Kinetic mechanism and inhibition of

Mycobacterium tuberculosis D-alanine:D-alanine ligase by

the antibiotic D-cycloserine
Gareth A. Prosser and Luiz Pedro S. de Carvalho
Mycobacterial Research Division, Medical Research Council National Institute for Medical Research, London, UK

Keywords D-cycloserine (DCS) is an antibiotic that is currently used in second-line

D-amino acid; peptidoglycan biosynthesis;
Rv2981c; tuberculosis
Correspondence
L. P. S. de Carvalho, Medical Research
Council National Institute for Medical
Research, The Ridgeway, Mill Hill, London
NW7 1AA, UK
Fax: +44 20 8816 2730
Tel: +44 20 8816 2358
E–mail: luiz.pedro@nimr.mrc.ac.uk
(Received 14 November 2012, revised 17
December 2012, accepted 19 December
2012)
doi:10.1111/febs.12108
treatment of tuberculosis. DCS is a structural analogue of D-alanine, and
targets two enzymes involved in the cytosolic stages of peptidoglycan syn-
thesis: alanine racemase (Alr) and D-alanine:D-alanine ligase (Ddl). The
mechanisms of inhibition of DCS have been well-assessed using Alr and
Ddl enzymes from various bacterial species, but little is known regarding
the interactions of DCS with the mycobacterial orthologues of these
enzymes. We have over-expressed and purified recombinant Mycobacteri-
um tuberculosis Ddl (MtDdl; Rv2981c), and report a kinetic examination
of the enzyme with both its native substrate and DCS. MtDdl is activated
by K+, follows an ordered ter ter mechanism and displays distinct affinities
for D-Ala at each D-Ala binding site (Km,D-Ala1 = 0.075 mM, Km,D-Ala2 =
3.6 mM). ATP is the first substrate to bind and is necessary for subsequent
binding of D-alanine or DCS. The pH dependence of MtDdl kinetic param-
eters indicate that general base chemistry is involved in the catalytic step.
DCS was found to competitively inhibit D-Ala binding at both MtDdl
D-Ala sites with equal affinity (Ki,DCS1 = 14 lM, Ki,DCS2 = 25 lM); however,
each enzyme active site can only accommodate a single DCS molecule at a
given time. The pH dependence of Ki,DCS2 revealed a loss of DCS binding
affinity at high pH (pKa = 7.5), suggesting that DCS binds optimally in the
zwitterionic form. The results of this study may assist in the design and
development of novel Ddl-specific inhibitors for use as anti-mycobacterial
agents.

Introduction
D-cycloserine (DCS; (R)-4-amino-1,2-oxazolidin-3-one)
is an antibiotic approved by the US Food and Drug
Administration that acts at the level of cell-wall bio-
synthesis to inhibit bacterial growth. As a cyclic ana-
logue of D-alanine, it targets two conserved enzymes
that are involved in the cytosolic stages of peptidogly-
can synthesis: alanine racemase (Alr) and D-alanine:
D-alanine ligase (Ddl) [1,2]. Alr is a pyridoxal
5-phosphate-dependent enzyme that converts L-alanine
to D-alanine, while Ddl catalyses ATP-dependent
peptide bond formation between two molecules of
D-alanine, generating the D-alanine-D-alanine dipeptide
that forms the C-terminus of the pentapeptide chain of
mature peptidoglycan monomers [3,4]. Although it
exhibits broad-spectrum antibiotic activity, DCS is
currently only recommended for second-line therapeu-
tic intervention for multi-drug resistant and extensively
drug resistant strains of Mycobacterium tuberculosis,

Abbreviations
Alr, alanine racemase; AMP-PNP, adenylyl-imidodiphosphate; ATP-c-S, adenosine 5′-[c-thio]triphosphate; DCS, D-cycloserine; Ddl,
D-alanine:D-alanine ligase; MtDdl, Mycobacterium tuberculosis D-alanine:D-alanine ligase; Pi, inorganic phosphate.

1150 FEBS Journal 280 (2013) 1150–1166 ª 2013 The Authors Journal compilation ª 2013 FEBS

G. A. Prosser and L. P. S. de Carvalho
the causative agent of tuberculosis [5]. The high preva-
lence of neurological side-effects associated with DCS
treatment, assumed to be a consequence of partial
agonism of the drug with the mammalian neuronal
N-methyl-D-aspartate receptor, is the primary reason
for the limited therapeutic appeal of this drug [6,7].
Despite off-target toxicity, the efficacy of DCS as an
anti-mycobacterial agent has generated significant
interest in its biological and chemical properties. DCS
further benefits from high gastric tolerance, and, as the
only clinically employed drug targeting Alr and Ddl,
shows no cross-resistance with other front- and sec-
ond-line tuberculosis drugs [5]. Clinical resistance to
DCS has also yet to become a serious issue [5]. Under-
standing the mechanism of action of DCS may there-
fore assist in development of improved tuberculosis
therapeutics.
To date, ambiguity still exists as to whether Alr or
Ddl is the lethal target of DCS. Both enzymes appear
to be essential for M. tuberculosis growth and patho-
genesis, according to transposon mutagenesis and gene
deletion studies [8–10]. Drug sensitivity assays in
M. tuberculosis have demonstrated that supplementa-
tion with D-alanine confers higher resistance to DCS
than supplementation with the dipeptide D-Ala-D-Ala,
supporting a more important role for Alr in the DCS
mechanism of inhibition [11]. However, these results
may be explained by easier penetration of D-alanine
compared to the dipeptide. Similarly, in Mycobacte-
rium smegmatis, Alr over-expression induces higher lev-
els of DCS resistance than Ddl over-expression does,
relative to the wild-type [12,13]. Unfortunately, as inhi-
bition of Alr is irreversible, its over-expression lowers
the DCS intracellular concentration, making interpreta-
tion complicated. On the other hand, the metabolomic
profiles of DCS-inhibited bacteria do not match those
of Alr mutants, suggesting greater importance of a sec-
ondary target, e.g. Ddl, in DCS inhibition [10,14]. It is
therefore likely that both targets contribute, at least in
part, to DCS susceptibility, and that a deeper under-
standing of the molecular mechanisms of inhibition of
both is required.
At the molecular level, most studies of DCS inhibi-
tion have focused on Alr as the target, for which the
mechanism of DCS inhibition is now well character-
ized. These studies, employing Alr enzymes from
diverse bacterial species, have shown that inhibition of
Alr is competitive with respect to D- or L-alanine, time-
dependent and irreversible, with structural data reveal-
ing that the mechanism involves formation of a stable
hydroxyisoxazole adduct between the drug and the
pyridoxal 5′-phosphate co-factor [15–17]. A Ki value of
9 lM has been reported for DCS with the M. tuberculosis
FEBS Journal 280 (2013) 1150–1166 ª 2013 The Authors Journal compilation ª 2013 FEBS
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D-Ala:D-Ala ligase inhibition by cycloserine
and Mycobacterium avium Alr orthologues [18].
Rational design of Alr inhibitors based on this knowl-
edge is on-going [19–21].
In comparison, little work has been performed on
DCS inhibition of Ddls. Ddl is proposed to catalyse
D-Ala-D-Ala formation through two distinct half-
reactions: an initial attack on the c-phosphate group
of ATP by the carboxylate oxygen of D-Ala1 (N-termi-
nal) to form a D-alanyl-phosphate intermediate and
ADP, followed by a second attack on the acyl-phos-
phate by the amino group of the incoming D-Ala2
(C-terminal) to form the dipeptide product and
inorganic phosphate (see Scheme 1). Working with
Streptococcus faecalis (now Enterococcus faecalis) Ddl,
Neuhaus & Lynch [2] showed in the early 1960s that
DCS is a competitive inhibitor against both D-alanine
substrates (N- and C-terminal), with micromolar affin-
ity. Based on the magnitudes of the calculated inhibi-
tion constants and corresponding bacterial growth
inhibition data, Neuhaus & Lynch proposed that DCS
inhibition at the N-terminal site was the principal site
of DCS antibiotic action [2]. Several other studies have
reported inhibition parameters for DCS with various
Ddls, although only inhibition of the C-terminal
D-Ala substrate has been assessed in these cases, pre-
sumably on the basis of experimental and mathemati-
cal simplicity. Furthermore, no additional mechanistic
details have been discussed [22–25]. Crystal structures
of many Ddls in apo form or in complex with ATP
analogues and D-Ala or phosphonic/phosphinic acid
transition state-like analogues have also been solved,
but none so far with DCS [24,26–31].
Surprisingly, no in-depth analysis of M. tuberculosis
Ddl (MtDdl), the only clinically relevant target of
DCS, has yet been performed. Using a partially puri-
fied enzyme preparation, David et al. [32] demon-
strated that MtDdl was indeed inhibited by DCS, and
reported a tentative Ki value (30 lM) of similar magni-
tude to other Ddl orthologues [2,33]. More recently,
Bruning et al. measured IC50 values and binding affini-
ties of MtDdl with D-Ala and DCS and solved the
crystal structure of MtDdl in apo form [29]; however,
no details on the kinetic or chemical mechanism of
either the native enzymatic reaction or DCS inhibition
were reported. In this study, we have explored the
native MtDdl-catalysed reaction and the interaction
between MtDdl and DCS through a combination of
steady-state kinetics, pH rate studies and equilibrium
binding techniques. We present a description of the
kinetic mechanism of MtDdl in the presence or
absence of DCS, ligand binding and inhibition con-
stants, the stoichiometry of inhibition, and pH effects
on kinetic and inhibition parameters. Based on these
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D-Ala:D-Ala ligase inhibition by cycloserine G. A. Prosser and L. P. S. de Carvalho

