Kinetics and stereochemistry of hydrolysis of an N-(phenylacetyl)-α- hydroxyglycine ester catalyzed by serine β-lactamases and dd-peptidases

Article abstract of DOI:10.1039/c2ob25585e

The α-hydroxydepsipeptide 3-carboxyphenyl N-(phenylacetyl)-α- hydroxyglycinate (5) is a quite effective substrate of serine β-lactamases and low molecular mass dd-peptidases. The class C P99 and ampC β-lactamases catalyze the hydrolysis of both enantiomers of 5, although they show a strong preference for one of them. The class A TEM-2 and class D OXA-1 β-lactamases and the Streptomyces R61 and Actinomadura R39 dd-peptidases catalyze hydrolysis of only one enantiomer of 5 at any significant rate. Experiments show that all of the above enzymes strongly prefer the same enantiomer, a surprising result since β-lactamases usually prefer l(S) enantiomers and dd-peptidases d(R). Product analysis, employing peptidylglycine α-amidating lyase, showed that the preferred enantiomer is d(R). Thus, it is the β-lactamases that have switched preference rather than the dd-peptidases. Molecular modeling of the P99 β-lactamase active site suggests that the α-hydroxyl of 5 may interact with conserved Asn and Lys residues. Both α-hydroxy and α-amido substituents on a glycine ester substrate can therefore enhance its productive interaction with the β-lactamase active site, although their effects are not additive; this may also be true for inhibitors.

Full text of DOI:10.1039/c2ob25585e

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PAPER
Kinetics and stereochemistry of hydrolysis of an N-(phenylacetyl)-
α-hydroxyglycine ester catalyzed by serine β-lactamases and DD-peptidases†
Ryan B. Pelto and R. F. Pratt*
Received 19th March 2012, Accepted 13th July 2012
DOI: 10.1039/c2ob25585e
The α-hydroxydepsipeptide 3-carboxyphenyl N-(phenylacetyl)-α-hydroxyglycinate (5) is a quite effective
substrate of serine β-lactamases and low molecular mass DD-peptidases. The class C P99 and ampC
β-lactamases catalyze the hydrolysis of both enantiomers of 5, although they show a strong preference for
one of them. The class ATEM-2 and class D OXA-1 β-lactamases and the Streptomyces R61 and
Actinomadura R39 DD-peptidases catalyze hydrolysis of only one enantiomer of 5 at any significant rate.
Experiments show that all of the above enzymes strongly prefer the same enantiomer, a surprising result
since β-lactamases usually prefer L(S) enantiomers and DD-peptidases D(R). Product analysis, employing
peptidylglycine α-amidating lyase, showed that the preferred enantiomer is D(R). Thus, it is the
β-lactamases that have switched preference rather than the DD-peptidases. Molecular modeling of the P99
β-lactamase active site suggests that the α-hydroxyl of 5 may interact with conserved Asn and Lys
residues. Both α-hydroxy and α-amido substituents on a glycine ester substrate can therefore enhance its
productive interaction with the β-lactamase active site, although their effects are not additive; this may
also be true for inhibitors.
α to the scissile amide or ester bond of β-lactamase and DD-pepti-
dase substrates and inhibitors has quite a long history, beginning
Introduction
The bacterial DD-peptidases remain well-validated antibiotic
targets and the β-lactams remain their major weakness.1 Resist-
ance to β-lactam antibiotics is largely associated with bacterial
production of β-lactamases, enzymes that catalyze β-lactam
destruction by hydrolysis. β-Lactamase inhibitors are therefore of
considerable interest in that they may be used to protect
β-lactams in their assault on bacteria and thus lead to therapeutic
success.2–4 The search for new chemical entities that interact
specifically with DD-peptidases or β-lactamases is thus an
ongoing enterprise.
