of penicillin-binding protein 2x from Streptococcus pneumo-
niae. Biochemistry 40, 15811–15823.
20. Oliva, M., Dideberg, O., and Field, M. J. (2003) Understanding the
acylation mechanisms of active site serine penicillin-recognizing
proteins: a molecular dynamics simulation study. Proteins: Struct.,
Funct., Genet. 52, 88–100.
ACKNOWLEDGMENT
We thank the staff of beamline FIP/BM30a at ESRF for
assistance in X-ray data collection. We also thank R. Herman for
expert work in protein crystallization.
ꢀ
21. Dıaz, N., Sordo, T. L., and Suarez, D. (2005) Insights into the base
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catalysis exerted by the DD-transpeptidase from Streptomyces K15: a
molecular dynamics study. Biochemistry 44, 3225–3240.
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S. (2007) Catalytic mechanism of penicillin-binding protein 5 of
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