Scheme 1. Ddl catalytic mechanism.

results, a model of DCS inhibition is proposed that A

may assist in the development of novel, Ddl-targeted
anti-mycobacterial agents.

Results

K+ activation of MtDdl
Preliminary experiments aimed at optimizing assay
conditions revealed a dependence of MtDdl kinetic
parameters on K+ concentration. Further investiga- B
tion demonstrated that Km,D-Ala2 decreased threefold
between 10 and 100 mM K+, but only a modest drop
was observed for kcat across the same concentration
range (Fig. 1 and results not shown). Km,ATP was simi-
larly unaffected (data not shown). Na+ was unable to
substitute for K+ in activation of MtDdl (data not
shown). Overall, maximal activity (lowest Km,D-Ala2
and highest kcat/Km,D-Ala2) was detected between 50
and 100 mM K+, and therefore an intermediate con-
centration of 80 mM K+ was chosen for subsequent
Fig. 1. MtDdl activation by K+. Values for (A) Km,D-Ala2 and (B) kcat/
enzyme assays.
Km,D-Ala2 were determined at various concentrations of K+.
Initial rates were measured at pH 7.3/37 °C. Reaction conditions
were 50 mM HEPES, 10 mM MgCl2, 3 mM ATP, 2 mM
Initial velocity patterns and kinetic parameters
phosphoenolpyruvate, 0.2 mM NADH, 9–14 U
mL1 lactate
Kinetic analysis of Ddl activity is complicated by use dehydrogenase, 6–10 U mL1 pyruvate kinase and 57 nM MtDdl.
of the same substrate (D-Ala) in the first and second
partial reactions (Scheme 1). As such, Ddl catalysis
does not follow classical Michaelis–Menten kinetics investigation. However, all Ddls studied to date exhibit
when D-Ala is the varied substrate, and any mathemat- a conserved pattern of relative affinities between the suc-
ical assessment of kinetic parameters must consider the cessive D-Ala binding steps, i.e. the first (N-terminal)
full rate equation for the kinetic mechanism under site has a 20–400-fold higher affinity for D-Ala than

1152 FEBS Journal 280 (2013) 1150–1166 ª 2013 The Authors Journal compilation ª 2013 FEBS

G. A. Prosser and L. P. S. de Carvalho
the second (C-terminal) site [23,24,31,34]. Such behav-
iour implies that, in the presence of D-Ala
concentrations approximately equal to Km,D-Ala2, the
N-terminal site is saturated ([D-Ala] ≫ Km,D-Ala1), and
therefore, under these conditions, the rate equation
may be simplified by ignoring any terms in the denom-
inator involving Km,D-Ala1. The rate equation thus
reduces to the Michaelis–Menten equation, and
parameters for Km,D-Ala2 and kcat may be calculated
from standard steady-state velocity data [35]. Indeed,
we found that double-reciprocal plots of MtDdl activ-
ity at D-Ala concentrations  2 mM were linear
(Fig. 2A), supporting the validity of the assumptions
described above. Initial velocities of MtDdl activity at
varying D-Ala concentrations (  2 mM) and multiple
fixed ATP concentrations generated an intersecting
A ing an initial high-affinity binding event followed by a
B dently by steady-state kinetic methods, we were unable
Fig. 2. MtDdl initial velocity patterns. (A) Double-reciprocal plot of
MtDdl initial rates at varying concentrations of D-Ala (  2 mM) and
several fixed concentrations of ATP: 0.12 mM (closed square),
0.3 mM (open triangle), 0.6 mM (closed triangle), 1.2 mM (open
circle) and 3 mM (closed circle). (B) Double-reciprocal plot of MtDdl
initial rates at varying concentrations of D-Ala (< 2 mM) and a fixed
saturating concentration of ATP (3 mM). Symbols represent
experimental data, and solid lines are fits of the data to the
relevant equations: Eqn (1) for (A) and Eqn (2) for (B).
FEBS Journal 280 (2013) 1150–1166 ª 2013 The Authors Journal compilation ª 2013 FEBS
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D-Ala:D-Ala ligase inhibition by cycloserine
Table 1. Steady-state kinetic parameters for recombinant MtDdl.
Parameter Value
1
kcat (s ) 9.7  0.3
Km,ATP (mM) 0.31  0.05
Km,D-Ala1app (mM) 0.075  0.010
Km,D-Ala2 (mM) 3.6  0.6
kcat/Km,D-Ala2 (M1
s1) 2690  450
pattern on a double-reciprocal plot, consistent with
MtDdl following a sequential kinetic mechanism
(Fig. 2A). True values for Km,D-Ala2, Km,ATP and kcat
were obtained by fitting this data to Eqn (1), and these
results are presented in Table 1. When D-Ala concen-
trations were varied below 2 mM (at a fixed, saturating
ATP concentration), enzyme velocity data presented in
double-reciprocal format displayed parabolic curvature
(Fig. 2B), characteristic of a kinetic mechanism involv-
lower-affinity binding event for the same substrate
[34], i.e. D-Ala. Fitting this data to Eqn (2) generated
an apparent value of 0.075 mM for the Km of D-Ala
binding to the N-terminal site (Table 1).
Product inhibition patterns
Next, we used product inhibition to investigate
the order of MtDdl substrate binding and product
release. The results are showed in Table 2. Due to the
impracticality of assessing product inhibition versus
each D-Ala substrate (N- and C-terminal) indepen-
to obtain patterns for the N-terminal D-Ala substrate.
Overall, the results were consistent with MtDdl follow-
ing a ordered sequential kinetic mechanism. The only
competitive inhibition pattern obtained was for ADP
versus ATP, at sub-saturating D-Ala concentrations,
suggesting that ATP and ADP are the first substrate
to bind and the last product to dissociate, respectively.
However, at saturating D-Ala concentrations, the ADP
versus ATP inhibition pattern became non-competi-
tive, potentially implying non-productive binding of
D-Ala to the MtDdl–ADP complex. The non-competi-
tive inhibition patterns for inorganic phosphate (Pi)
versus both ATP and D-Ala provide evidence of reac-
tion reversibility (enzyme forms are reversibly con-
nected through the chemical step) and indicate that Pi
is the first product to dissociate. An uncompetitive
pattern for D-Ala-D-Ala versus ATP is consistent with
D-Ala-D-Ala being the second product to dissociate;
however, a non-competitive pattern for D-Ala-D-Ala
versus D-Ala suggests a more complex binding
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D-Ala:D-Ala ligase inhibition by cycloserine G. A. Prosser and L. P. S. de Carvalho

Table 2. Product inhibition patterns and inhibition constants for recombinant MtDdl.