with the example of the cephamycins, a class of β-lactam natural
products containing the 7-α-methoxy group, 2.6 Synthetic ana-
logues of these, such as cefoxitin, 3, soon followed.7 Often, on
interaction with β-lactamases, these substituents lead to quite
inert acyl–enzyme complexes and thus to inhibitory
substrates.8–14 The effects of α-substituents (X = Me, OMe,
+
CH2OH, CH2NH3 , CO2−) on the reactivity of acyclic β-lacta-
mase and DD-peptidase substrates such as 4 have also been
examined.15
Recently, we have shown that α-hydroxyalkyl esters such as 1
can be good substrates of class A and C β-lactamases.5 The
incorporation of substituents
The effect of the α-hydroxy substituent on β-lactamase and
DD-peptidase substrates and inhibitors has not, to our knowledge,
been studied, although 7-α-hydroxy-penicillins and cephalospor-
ins have been synthesized.16–18 In this paper we describe the
preparation of the α-hydroxy-depsipeptide 5 and its reactivity
with typical β-lactamases and DD-peptidases. In particular, an
unusual specificity of β-lactamases for the α-D(R)-enantiomer
was observed. [Throughout the paper we refer to the enantiomers
of 5 as D and L in analogy to familiar amino acid terminology,
where the OH group of 5 is seen as replacing the usual amino
Department of Chemistry, Wesleyan University, Lawn Ave., Middletown,
CT 06459, USA. E-mail: rpratt@wesleyan.edu
†Electronic supplementary information (ESI) available: NMR spectra
of 5 and its reaction products, and HPLC experiments. See DOI:
10.1039/c2ob25585e
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acid side chain. We do this to enable the discussion to be easily
understood in the framework of the β-lactamase literature where
acyclic substrates are amino acid derivatives. In the present text,
however, we also give the formal RS descriptor when enantio-
mers of 5 are referred to.] The specificity of the enzyme pepti-
dylglycine α-amidating lyase (PAL) for α-hydroxyglycine
derivatives was employed to make this stereochemical assign-
ment. Molecular modeling was employed to help understand this
result.
10−4 s−1 15 (although the latter represents a value in 100 mM
MOPS buffer). The same products were obtained from the enzyme-
catalyzed hydrolyses (Fig. S8, ESI†). Fig. 1A shows spectrophoto-
metric data for turnover of 5 by the class C P99 β-lactamase.
Two equal phases of reaction are seen, both faster than the spon-
taneous hydrolysis. It is clear, therefore, that this enzyme cata-
lyzes the hydrolysis of both enantiomers of 5, unless the slower
phase represents the rate of racemization of the enantiomers (see
below). The class C ampC β-lactamase behaved similarly. On
the other hand, Fig. 1B shows that the class A TEM β-lactamase
only measurably catalyzes the hydrolysis of one enantiomer of 5.
Since the second slower phase of reaction in this case represents
spontaneous hydrolysis, it is clear that the second phase of the
P99 reaction of Fig. 1A must represent enzyme-catalyzed
hydrolysis of the second enantiomer rather than racemization.
Addition of the P99 enzyme to the reaction mixture directly
after the TEM enzyme had hydrolyzed its preferred enantiomer
led to the data of Fig. 1C. The second phase shows a hydrolysis
rate equal to that of the slower enantiomer in reaction with the
P99 enzyme, i.e. P99 and TEM enzymes preferentially hydro-
lyze the same enantiomer of 5. The converse experiment (not
shown) where the TEM enzyme was added directly after the first
phase of the P99 reaction was also performed. No acceleration in
the rate of hydrolysis, above that of P99 catalysis, of the remain-
ing enantiomer was then observed, confirming the conclusions
reached above.
Results and discussion
The synthesis of 5 was straightforward, following well-esta-
blished precedents (Scheme 1). Preliminary experiments showed
that at least one enantiomer of 5 was a substrate of several β-lac-
tamases and DD-peptidases. These enzymes catalyzed its
hydrolysis to a mixture of the α-hydroxy acid and m-hydroxy-
benzoic acid (see below). The issues of interest were then the
stereochemical preferences of the various enzymes for the enan-
tiomers of 5 and the magnitudes of the steady state parameters
for turnover of these enantiomers. The former of these is first
addressed below.
Similar experiments were carried out with the class D OXA-1
β-lactamase and the R61 and R39 DD-peptidases, with the same
result as that obtained with the TEM β-lactamase, viz. all three
enzymes catalyzed the hydrolysis of only one enantiomer of 5,
and that enantiomer was the same as that preferred by the P99
and TEM enzymes.
In a buffered solution at pH 7.5, 5 spontaneously hydrolyzes
to a mixture of N-(phenylacetyl)-α-hydroxyglycine, 6, and
1
m-hydroxybenzoate (Scheme 2), as demonstrated by H NMR
These results were rather interestingly different to those
obtained previously for 4 (X = OMe, CH2OH, Me). In those
cases, as above, only one enantiomer was a substrate of the R61
and R39 DD-peptidases. On the basis of the structure of the likely
(Fig. S4–S7, ESI†) and HPLC experiments (see below). The
pseudo-first order rate constant for this reaction in 20 mM
MOPS buffer, pH 7.5, was found to be (1.1 0.1) × 10−4 s−1
,
a value quite comparable to that of 4 (X = OMe), viz. 2.5 ×
Scheme 1 Synthesis of 5.
Scheme 2 Hydrolysis of the depsipeptide 5.
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The dilemma presented by the current results is now clear. If
the DD-peptidases prefer the D(R)-enantiomer of 5, then the
enantio-preference of the β-lactamases has switched. But do the
DD-peptidases still prefer (absolutely) the D(R)-enantiomer? Or is
it possible that, because of the very different chemical properties
of a methyl and a hydroxyl group, the DD-peptidase preference is
switched instead? We felt that a direct experimental resolution of
these questions was required.
The enzyme peptidylglycine α-amidating lyase (EC 4.3. 2.5)
(PAL, also known as peptidylglycolate lyase or PGL) catalyzes
the reaction of Scheme 3, with an absolute preference for the (L)
S configuration at the α-position of the substrate.23 It has been
used previously to determine the absolute configuration of
α-hydroxyglycine derivatives, including N-(phenylacetyl)-
α-hydroxyglycine, 6.24 We have therefore employed it to deter-
mine the absolute configuration of 6 produced on hydrolysis of 5
by the β-lactamases and DD-peptidases (Scheme 1). First, a
sample of PAL was added to a solution of 6 in deuterated buffer
1
and the ensuing reaction monitored by H NMR spectroscopy.
One half of 6 was converted to phenylacetamide and glyoxylate,
the identity of the products was confirmed by the addition of
authentic samples. PAL therefore behaved in our hands as
expected. It was then added to the products of the TEM and P99
β-lactamase catalyzed hydrolysis of 5.
In one experiment, solid TEM (ca. 0.1 mg) was added to a
solution of 5 (1.0 mM) and the subsequent reaction monitored
Scheme 3 The PAL reaction.
Fig. 1 Total progress curves (absorption at 290 nm) for the β-lacta-
mase-catalyzed turnover of 5 (0.5 mM). A: P99 β-lactamase (35 nM). B:
TEM β-lactamase (0.25 μM). C: On addition of the P99 β-lactamase (70 nM)
immediately after (arrow) the TEM (1.0 μM)-catalyzed reaction.
in vivo substrate of these enzymes, a carboxyl-terminating
D-alanyl-D-alanine peptide,19 it was assumed that the reactive
isomer was the D(R)-enantiomer. Given this assignment, the P99
and TEM β-lactamases therefore preferred the L(S)-enantiomer,
although, as in the present case, the P99 enzyme also catalyzed
more slowly the hydrolysis of the other [D(R)] enantiomer. The
structural basis of these preferences has been discussed,5 where
the major point is the presence in DD-peptidases of a small
hydrophobic binding pocket for the penultimate D-alanine
methyl group, which is not present in β-lactamases. Indeed, the
deletion of such a pocket has been proposed to be a major
element of β-lactamase evolution.20–22
Fig. 2 HPLC-generated progress curves for the PAL-catalyzed turn-
over (monitoring the appearance of phenylacetamide) of reaction
product 6 from hydrolysis of 5 by the P99 (closed diamonds) and TEM
(closed circles) β-lactamases. The open circles describe the disappear-
ance, by spontaneous reaction, of the single enantiomer of 5 left intact
by the TEM β-lactamase. The lines represent exponential fits to the data.