Varied substrate Product Fixed substratea Inhibition pattern Kis (mM) Kii (mM)

D-ala D-Ala-D-Ala 0.088  0.43 

b
Saturating ATP NC 0.010 0.05
Pi c Saturating ATP NC 37  6 87  8
ADP 0.3 mM ATP NC 5.2  0.7 3.7  0.2
ATP D-Ala-D-Ala Saturating D-Ala UC – 0.95  0.08
D-Ala-D-Ala 0.2 mM D-Ala UC – 0.071  0.004
Pi Saturating D-Ala NC 45  6 81  8
Pi 0.2 mM D-Ala NC 53  10 34  5
ADP Saturating D-Ala NC 2.2  0.4 4.0  0.7
ADP 0.2 mM D-Ala C 3.9  0.4 –
a
Saturating substrate concentrations were 3 mM for ATP and 100 mM for D-Ala. b NC, non-competitive; C, competitive; UC, uncompetitive.
c
The K+ concentration was increased to 175 mM when Pi was used as a product inhibitor.

Table 3. MtDdl–ligand dissociation constants.

Ligand Nucleotide co-factora Kd (mM)

D-Ala None ≫20b

ADP 11  1
AMP-PNP 3.8  1
ATP-c-S ≫20
D-Ala-D-Ala None ≫20
Scheme 2. MtDdl kinetic mechanism. ATP 0.012  0.001
ADP 0.35  0.04
DCS None ≫20
behaviour for this product, most likely formation of a ATP 0.0094  0.0007
dead-end Ddl–ATP–D-Ala-D-Ala complex, as previ- ADP 2.7  0.6
ously described for other Ddls [23,25]. The consensus AMP-PNP 0.46  0.04
kinetic mechanism inferred from these results is ATP-c-S ≫20
illustrated in Scheme 2. a
Co-substrate concentrations used: ATP, 5 mM; AMP-PNP, 5 mM;
ATP-c-S, 5 mM; ADP, 10 mM. b Highest concentration tested.

Binding studies: D-Ala and D-Ala-D-Ala

We sought further proof of an ordered kinetic mecha-
nism by assessment of MtDdl–ligand binding affinities,
using intrinsic tryptophan fluorescence quenching as a
measure of protein–ligand interactions. MtDdl was
titrated against D-Ala or D-Ala-D-Ala in the presence
or absence of saturating levels of various nucleotide
co-substrates, and changes in intrinsic tryptophan fluo-
rescence were recorded. We were unable to assess
nucleotide binding parameters due to an unresolvable
inner-filter effect (data not shown). For both D-Ala
and D-Ala-D-Ala as titrant, fluorescence changes were
only observed in the presence of the nucleotide co-sub-
strate; no changes were detected with apo-enzyme at
any ligand concentration tested (up to and including
20 mM; data not shown). Dissociation constants calcu-
lated from these results are listed in Table 3. Despite
rigorous testing over a wide range of ligand concentra-
tions, we were unable to detect a second binding event
when titrating D-Ala (data not shown), in contrast to
previous results for MtDdl [29]. Therefore, only one
Kd value for each MtDdl–nucleotide pair is recorded
for this substrate.
Three main results are worth emphasizing here.
First, the Kd for D-Ala obtained in the presence of
adenylyl-imidodiphosphate (AMP-PNP) is identical to
the Km,D-Ala2, indicating that either the low-affinity site
is being titrated under these conditions, or that substi-
tution of AMP-PNP for ATP decreases the affinity of
D-Ala at the N-terminal site by almost 50-fold (Kd
with AMP-PNP compared to Km,D-Ala1). Second,
D-Ala-D-Ala binds 30-fold more tightly to MtDdl in
the presence of ATP versus ADP, in agreement with
formation of the dead-end complex MtDdl–ATP–D-
Ala-D-Ala. Third, of the two non-cleavable ATP
analogues used in this study to investigate D-Ala bind-
ing [adenosine 5′-[c-thio]triphosphate (ATP-c-S) and
AMP-PNP], ligand-induced fluorescence changes were
only detectable with AMP-PNP. Similarly, by steady-
state kinetic methods, only AMP-PNP displayed a
competitive inhibition pattern versus ATP (Kis =

1154 FEBS Journal 280 (2013) 1150–1166 ª 2013 The Authors Journal compilation ª 2013 FEBS

G. A. Prosser and L. P. S. de Carvalho
0.3 mM; data not shown), and no inhibition was
detected with ATP-c-S (data not shown). These results
indicate that ATP-c-S is a poor substitute of ATP for
MtDdl.
pH dependence of kinetic parameters
Next, we investigated the effect of pH on the magnitude
of MtDdl kinetic parameters, in order to assess the role
of general acid-base chemistry in MtDdl catalysis and
substrate recognition. kcat and kcat/Km,D-Ala2 values were
measured at multiple pH levels between pH 6.0 and 9.0,
and plotted as a function of pH (Fig. 3). The plot for
kcat displayed a slope of +1 at low pH and pH indepen-
dence at high pH; this data was best fitted to an equa-
tion describing involvement of a single ionizable group,
which must be deprotonated for maximal activity
(Eqn 12) and permitted calculation of a pKa value of
6.5  0.1 for ionization of this putative residue.
Similarly, kcat/Km,D-Ala2 values increased, with a slope
of +2, at low pH, and displayed pH independence at
high pH. Fitting of this data to an equation describing
A DCS on Km,D-Ala2 (see Experimental procedures); in
B N-terminal site, and the precise mechanism utilized by
Fig. 3. pH dependence of MtDdl kinetic parameters. Values for
MtDdl kcat (A) and kcat/Km,D-Ala2 (B) were measured at various pH
levels, as described in Experimental procedures, and plotted as a
function of pH. Buffers used for each pH are listed in Experimental
procedures. pH values were recorded directly after the completion
of each time course, and the mean value is shown. Symbols
represent experimental data, and solid lines are fits of the data to
the relevant equations: Eqn (12) for (A) and Eqn (13) for (B).
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D-Ala:D-Ala ligase inhibition by cycloserine
the role of two (non-resolvable) ionizable groups, which
must be deprotonated for maximal activity (Eqn 13)
generated a pKa value of 7.2  0.1 for these groups.
Km,ATP was not substantially affected across the pH
range tested (data not shown).
Steady-state kinetics of DCS inhibition
Addition of DCS to reaction mixtures caused a con-
centration-dependent decrease in enzymatic rate, con-
sistent with its action as an inhibitor of MtDdl
catalysis. No ATP hydrolysis was detected in reactions
containing only DCS, MtDdl and ATP, confirming
that DCS is not a substrate of the enzyme (data not
shown). When DCS inhibition was tested at a fixed
saturating D-Ala concentration and varying ATP con-
centrations, a linear uncompetitive inhibition pattern
was observed, with a Kii value of 600  30 lM
(Fig. 4A). In contrast, when D-Ala was the varied sub-
strate at concentrations  2 mM, addition of DCS
generated a linear competitive inhibition pattern, with
a Kis of 25  3 lM (Fig. 4B). As the D-Ala concentra-
tions tested in this instance were ≫ Km,D-Ala1app, the
extrapolated Ki value exclusively indicates the effect of
other words, it defines the affinity of DCS for the
C-terminal D-Ala site (Ki,DCS2). In order to evaluate
the effect of DCS on N-terminal D-Ala binding, we
measured DCS-inhibited rates at D-Ala concentrations
approximately equal to Km,D-Ala1app (50–1000 lM). The
double-reciprocal plot of this data is presented in
Fig. 4C. As illustrated in Scheme 3, a number of
possible inhibition mechanisms exist for DCS at the
MtDdl is unknown. Attempts to fit these data to any
of the rate equations describing the possible DCS
binding mechanisms were unsuccessful, even when rate
equations were simplified by constraining known
parameters to values obtained from earlier experi-
ments (i.e. kcat, Km,D-Ala2, Km,D-Ala1 and Ki,DCS2).
Instead, the data were re-plotted to a rearranged form
of the rate equation ([D-Ala] 9 [1/v  1/kcat] versus
1/[D-Ala]) (Fig. 4D), and the patterns formed by the
resulting datasets were used to acquire non-quantita-
tive mechanistic details regarding DCS inhibition (as
described in Experimental procedures, and previously
by Neuhaus & Lynch [2]). Briefly, changes in the ordi-
nate intercept indicated binding of DCS to the MtDdl
–ATP–D-Ala complex (C-terminal site binding) and
slope changes indicated binding of DCS to the MtDdl
–ATP complex (N-terminal site binding). Secondary
plots of slopes (taken from linear regressions of indi-
vidual datasets) versus DCS concentration were linear
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D-Ala:D-Ala ligase inhibition by cycloserine G. A. Prosser and L. P. S. de Carvalho