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spectrophotometrically as described above (Fig. 1B). Immedi-
ately after the first phase (when the enzyme-catalyzed reaction
was complete), PAL (2 μM) was added and the subsequent reac-
tion monitored by HPLC with the results shown in Fig. 2 (an
example of the raw data is shown in Fig. S3 of the ESI†). The
PAL-catalyzed appearance of phenylacetamide occurred at the
same rate as disappearance of the unreacted isomer of 5 from
spontaneous hydrolysis. This proves that the TEM β-lactamase
has absolute specificity for the D(R) enantiomer of 5, yielding
the D(R) enantiomer of 6, which does not react with PAL. To
support this conclusion, PAL (2 μM) was added to the products
of the P99-catalyzed reaction of 5. In this case, both enantiomers
are hydrolyzed (Fig. 1A). Addition of PAL then led to the initial
appearance of phenylacetamide (0.5 mM) at a much faster rate
than from the TEM experiment. This result shows that if the L(S)
enantiomer of 5 were hydrolyzed by TEM, the PAL catalyzed
formation of 6 would be observed at a rate faster than that of the
spontaneous hydrolysis of 5.
between the enzyme and the depsipeptides 4 (X = H) and D(R)-4
(X = Me), previously constructed and described;15,20,22 adducts
of 7 rather than 5 were constructed since we have more experi-
ence with the conformation at the active site of the D-Ala leaving
group than the m-hydroxybenzoate.25 Energy-minimized struc-
tures of tetrahedral intermediates 8, derived from attack of the
enzyme on 7, were obtained, following molecular dynamics
exploration. Two conformations of the ligand were examined,
corresponding to those previously identified, A and B, the
former with the α-C directed into the enzyme and the latter with
it out.15,20
A combination of the above results shows that all of the above
enzymes prefer the D(R) enantiomer of 5 as a substrate. The L(S)-
enantiomer is only hydrolyzed at a significant rate by the class C
β-lactamases, but even these enzymes prefer the D(R) enantio-
mer. As noted above, this stereo-preference by the β-lactamases
(but not the DD-peptidases) is contrary to most precedent with
depsipeptide substrates of structure 4. It is, however, in agree-
ment with the stereo-preference of β-lactamases for the α-hydro-
xyalkyl esters 1. It may be, therefore, that the
α-hydroxydepsipeptides 5 bind and react at the β-lactamase
active site in a conformation more closely resembling that of 1
than that of 4. The occupancy of the active site by 15 and by 415
has been previously discussed. The interaction of 5 with the
β-lactamase active site is explicitly addressed below.
An apparently stable complex, with active site functional groups
strongly interacting with the ligand in familiar ways, was
obtained when molecular dynamics runs were started from con-
formation A but not from B. Thus the complex derived from A,
shown in Fig. 3, may be a good approximation to the tetrahedral
complex 8, and also to that derived from reaction of 5 with the
enzyme. Much intramolecular hydrogen bonding is seen in this
complex, as expected of a reactive intermediate in the β-lacta-
mase active site,15,20 including hydrogen bonds between the
+
α-hydroxyl group (hydrogen bond acceptor) and Lys67-NH3
and also the Asn152 side chain carbonyl (here the hydroxyl is a
hydrogen bond donor). Interestingly, these same interactions
were observed in one of the conformations that may occur on
reaction of the P99 β-lactamase with 1.5 This structure is prob-
ably even more likely in this than in the latter case because of
the presence in 5 of the amido side chain which is seen in Fig. 3
Computational models were constructed of complexes
between (R)-7, which contains the α-hydroxyacyl group of 5,
and the P99 β-lactamase. These were based on model complexes
Fig. 3 Stereoview of an energy-minimized tetrahedral intermediate structure 8 formed on reaction of 7 with the P99 β-lactamase. Only heavy atoms
are shown.