A B

C D

Fig. 4. DCS inhibition of MtDdl catalytic activity. (A–C) Double-reciprocal plots of MtDdl initial rates at varying concentrations of one
substrate, a fixed saturating concentration of the second substrate, and multiple fixed concentrations of DCS. (A) ATP as varied substrate,
D-Ala fixed at 100 mM. DCS concentrations were 0 mM (closed circle), 0.5 mM (open circle), 1 mM (closed triangle) and 2 mM (open triangle).
(B) D-Ala as varied substrate (  2 mM), ATP fixed at 3 mM. DCS concentrations were 0 mM (closed circle), 0.02 mM (open circle), 0.05 mM
(closed triangle), 0.1 mM (open triangle) and 0.2 mM (closed square). (C) D-Ala as varied substrate, with the graph centred on concentrations
< 2 mM, ATP fixed at 3 mM. DCS concentrations were 0 mM (closed circle), 0.02 mM (open circle), 0.05 mM (closed triangle), 0.1 mM (open
triangle) and 0.2 mM (closed square). (D) Rearranged plot of data represented in (C). The inset in (D) shows the slope values for each
linearly regressed dataset versus DCS concentration (individual linear regressions not shown). Symbols in all cases are experimental data,
and solid lines in (A), (B) and (D) are simultaneous fits of all data points to the relevant equations: Eqn (6) for (A), Eqn (4) for (B) and
Eqn (10) for (D). The solid lines in (C) represent simulated data obtained by substituting the kinetic and inhibition constants in Eqn (11) for
the relevant experimentally measured values.

Scheme 3. Possible mechanisms of DCS binding to MtDdl.

(see inset to Fig. 4D), suggesting no (or insignificant) matical analysis of the extrapolated ordinate intercept
formation of MtDdl–ATP–DCS–DCS complexes values implied negligible binding of D-Ala to the
(simultaneous binding of DCS to both sites, for which C-terminal site of the MtDdl–ATP–DCS complex (see
a parabolic secondary plot is expected). Finally, mathe- Experimental procedures; data not shown). Therefore,

1156 FEBS Journal 280 (2013) 1150–1166 ª 2013 The Authors Journal compilation ª 2013 FEBS
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G. A. Prosser and L. P. S. de Carvalho D-Ala:D-Ala ligase inhibition by cycloserine

Table 4. DCS inhibition constants. A

Parameter Varied substrate Inhibition pattern Value (lM)

Kii ATP Uncompetitive 600  30

Ki,DCS1 D-Ala (C-terminal) Competitive 25  3
Ki,DCS2 D-Ala (N-terminal) Competitive 14  1

we fitted the re-arranged data to the rate equation

describing the DCS binding mechanism illustrated in
black in Scheme 3, with all relevant parameters con-
strained to previously measured values to simplify cal-
culation. The value for Ki,DCS1 calculated by this
method (14  0.9 lM) was then combined with the
B
other previously determined kinetic parameters and
applied to the full rate equation for the established
mode of DCS inhibition (Eqn 11). The resulting simu-
lated primary rate data were then compared to the
experimental data in order to test the accuracy of the
mathematical analysis used. As illustrated in double-
reciprocal format (solid lines in Fig. 4C), the simu-
lated data correlate closely with the experimental data,
confirming that the calculated Ki,DCS1 parameter is an
accurate estimate of DCS binding affinity for the
N-terminal site. The DCS inhibition constants mea-
sured in this study are summarized in Table 4.

Fig. 5. Stoichiometry of DCS and D-Ala-D-Ala binding to MtDdl.

Binding studies: DCS
Results from fluorescence quenching experiments
revealed a single dissociation constant of 9.4 
0.7 lM for DCS and MtDdl–ATP (Table 3), in good
agreement with the calculated Ki,DCS1 of 14 lM. No
fluorescence changes were detected when apo-MtDdl
was titrated against DCS (up to and including 20 mM
ligand; data not shown), consistent with the results
obtained using D-Ala as titrant. When AMP-PNP was
used as the nucleotide co-substrate analogue, the Kd
for DCS increased almost 50-fold to 460  44 lM. In
summary, DCS inhibition is highly dependent on the
presence and identity of the nucleotide bound.
Changes in affinity for DCS of up to 280-fold are
caused by changes in the nucleotide bound.
Next, we employed a fluorescence quenching-based
method (see Experimental procedures) to determine
ligand-binding stoichiometries. We were unable to
determine the stoichiometry of D-Ala binding to
MtDdl using this method, as protein concentrations
 109 the enzyme ligand dissociation constant were
required for accurate stoichiometry calculation (Kd for
D-Ala with AMP-PNP as co-substrate = 3800 lM).
The value obtained for DCS from this study was 0.80
(Fig. 5A), indicative of a binding stoichiometry of 1:1.
Plots illustrate changes in MtDdl intrinsic tryptophan fluorescence
as a function of (A) DCS or (B) D-Ala-D-Ala concentration. The value
for the x axis point at which the horizontal asymptote and tangent
to the initial data points meet is shown. Excitation and emission
wavelengths used for fluorescence measurements were 290 and
350 nm, respectively. Stoichiometry was determined as described
in Experimental procedures.
As a positive control, we tested the stoichiometry of
binding of D-Ala-D-Ala (Kd for D-Ala-D-Ala with ATP
as co-substrate = 12  0.7 lM), a ligand that is
expected by structural and kinetic considerations to
bind with a stoichiometry of one to each MtDdl active
site. Indeed, the experimentally determined value for
this ligand was 0.85 (Fig. 5B), consistent with a 1 : 1
binding ratio and a stoichiometry of one molecule per
enzyme monomer for DCS.
pH dependence of DCS inhibition
We next measured Ki,DCS2 values for MtDdl between
pH 6.0 and 9.0, in order to assess the importance of
enzyme/inhibitor protonation states on the DCS bind-
ing affinity to the C-terminal site. Calculated values
of pKi,DCS2 (log10 Ki,DCS2) and pKm,D-Ala2 (log10
Km,D-Ala2) as functions of pH are shown in Fig. 6.

FEBS Journal 280 (2013) 1150–1166 ª 2013 The Authors Journal compilation ª 2013 FEBS 1157