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Table 1 Steady state kinetics parameters for the enzyme-catalyzed hydrolysis of 5 and of 1 and 4 (X = OMe) for comparison
Enzyme
P99a
Substrate
Enantiomer
kcat (s−1
)
Km (mM)
kcat/Km (s−1 M−1
)
5
D(R)
L(S)
D(R)
D(R)
L(S)
D(R)
L(S)
D(R)
L(S)
D(R)
D(R)
L(S)
D(R)
L(S)
D(R)
D(R)
L(S)
D(R)
L(S)
D(R)
L(S)
D(R)
L(S)
137 13
0.13 0.03
0.84 0.09
0.17
1.3
0.81
1.0 × 106
7.1 × 104
3.9 × 104
1.1 × 104
3.8 × 105
59
4
1 (R = CH2Ph)5
4 (X = OMe)15
6.9
13.7
310
AmpCb
TEM-2c
5
5
≥510
≥1.5
(3.4 0.2) × 105
(9.4 0.1) × 103
1.7 × 104
≥14
≥1.5
46 12
2.7 0.9
No reaction observed
≥0.46
1 (R = CH2Ph)5
4 (X = OMe)15
≥1
460
No reaction observed
≥0.02
≥0.5
≥2.5
47
OXA-1d
R61e, f
5
≥4.5
(1.8 0.17) × 103
No reaction observed
No reaction observed
6.9 0.4
1 (R = CH2Ph)5
5
0.36 0.05
0.45
1.9 × 104
9.8 × 104
2.4 × 103
5.7 × 105
No reaction observed
44
No reaction observed
10.4 0.5
4 (X = OMe)15
5
R39 f,g
4.3 0.3
0.20 0.03
No reaction observed
4 (X = OMe)15
114
7
No reaction observed
a Class C β-lactamase of Enterobacter cloacae P99. b Class C β-lactamase from E. coli. c Class A β-lactamase from the TEM-2 plasmid in E. coli.
d Class D β-lactamase from E. coli. e LMM class B DD-peptidase from Streptomyces R61. f Compound 1 did not react with either the R61 or R39
DD-peptidase.5 g LMM class C DD-peptidase from Actinomadura R39.
to hydrogen bond into the active site in just the same way as it is
thought to do in natural substrates.26 The interactive features of
the structure of Fig. 3, therefore, may lead to the reactivity of
both 1 and 5 with the P99 β-lactamase. The hydrogen bonding
interaction of the α-hydroxyl group with Lys67 must overcome
any unfavorable steric interaction that it might have with the β-C
of Tyr221. Such a steric interaction has been proposed to reduce
the reactivity of D-alanyl derivatives with the P99
β-lactamase.20–22
Steady state kinetics parameters for the turnover of 5 by the
enzymes alluded to above are presented in Table 1, along with
those of 1 (R = CH2Ph) and 4 (X = OMe). It can be seen at once
that 5 is quantitatively comparable to the latter compounds as a
substrate of these enzymes. The weak apparent binding (high Km
values) of 5 to several of these enzymes is offset by high turn-
over numbers (kcat), thus yielding quite large values for kcat/Km.
Many β-lactams are turned over at comparable rates although the
most specific β-lactams do have higher kcat/Km values (106–
107 s−1 M−1).27,28
Comparison of the kinetics results with those from the com-
pound 4 (X = OMe), bearing the electronically similar but steri-
cally larger α-OMe group, is also interesting. In general, the
D(R)-enantiomer of 4 is less reactive as a β-lactamase substrate
than 5. This difference presumably reflects the limited space
available for bulky α-substituents in a substrate of a serine
β-lactamase;20–22 in fact, the L(S)-enantiomers of 4 are more
reactive with the P-99 and TEM β-lactamases than the D(R)
(Table 1, ref. 15). Strikingly, however, with both DD-peptidases,
the D(R)-enantiomer of 4 (X = OMe) is more reactive than that
of 5. This may reflect the better fit of the bulkier OMe group
into the methyl pocket of DD-peptidases, and perhaps, also, a
negative effect of the α-hydroxyl group on catalysis, by its
hydrogen bonding to the catalytically necessary active site lysine
side chain (Lys67 of the P99 enzyme – see Fig. 3). Thus, incor-
poration of the D-α-hydroxyl substituent into a classical depsi-
peptide, 4 (X = H), does not enhance reactivity to the extent that
it does with alkyl side chains, 1, but the resulting molecule, 5,
certainly does maintain significant active site affinity.
The comparison of kinetic data from other m-hydroxybenzo-
ates, 1 (R = CH2Ph) and 4 (X = OMe), with those of 5 shows
that the latter compound is a quite effective substrate of both
serine β-lactamases (classes A, C and D) and DD-peptidases (low
molecular mass, classes B and C). In all cases, the classical
amido side chain of 5 leads to a better (kcat/Km) substrate than
the alkyl (benzyl) side chain of 1. Indeed, it has already been
established that the presence of the α-hydroxyl group is essential
to any substrate activity in 1.5 The effect of the amido side chain
is particularly striking in the class A and D β-lactamases
(TEM-2 and OXA-1) and even more so in the DD-peptidases
where no activity is seen at all with 1.
The D-α-hydroxyl group, therefore, appears to activate certain
β-lactamase (classes A and C) active sites by interaction with
functional groups in a similar way to the classical amido side
chain, thus inducing a reactive conformation. The effects of the
two groups are not, however, additive, probably because they
compete for the attention of the active site asparagine (Asn 152
in the P99 enzyme) (Fig. 3). The D-α-hydroxyl group appears to
have less effect on the DD-peptidase active site despite its very
similar structure. Incorporation of the D-α-hydroxyl group,
instead of the L-amido group, into a molecule may therefore be a
useful strategy to design new substrates and substrate and tran-
sition state analog inhibitors for serine β-lactamases.
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Sciences, University of Illinois. Absorption spectra and spectro-
photometric reaction rates were obtained from a Hewlett-Packard
8453 UV-VIS spectrophotometer. All enzyme concentrations
were determined spectrophotometrically.
Experimental
3-Carboxyphenyl N-(phenylacetyl)-α-D,L-hydroxyglycinate (5)
This compound was synthesized (Scheme 1) based on the pro-
cedures reported by Adediran et al.15 A solution of phenylaceta-
mide (13.0 g, 0.10 mol) and glyoxylic acid (11.5 g, 0.11 mol) in
acetone (75 ml) was heated under reflux for 17 h. The solvent
was evaporated and the residue dried overnight on an oil pump.
The solid was then recrystallized from chloroform–dioxane
(1 : 1), yielding N-(phenylacetyl)-α-hydroxyglycine as pale
yellow crystals. This acid (4.2 g, 20 mmol), 3-(p-methoxy-ben-
zyloxycarbonyl) phenol (5.09 g, 20 mmol) and DMAP (220 mg,
1.8 mmol) were dissolved in methylene chloride (50 ml). The
stirred solution was cooled to 0 °C and DCC (5.01 g,
24.2 mmol) was added. The mixture was allowed to warm to
room temperature as it was stirred overnight. It was then filtered
and the filtrate washed sequentially with water, 10% citric acid,
water, saturated sodium bicarbonate, and water again. The
organic layer was dried over MgSO4, filtered, and the solvent
removed by rotary evaporation. The resulting crude residue was
then purified by chromatography on silica gel with methylene
chloride–ethyl acetate (3 : 1) as the eluent, yielding 560 mg (6%)
of the p-methoxybenzyl ester of the required product. This pro-
tected product (560 mg, 1.2 mmol) was dissolved in methylene
chloride (8 ml) and trifluoroacetic acid (10 ml) with anisole
(135 μl, 1.2 mmol) and the solution stirred for 1 h at room temp-
erature. The solvent was then removed and the crude product
washed with benzene. The insoluble material was recrystallized
from acetonitrile yielding 256 mg (62%) of colorless crystals
Hydrolysis kinetics. These studies were carried out at 25 °C,
buffered in 20 mM 3-morpholinopropanesulfonic acid (MOPS)
at a pH of 7.5, except in the case of the OXA-1 enzyme where
the buffer also included 50 mM NaHCO3 and 0.1% gelatin. The
substrate was prepared in concentrated acetonitrile stock solu-
tions and diluted to ≤5% acetonitrile in assays.
Hydrolysis of 5 was monitored spectrophotometrically (m-
hydroxybenzoate release) at 290 nm (Δε = 1972 M−1 cm−1),
300 nm (Δε = 1100 M−1 cm−1), or 305 nm (Δε = 554 M−1
cm−1) depending on the concentration employed. The spon-
taneous hydrolysis total progress curves were fitted to a first
order rate equation by means of a nonlinear least-squares
program and the rate constants from several runs thus obtained
averaged.