D-Ala:D-Ala ligase inhibition by cycloserine
Fig. 6. pH dependence of Ki,DCS2 and Km,D-Ala2. Ki,DCS2 and Km,D-Ala2
values were measured at various pH levels, as described in
Experimental procedures. The negative logarithms of these values
(pKi,DCS2, closed triangle; pKm,D-Ala2, closed circle) are plotted as a
function of pH. Open triangles represents pKi,DCS2 values (pKi,DCS2′)
adjusted for the true concentration of the zwitterionic form of DCS
in solution (calculated based on a pKa of 7.5 for the DCS
zwitterion/free base pair). Buffers used for each pH are listed in
Experimental procedures. Symbols are experimental data, and solid
or dashed lines are fits of the data to the relevant equations:
Eqn (14) for pKi,DCS2; Eqn (13) for pKm,D-Ala2 and pKi,DCS2′. pKa
values calculated by these methods were as follows: pKm,D-Ala2
pKa1 (1) = 7.2  0.1; pKi,DCS2 pKa1 (2) = 7.1  0.4, pKa2 (3) =
7.5  0.2; pKi,DCS2′ pKa1 (4) = 7.4  0.1.
Both pKi,DCS2 and pKm,D-Ala2 values increased linearly
at low pH with a slope of +2, with pKa1 values of
7.2  0.4 and 7.2  0.1 calculated for the pKi,DCS2
and pKm,D-Ala2 plots, respectively. In contrast, at high
pH (> 7.5), pKi,DCS2 values decreased linearly with a
slope of –1, but pKm,D-Ala2 values were unaffected. A
pKa2 value of 7.5  0.2 was assigned to the second
ionization event apparent in the pKi,DCS2 plot.
Discussion
MtDdl kinetic mechanism
In this work, we have characterized the M. tuberculosis
Rv2981c gene product, D-alanine:D-alanine ligase,
assessing its interactions and activities with native
substrates and the inhibitor DCS. Similar to ortholo-
gous Ddls, MtDdl catalytic activity was dependent on
the presence of K+ and Mg2+. Very little investigation
has been performed on the K+ dependence of Ddl
1158 FEBS Journal 280 (2013) 1150–1166 ª 2013 The Authors Journal compilation ª 2013 FEBS
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G. A. Prosser and L. P. S. de Carvalho
enzymes, with almost all previous studies using a con-
centration close to 10 mM [22,23,29,31,36,37], presum-
ably based on the early observations of Neuhaus [3].
However, we have shown that the optimal concentra-
tion of K+ for MtDdl (maximal kcat/Km,D-Ala2) is
between 50 and 100 mM, indicating that either MtDdl
has a higher requirement for the cation, or, assuming
that other Ddls exhibit similar profiles, that previously
reported kinetic parameters for orthologous Ddls have
been under-estimated. Experimentally determined bac-
terial intracellular concentrations of K+ range from
0.3 to 0.8 M [38], implying that use of higher K+ con-
centrations, such as those used here, is more physio-
logically appropriate for future Ddl activity testing.
Despite these discrepancies, our measured kinetic
parameters for MtDdl were of similar magnitude to
those for other native Ddls, in particular kcat (reported
values of 100–1000 min1) and Km,D-Ala2 (reported val-
ues of 0.5–10 mM) [3,23–25]. Our Km,D-Ala2 value of
3.6 mM at pH 7.3 agreed well with the value reported
by David et al. (2 mM) for partially purified M. tuber-
culosis Ddl [32]. Km,D-Ala1 values appear to be more
variable (2–660 lM), although this may be a conse-
quence of the higher level of error associated with
measurement and calculation of this parameter. None-
theless, our recorded MtDdl Km,D-Ala1app value of
75 lM is well within this range.
Product inhibition patterns were most consistent
with an ordered ter ter kinetic mechanism, with
additional formation of a dead-end complex between
D-Ala-D-Ala and MtDdl–ATP, in agreement with
results obtained with Escherichia coli DdlB (see
Scheme 2) [39]. However, we observed an additional
pattern (not seen with E. coli DdlB) of non-competi-
tive inhibition between ADP and ATP at high D-Ala
concentrations. A possible explanation is non-produc-
tive binding of D-Ala to the enzyme–ADP complex;
however, our binding data (high Kd for D-Ala with the
MtDdl–ADP complex; see Table 3) do not readily
support this hypothesis. The identity and significance,
if any, of this additional pattern remain unknown.
The predicted order of substrate binding of MtDdl
was confirmed by fluorescence-quenching binding
studies: briefly, nucleotide binding was found to be
essential for subsequent D-Ala or D-Ala-D-Ala binding.
This agrees with our kinetic model, as well as that
described for E. coli DdlB [39], and is also consistent
with structural data from orthologous Ddls that
indicate a strict requirement for conformational
changes within the apoprotein, induced by nucleotide
binding, prior to accommodation of D-alanyl ligands
[28,40]. However, our results are in stark contrast to
those previously reported for MtDdl, where nucleotide

G. A. Prosser and L. P. S. de Carvalho
binding was shown not to be a prerequisite for subse-
quent ligand binding [29]. In addition, two binding
events (two distinct Kd values) were reported per
ligand (D-Ala and DCS) in the previous study, com-
pared to a single measurable Kd for each in this study.
We are uncertain as to how such disparate results were
obtained with the same enzyme; however, both our
kinetic data and other published mechanistic details of
Ddl catalysis [28,39] are consistent with the binding
measurements obtained in this study and in contrast
to those of Bruning et al. [29]. Also, the nucleotide
used for measuring dissociation constants in the pre-
vious study [29] was ATP-c-S, a compound that our
results clearly show was unable to be utilized by
MtDdl as an ATP analogue. The lack of detectable
binding of ATP-c-S to MtDdl in our study may
explain why no effect was observed on D-Ala or DCS
binding by Bruning et al. [29].
It is noteworthy that AMP-PNP, but not ATP-c-S,
was able to substitute ATP as the nucleotide co-
substrate of MtDdl in our binding studies, although
ligand affinity (both D-Ala and DCS) was reduced
50-fold in the presence of AMP-PNP compared to
ATP. The differences in chemical and structural
properties of the two species may have implications in
crystallization trials and/or design of drugs targeting
the ATP-binding site.
pH dependence of kinetic parameters
As mentioned earlier, the chemical mechanism of Ddl
catalysis is proposed to involve two stages, the second
of which is nucleophilic attack on the acyl-phosphate
intermediate by the amino group of the incoming
C-terminal D-Ala substrate (see Scheme 1) [39]. For
this step to be successful, the D-Ala2 amino group
must exist in the electron-rich free base form (NH2).
However, at physiological pH, D-Ala exists primarily
as the zwitterion (COO, NH3+), and therefore de-
protonation of the a-amino group, presumably via an
active-site catalytic base, is the first logical chemical
step of the second reaction stage. Our pH profile data
for kcat demonstrate that at least one enzymic residue,
with a pKa of 6.5 and whose protonation negatively
affects enzymatic activity, is involved in the rate-
determining step (assumed to be catalysis), in agree-
ment with the consensus mechanism described above.
The two un-resolvable ionizations observed in the
kcat/Km,D-Ala2 pH profile most likely relate to the same
catalytic residue mentioned above and an additional
residue involved in productive substrate binding. The
equivalent pH profile for E. coli DdlB also exhibited
a slope increase from low to high pH; however, slope
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D-Ala:D-Ala ligase inhibition by cycloserine
magnitudes were ambiguous and pKa values were not
assigned [41]. However, the inflection point of the
E. coli DdlB kcat pH profile appeared to occur at a
substantially lower pH than that for MtDdl, suggest-
ing either a unique identity of the residue involved in
E. coli DdlB catalysis, or a lower pKa for this residue.
As almost all significant active-site residues are con-
served between the two enzymes [29,42], the latter is
more likely. The identity of this catalytic base, assum-
ing it is conserved across all Ddls, has so far
remained elusive, despite comprehensive mutagenesis
testing with E. coli DdlB [42–44]. Assuming the pKa
of the catalytic group is similar to that of the free
amino acid, although this is often not the case in
enzyme active sites, the pKa value reported here
suggests the involvement of a His residue; however,
the only conserved His observed within the Ddl active
site (H118 in MtDdl) is too remote from the
proposed position of the D-Ala2 amino group for it to
be functional in proton abstraction [28,29]. However,
this His residue may be involved in productive D-Ala2
binding [42], and may therefore represent the second
residue observed in the kcat/Km,D-Ala2 profile (mean
pKa of the two residues is 7.2). Mutagenesis and
structural studies are currently underway to determine
the identity of these ionizable groups and their exact
role in the chemical mechanism of MtDdl.
Mechanism of DCS inhibition
DCS is a broad-spectrum antibiotic that is currently
only prescribed for the treatment of multi-drug
resistant and extensively drug resistant tuberculosis.
Therefore, the only clinically relevant drug targets of
DCS are those specific to M. tuberculosis, i.e. MtAlr
and MtDdl. However, this study represents the first
in-depth kinetic analysis of the interaction between
MtDdl and DCS.
Unlike the mechanism of inhibition described for
Alr, inhibition of Ddl was found to be reversible.
Consistent with all studies to date [2,23,25,32], we
found DCS to be a competitive inhibitor against
D-Ala2. We also showed, for the first time, that inhibi-
tion of DCS is uncompetitive versus ATP, indicating a
strict requirement for nucleotide binding prior to
inhibitor binding. This is consistent with our equilib-
rium binding results, and is overall in accordance with
a shared mechanism of binding between DCS and
D-Ala. Our kinetic and binding data contradict results
previously reported for MtDdl, where DCS was shown
to bind with equal affinity to both apo and nucleo-
tide-bound enzyme forms [29]. We are currently
unable to explain these discrepancies.
1159