Initial rates of hydrolysis of 5 by the P99 enzyme (35 nM),
measured spectrophotometrically at a number of concentrations
(0–1.0 mM), were fitted to the Henri–Michaelis–Menten
equation by a non-linear least squares procedure to obtain the
steady state kinetics parameters. This procedure was also used
for the slower second phase from rates estimated after com-
pletion of the first phase. In both instances, the initial rates were
corrected for spontaneous hydrolysis. Kinetic parameters for the
hydrolysis of a single enantiomer by the R61 DD-peptidase
(0.25 μM) and the TEM β-lactamase (0.25 μM) were also
obtained from initial rate measurements as described above.
Total progress curves for hydrolysis of 5, catalyzed by the
AmpC β-lactamase (69 nM) and the OXA-1 β-lactamase
(0.25 μM), were fitted by means of the Dynafit program29 to a
two-substrate model (beginning with equal amounts of the D and
L enantiomers). Total progress curves generated by the R39
DD-peptidase (0.25 μM) were treated in the same way. Substrate
concentrations in these experiments were in the 0.1–2.5 mM
range.
1
(mp 176–178 °C). NMR: H (DMSO): δ 13.21 (s, 1H), 9.29 (d,
1H, J = 8.3 Hz), 7.83 (d, 1H, J = 7.6 Hz), 7.67 (s, 1H), 7.56 (t,
1H, J = 7.9 Hz), 7.29 (m, 6H), 6.79 (d, 1H, J = 7.6 Hz), 5.63 (t,
1H, J = 8.0 Hz), 3.52 (s, 2H); IR (cm−1): 1767, 1690, 1654. MS
Calc. 329.30 ES− 328.07. HRMS (TOF, ESI+) m/z [M + H+]
calc for C17H16NO6 330.0978; found 330.0977. Its purity is
indicated by the NMR spectrum and HPLC chromatogram
(Fig. S1 and S2, ESI†).
Enzymes
PAL assay. To a solution of the depsipeptide 5 (250 μl,
1.0 mM) in a buffer containing 150 mM MES, 1 mM Cd(NO3)2
and 0.02% Lubrol (Thesit), adjusted to pH 5.0, ca. 0.1 of solid
β-lactamase, either the P99 or TEM-2 β-lactamase, was added
and the rapid hydrolysis of 5 monitored at 290 nm. Upon com-
pletion of the hydrolysis reaction the enzyme PAL was added to
a final concentration of 2 μM. Aliquots (20 μl) of the resulting
solution, taken at suitable times, were then analyzed by HPLC to
monitor progress of the subsequent reaction.
The class C P99 β-lactamase from Enterobacter cloacae and the
class A TEM-2 β-lactamase from Escherichia coli W3310 were
purchased from the Centre for Applied Microbiology and
Research (Porton Down, Wiltshire, UK). The ampC enzyme was
provided by Dr Brian Shoichet of the University of California at
San Francisco. The class D OXA-1 β-lactamase was generously
provided by Dr Michiyoshi Nukaga, Jyosai International Univer-
sity, Japan. Purified samples of the Streptomyces R61 DD-pepti-
dase and Actinomadura R39 DD-peptidase were generous gifts
from Dr J.-M. Frère and Dr P. Charlier of the University of
Liege, Liege, Belgium.
HPLC assay. An isocratic reverse phase HPLC separation of
the β-lactamase/PAL reaction products was carried out using a
Machery-Nagel C18 analytical column with a mobile phase con-
sisting of 80% potassium phosphate solution (50 mM), adjusted
to pH 6.0, and 10% methanol. The effluent of the column was
monitored at 258 nm. Calibration curves for quantitation were
obtained from authentic samples of the starting materials and
products. Retention times were 9.0 min for 5, 3.8 min for 6, and
6.9 min for phenylacetamide.
Analytical and kinetic methods
A Varian Gemini-300 MHz NMR spectrometer was used to
collect 1H NMR spectra.
The high resolution electrospray mass spectrum was obtained
from the Mass Spectrometry Laboratory, School of Chemical
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The structure of Fig. 3 was derived from computational models
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Acknowledgements
We are grateful to Professor Betty Eipper of the University of
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as to its use. This research was supported by National Institutes
of Health Grant AI-17986 to RFP.
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7362 | Org. Biomol. Chem., 2012, 10, 7356–7362
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