D-Ala:D-Ala ligase inhibition by cycloserine
We also proved through kinetic and binding (stoi-
chiometry) analysis that the MtDdl active site is only
capable of accommodating a single DCS molecule at
a time, in either the N- or C-terminal sites. The
inability of DCS to occupy both sites simultaneously
is a point worth considering, particularly from a drug
development perspective. A possibility is that phos-
phorylation of the N-terminal site ligand is necessary
prior to binding of the C-terminal substrate, perhaps
due to steric constraints, and that DCS cannot be
phosphorylated at this site. However, although we
found no evidence of ATP hydrolysis in the presence
of DCS and enzyme alone, this does not discount the
possibility of a DCS phosphorylation/dephosphoryla-
tion cycle occurring within the N-terminal site upon
DCS binding, as was shown to take place with D-Ala
and ATP within the active sites of enterococcal D-ala-
nine:D-lactate (VanA) and D-alanine:D-serine (VanC2)
ligases [45]. Other potential scenarios include steric
hindrance between adjacent DCS molecules within the
two sites, or prevention of active site rearrangements
that are necessary for C-terminal site binding due to
the bulkier nature of the DCS molecule compared to
the native D-Ala substrate. Further experimentation is
required to distinguish the precise mechanisms
involved. For example, co-crystal structures of MtDdl
with AMP-PNP, D-Ala and DCS would shed light on
the structural basis for the stoichiometry observed
during binding and inhibition experiments.
Contribution of each D-Ala binding site in DCS
inhibition in vivo
Our inhibition data reveal that, at pH 7.3 at least, DCS
has approximately equal affinity for both the N- and
C-terminal sites of MtDdl (Ki,DCS1 14 lM, Ki,DCS2
25 lM). This differs considerably from the properties of
S. faecalis Ddl, for which Ki,DCS1 was reported to be
seven times lower than Ki,DCS2 (22 lM compared to
140 lM). As these are the only published reports com-
paring DCS affinity at the different sites, it is difficult
to establish any consensus mechanistic or structural
explanation for these differences. Nevertheless, our
results have profound implications regarding the rela-
tive contributions and importance of each Ddl site in
DCS antibiotic activity and therefore future drug
design. As already mentioned, the current prevailing
hypothesis is that N-terminal site binding of DCS is
the primary cause of the antibiotic action of this drug,
based primarily on the higher observed affinity of this
site for DCS [2,23]. However, for MtDdl, for which
our results demonstrate similar affinities at both sites,
the intracellular DCS concentration is irrelevant with
1160 FEBS Journal 280 (2013) 1150–1166 ª 2013 The Authors Journal compilation ª 2013 FEBS
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G. A. Prosser and L. P. S. de Carvalho
regard to the relative importance of each site, and the
principal determinants are instead the intracellular
D-Ala concentration and the Km for each site. An exami-
nation of the rate equation (Eqn 11) for MtDdl reveals
that, as D-Ala concentrations decrease below Km,D-Ala1,
binding of DCS to the N-terminal site becomes the pri-
mary source of inhibition [the denominator reduces to
Km,D-Ala1 9 Km,D-Ala2 9 (1 + [DCS]/Ki,DCS1)], while at
D-Ala concentrations that saturate the N-terminal site,
DCS binding to the C-terminal site predominates
[Km,D-Ala1 9 Km,D-Ala2 9 (1 + [DCS]/Ki,DCS1) becomes
negligible]. Although we were unable to find explicit
references to mycobacterial intracellular D-Ala concen-
trations in the literature, we were able to predict a
concentration of 3–5 mM in wild-type bacteria by
consideration of data for M. tuberculosis glutamate
concentrations [46] and the concentration ratios
between D-Ala and glutamate in M. smegmatis [13,47].
This concentration range is well above saturation of
the N-terminal site. However, the presence of DCS is
expected to cause a decrease in the D-Ala pool through
inhibition of Alr. Although D-Ala depletion is indeed
observed upon challenge of M. smegmatis at the mini-
mal inhibitory concentration of DCS [13,47], the over-
all D-Ala concentration was tentatively calculated to
remain higher than 1 mM and therefore still well above
the threshold for N-terminal site saturation. Although
more work is required to confirm these hypotheses,
they suggest, in contrast to previous arguments, that
binding of DCS to the C-terminal site of MtDdl is the
principal contributor to DCS antibiotic action under
physiological conditions.
It is noteworthy that the two substrate binding sites
of MtDdl show a 50-fold difference in affinity for
D-Ala, but little to no difference in affinity for DCS.
For an inhibitor whose activity is dependent on its
resemblance to the native substrate, such a contrast in
relative affinities is unexpected. Thus, there is a clear
preference for binding of DCS at the C-terminal site
that is not mirrored at the N-terminal site. This sug-
gests the existence of additional structural features
within the C-terminal site that assist in DCS binding,
but not D-Ala binding, such as hydrogen bonding
between a suitable enzymic residue(s) and the isoxaz-
olidone ring oxygen of DCS, a component that is not
present in D-Ala. It is also possible that this difference
is related to the structural rigidity and planar confor-
mation of the DCS molecule itself, as suggested by the
very poor inhibitory activity of D-serine amide, a ring-
cleaved, and therefore much more flexible, derivative
of DCS, against S. faecalis Ddl [2]. Enzyme mutagene-
sis and DCS structure–activity relationship studies are
required to assess these theories further.

G. A. Prosser and L. P. S. de Carvalho
Ionic state of DCS bound to MtDdl
Both DCS and D-Ala affinity at the C-terminal site (as
measured by Ki,DCS2 and Km,D-Ala2, respectively) were
found to be pH-dependent between pH 6.5 and 9. At
pH values lower than 7.5, both parameters increased
at a similar rate (illustrated by a decrease in pKi,DCS2
and pKm,D-Ala2 in Fig. 6), most likely representing ion-
izable enzymic residues that must be deprotonated for
binding of both ligands. At pH values > 7.5, the two
profiles diverge, with pH independence observed for
Km,D-Ala2 values, and a constant increase in magnitude
with pH observed for Ki,DCS2 values. The slope of 1
observed in the pKi,DCS2 versus pH profile at high pH
indicates the involvement of a single ionizable group
that must be protonated for efficient DCS binding. It
is possible that this ionization refers to an enzymic
group that is specific for DCS binding; however, it is
more likely that it represents ionization of the amino
group of DCS itself, as shown in Scheme 4. This is
substantiated by the similarity of the calculated and
expected pKa values for this ionization event (both 7.5)
[48], as well as by the accepted mechanism of D-Ala
binding, whereby the zwitterion is preferred over the
free base form [43,44]. Taking into account the true
concentration of zwitterionic DCS in solution at each
pH tested, new Ki,DCS2′ values were calculated, reveal-
ing a pH profile that is almost identical to that of
Km,D-Ala2 (Fig. 6). These results are consistent with
those for DCS inhibition of S. faecalis Ddl, for which
exclusive binding of the zwitterionic form of DCS to
the C-terminal site was also proposed [2]. It remains to
be seen whether a similar pattern is observed for DCS
binding at the N-terminal site, and how these data
may be exploited for development of improved
MtDdl-specific analogues.
Summary
M. tuberculosis D-alanine:D-alanine ligase shows K+
and Mg2+ dependence, and follows an ordered ter ter
kinetic mechanism, with ATP as the first substrate to
bind and ADP as the final product to dissociate. The
Scheme 4. Ionization states of DCS.
FEBS Journal 280 (2013) 1150–1166 ª 2013 The Authors Journal compilation ª 2013 FEBS
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D-Ala:D-Ala ligase inhibition by cycloserine
first D-Ala substrate binds with 50-fold higher affinity
than the second D-Ala, consistent with previously stud-
ied orthologous Ddls. pH profile data support the exis-
tence of a general base catalysed step in the reaction
mechanism, presumably D-Ala2 amino group proton
abstraction in the second half-reaction, in order to
prime it for nucleophilic attack on the acyl-phosphate
intermediate generated in the first half reaction. DCS
is a competitive inhibitor against both D-Ala substrates
of MtDdl, and is uncompetitive versus ATP. DCS
binds with equal affinity to both substrate binding
sites, but cannot bind simultaneously to both. DCS is
proposed to bind to MtDdl exclusively in its zwitter-
ionic form. The mechanistic features of DCS inhibition
revealed by this work are invaluable for the future
rational design of novel Ddl inhibitors that may be
used in the treatment of tuberculosis.
Experimental procedures
Materials
All chemicals were reagent or analytical grade and
purchased from Acros Organics (Geel, Belgium), Fisher
Scientific (Loughborough, UK), or Sigma (Poole, UK).
The EnzChek Phosphate assay kit was purchased from
Invitrogen (Paisley, UK). Chromatographic columns for
protein purification were purchased from GE Healthcare
(Chalfont St. Giles, UK), and Ni–NTA resin was purchased
from EMD (Beeston, UK). Complete EDTA-free protease
inhibitor was purchased from Roche (Burgess Hill, UK).
Cloning, over-expression and purification of
MtDdl
The Rv2981c gene sequence from M. tuberculosis H37Rv
was codon-adapted to E. coli, and its nucleotide sequence
was synthetically prepared and ligated into the pJ411 plas-
mid (DNA 2.0, Menlo Park, CA, USA). The DNA
sequence was confirmed by full-length gene sequencing. This
construct contained a non-cleavable N-terminal hexahisti-
dine tag to facilitate purification. The plasmid was trans-
formed into competent E. coli BL21(DE3)pLysS cells, and
recombinant MtDdl was over-expressed by overnight induc-
tion with 0.2 mM isopropyl thio-b-D-galactoside at 18 °C in
Luria Broth (20 L total volume). Cells were harvested,
suspended in buffer A [20 mM triethanolamine (TEA),
300 mM NaCl and 50 mM imidazole, pH 7.8] containing
lysozyme and Complete EDTA-free protease inhibitor cock-
tail, and lysed by sonication. Soluble and insoluble cellular
fractions were separated by centrifugation (25 000 g,
30 min). MtDdl was purified from the soluble fraction by
nickel-affinity chromatography, with the recombinant
enzyme eluting as a single chromatographic peak (as moni-
1161

D-Ala:D-Ala ligase inhibition by cycloserine
tored at 280 nm) yielding roughly 20 mg recombinant pro-
tein per g of E. coli cell paste. SDS/PAGE analysis of the
purified enzyme resulted in a single major band at the
expected molecular weight of 40 kDa, with > 95% purity.
The protein identity was further confirmed by electrospray
ionization mass spectrometry (ESI–MS) at the National
Institute for Medical Research Mass Spectrometry Facility.
Measurement of enzymatic activity
Initial velocities of the MtDdl-catalysed forward reaction
were monitored continuously by UV–Vis spectrophotometry
using a Shimadzu UV–2550 UV–Vis spectrophotometer
(Shimadzu, Milton Keynes, UK), either by coupling ATP
hydrolysis to NADH oxidation (e340 = 6220 M1
cm1) via
pyruvate kinase and lactate dehydrogenase, or by measuring
inorganic phosphate (Pi) release using the commercially avail-
able EnzChek phosphate assay kit (e360 = 11 000 M1
cm1).
The latter was used exclusively for rate determination when
ADP was present in the initial reaction mixture (e.g. ADP
product inhibition analysis). All reactions, unless otherwise
stated, were performed in a 50 mM buffer (HEPES pH 7.3,
except when pH dependence was tested; see below) supple-
mented with 10 mM MgCl2 and 80 mM KCl. For pyruvate
kinase/lactate dehydrogenase-coupled assays, additional
reagents included 2 mM phosphoenolpyruvate, 0.2 mM
NADH, 9–14 U
mL1 lactate dehydrogenase and
6–10 U
mL1 pyruvate kinase. Additional reagents necessary
for the EnzChek assay were added according to the manufac-
turer’s instructions. D-Ala and ATP concentrations were var-
ied as necessary (‘saturating’ concentrations used in this
study were 3 mM ATP and 100 mM D-Ala, at pH 7.3). Indi-
vidual reactions were prepared in 1 mL cuvettes and allowed
to equilibrate to 37 °C prior to addition of MtDdl (typical
final concentration of 50–1000 nM). The MtDdl concentra-
tion used never exceeded 0.7% of the lowest recorded Km or
6% of the lowest recorded Ki. Initial velocities were extrapo-
lated from within the first 60 s of data of each time course.
Initial velocity patterns and Km,D-Ala1
determination
Initial velocity patterns for MtDdl were determined by
measuring steady-state rates at varying concentrations of
D-Ala (0.5 to > 209 Km,D-Ala2) and multiple fixed concen-
trations of ATP. Kinetic parameters (kcat, Km,ATP and
Km,D-Ala2) were calculated from this data by fitting to the
rate equation for an ordered sequential mechanism:
v ¼ kcat AB=ðKiA KB2 þ KB2 A þ KA B þ ABÞ (1)
where A is ATP, B is D-Ala, KA and KB2 are their respec-
tive Michaelis constants (KB2 specifically defines the Micha-
elis constant for the second D-Ala binding event, Km,D-Ala2),
and KiA is the inhibition constant for ATP. To calculate
1162 FEBS Journal 280 (2013) 1150–1166 ª 2013 The Authors Journal compilation ª 2013 FEBS
17424658, 2013, 4, Downloaded from https://febs.onlinelibrary.wiley.com/doi/10.1111/febs.12108, Wiley Online Library on [08/12/2023]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License
G. A. Prosser and L. P. S. de Carvalho
the apparent Km,D-Ala1, steady-state rates were measured at
a fixed saturating ATP concentration and varying D-Ala
concentrations < Km,D-Ala2 (50–1000 lM). These data were
combined with rates obtained previously at D-Ala concen-
trations  Km,D-Ala2 and applied to the reciprocal equation
describing the Ddl mechanism:
1=v ¼ 1=kcat þ KA2 =kcat A þ KA1 KA2 =kcat A2 (2)
where A is the D-Ala concentration and KA1 and KA2 are
the apparent Michaelis constants for D-Ala binding to the
first and second binding sites, respectively (i.e. Km,D-Ala1app
and Km,D-Ala2app). Prior to curve fitting, kcat and
Km,D-Ala2app were obtained by fitting initial velocities at high
D-Ala concentrations (0.5 to > 209 Km,D-Ala2) and saturat-
ing ATP concentrations to the Michaelis–Menten equation:
v ¼ kcat A=ðKA2 þ AÞ (3)
The relevant parameters in Eqn (2) were subsequently con-
strained to these values, in order to reduce error associated
with curve fitting to double-reciprocal plots.
Product inhibition patterns
Product inhibition patterns were determined by measuring
MtDdl activity in the presence of varying concentrations of
one substrate (for D-Ala, concentrations from 0.5 to > 209
Km,D-Ala2 only), a fixed concentration of the second sub-
strate, and multiple fixed concentrations of a single product
(ADP, D-Ala-D-Ala or Pi). Inhibition constants were calcu-
lated by fitting each dataset to one of three equations,
depending on the pattern observed in double-reciprocal
plots. These included equations for competitive inhibition
(lines intersecting on the y axis; Eqn 4), non-competitive
inhibition (lines intersecting to the left of the y axis; Eqn 5)
and uncompetitive inhibition (parallel lines; Eqn 6):
v ¼ kcat A=½Kð1 þ I=Kis Þ þ A (4)
v ¼ kcat A=½Kð1 þ I=Kis Þ þ Að1 þ I=Kii Þ (5)
v ¼ kcat A=½K þ Að1 þ I=Kii Þ (6)
where K is the apparent Michaelis constant of the varied
substrate, A is the concentration of the varied substrate,
I is the concentration of inhibitor, and Kii and Kis are inter-
cept and slope inhibition constants for the added inhibitor
versus the varied substrate, respectively.
DCS inhibition: versus ATP and D-Ala2
Inhibition constants for DCS were determined by measuring
steady-state rates of reactions containing varying
concentrations of one substrate, a fixed saturating concentra-
tion of the second substrate, and multiple fixed concentra-
 17424658, 2013, 4, Downloaded from https://febs.onlinelibrary.wiley.com/doi/10.1111/febs.12108, Wiley Online Library on [08/12/2023]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License
G. A. Prosser and L. P. S. de Carvalho D-Ala:D-Ala ligase inhibition by cycloserine

tions of DCS. Datasets obtained with ATP as varied sub-
strate were fitted to Eqn (6); datasets obtained with D-Ala as
the varied substrate, at concentrations 0.5 to > 209 Km,D-Ala2,
were fitted to Eqn (4). The calculated Kis value in this
instance corresponds to Ki,DCS2 (Ki2 in Scheme 3).
DCS inhibition versus D-Ala1
The data used for calculation of Ki,DCS2 were combined
with additional rate data measured with D-Ala concentra-
tions ranging from below Km,D-Ala1app to 0.59 Km,D-Ala2
(saturating ATP concentration, multiple fixed DCS concen-
trations) and analysed using the rationale described by
Neuhaus & Lynch [2]. These data were rearranged and
plotted as [D-Ala] 9 (1/v  1/kcat) versus 1/[D-Ala]. The
nature of DCS binding at the N-terminal site was deter-
mined by comparing the data patterns of this plot with pat-
terns expected from the various rate equations describing
the various DCS inhibition mechanisms. The rate equation
describing the most general mechanism (shown in
Scheme 3) is as follows:
   
1 1 KA2 KA1 I I
A  ¼ 1þ þ The effect of pH on MtDdl activity was measured using
v kcat kcat Ki1 Ki3 Ki2 
KA1 KA2 I I2
þ 1þ þ (7)
Akcat Ki1 Ki1 Ki4
where A is the D-Ala concentration, I is the DCS concen-
tration, KA1 and KA2 are the Michaelis constants for D-Ala
binding to the N- and C-terminal sites respectively, and
Ki1, Ki2, Ki3 and Ki4 are inhibition constants as indicated in
Scheme 3 (referred to in the text as Ki,DCS1, Ki,DCS2, Ki,
DCS1,D-Ala2 and Ki,DCS1,DCS2, respectively). The ordinate
intercept and slope of an individual dataset (a dataset being
all rates measured at a single DCS concentration) plotted
to the above equation may be defined as follows:
Intercept ¼ KA2 =kcat ð1 þ KA1 I=Ki1 Ki3 þ I=Ki2 Þ
nate intercepts and those expected for a mechanism where
this inhibition mechanism does not occur (i.e. Ki,DCS1:D-Ala2
is infinite; the remaining parameters were substituted for
their previously determined values). The difference in mag-
nitude of these values indicates the contribution of the term
that includes Ki,DCS1:D-Ala2 to the intercept value.
A Ki,DCS1 value was calculated by fitting the complete
dataset to Eqn (10):
     
1 1 KA2 I KA1 KA2 I
A  ¼ 1þ þ 1þ (10)
v kcat kcat Ki2 Akcat Ki1
Prior to curve fitting, kcat, KD-Ala1, KD-Ala2 and Ki,DCS2
parameters were constrained to values calculated using the
equations and methods previously described. Primary rate
data were simulated using a rearranged form of this equa-
tion, as defined below:
kcat
v¼     (11)
KA2 A 1 þ KIi2 þ KA1 KA2 1 þ KIi1 þ A2
pH studies
the steady-state methods described above, using a different
pH-defined buffer for each pH investigated. Buffers used
included PIPES (pH 6.3–6.5), MOPS (pH 6.6), HEPES
(pH 6.8–7.6), Tris (pH 7.9), TAPS (pH 8.2–8.5) and
N-cyclohexyl-2-aminoethanesulfonic acid (CHES; pH 8.7–
8.9). At pH levels > 8.2, pyruvate kinase and lactate
dehydrogenase concentrations were increased to 20–30 and
30–45 U
mL1, respectively. Appropriate controls were
run to ensure that MtDdl was stable over the range of
pHs tested for at least the duration required for a single
time course, and chemical and/or physical differences
between the buffers used had no significant effect on
MtDdl activity. Plots of kcat or kcat/Km,D-Ala2 versus pH
(8) were fitted to Eqns (12,13) respectively, and a plot of Ki,
DCS2 versus pH was fitted to Eqn (14).

Slope ¼ KA1 KA2 =kcat ð1 þ I=Ki1 þ I2 =Ki1 Ki4 Þ (9) Y ¼ C=ð1 þ H=Ka Þ (12)

Therefore, as a function of DCS concentration, the exis-
tence of a particular mode of binding of DCS has the fol-
lowing expected effects on the ordinate intercepts and
slopes of individual datasets: (a) Ki1 (Ki,DCS1): positive lin-
ear relationship between slope and DCS concentration; (b)
Ki2 (Ki,DCS2): positive linear relationship between ordinate
intercept and DCS concentration; (c) Ki3 (Ki,DCS1:D-Ala2):
positive linear relationship between ordinate intercept and
DCS concentration; (d) Ki4 (Ki,DCS1:DCS2): positive para-
bolic relationship between slope and DCS concentration.
Ordinate intercepts and slopes were obtained by subject-
ing each dataset to linear regression. The presence of a
Ki,DCS1,D-Ala2 inhibition constant was tested by calculating
the differences between the experimentally determined ordi-
Y ¼ C=ð1 þ H=Ka1 þ H2 =K2a1 Þ (13)
Y ¼ C=ð1 þ H=Ka1 þ H2 =K2a1 þ Ka2 =HÞ (14)
In these equations, Y is the parameter under investiga-
tion (kcat, kcat/Km,D-Ala2 or Ki2), C is the pH-independent
value of Y, H is the proton concentration, and Ka1 and Ka2
are acid dissociation constants for enzyme or ligand ioniz-
able groups.
Binding studies
Dissociation constants for MtDdl–ligand complexes were
determined by measuring alterations in the intrinsic

FEBS Journal 280 (2013) 1150–1166 ª 2013 The Authors Journal compilation ª 2013 FEBS 1163

D-Ala:D-Ala ligase inhibition by cycloserine
tryptophan fluorescence of MtDdl upon ligand binding.
Reactions were performed in either 1 or 3 mL quartz cu-
vettes, at 37 °C, using a temperature-controlled RF–
5301PC spectrofluorophotometer (Shimadzu). Samples
were excited at 290 nm, and emission spectra were
recorded between 300 and 400 nm. Samples were buffered
with 50 mM HEPES (pH 7.3) and contained 15 mM
MgCl2 and 80 mM KCl. MtDdl was added to a final con-
centration of 1 lM, and changes in fluorescence were mea-
sured upon titration of the desired ligand. The difference
between the initial volume of the reaction (minus ligand)
and the final volume (ligand at saturating levels or at its
highest possible concentration) did not exceed 2% of the
total sample volume, and therefore volume changes were
deemed to have no significant effect on overall fluores-
cence values over the course of each experiment. The
greatest difference in fluorescence between free MtDdl (no
substrate) and saturated MtDdl (maximum substrate) was
observed at 350 nm emission (data not shown), and there-
fore data at this wavelength were used to represent fluo-
rescence changes. Plots of change in fluorescence (DF)
versus ligand concentration were fitted to Eqn (15),
describing a rectangular hyperbola:
DF ¼ DFmax L=ðKd þ LÞ (15)
where L is the ligand being titrated, Kd is its apparent dis-
sociation constant, and DFmax is the maximum possible
fluorescence change inducible in the protein (at infinite
ligand concentration).
Stoichiometry
The stoichiometry of DCS binding to MtDdl was deter-
mined using a protocol described by Fersht [49]. Briefly,
3 mL samples composed of 50 mM HEPES buffer pH
7.3, 15 mM MgCl2, 80 mM KCl, 5 mM ATP and 80 lM
Ddl were placed in a quartz cuvette and allowed to
equilibrate to 37 °C. DCS (or D-Ala-D-Ala) was then
titrated in low micromolar increments (ligand concentra-
tion  [MtDdl]), with fluorescence readings (excitation at
290 nm, emission at 350 nm) taken after each addition.
After a linear dependence between the change in fluores-
cence and ligand concentration had been established,
higher ligand concentrations were added until a maxi-
mum fluorescence change was reached. After plotting the
change in fluorescence against ligand concentration, a
tangent was drawn to the initial linear portion of the
curve (at low ligand concentration) and an asymptote
was drawn parallel to the x axis at the DF value calcu-
lated to result from 100% bound enzyme (determined
using calculated Kd values). The point on the x axis at
which the tangent and asymptote intersected was divided
by the enzyme concentration to give the stoichiometry
ratio.
1164 FEBS Journal 280 (2013) 1150–1166 ª 2013 The Authors Journal compilation ª 2013 FEBS
17424658, 2013, 4, Downloaded from https://febs.onlinelibrary.wiley.com/doi/10.1111/febs.12108, Wiley Online Library on [08/12/2023]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License
G. A. Prosser and L. P. S. de Carvalho
Data analysis
Kinetic and binding parameters were calculated, unless
otherwise stated, from non-linear regression fitting of experi-
mental data to the relevant equation (as described in individ-
ual sections above), using SIGMAPLOT 12.0 (Systat Software
Inc., London, UK).
Acknowledgements
We thank Dr Steve Howell for ESI-MS analysis of
MtDdl and the National Institute for Medical
Research Large Scale Laboratory for E. coli growth.
We thank Dr Steve J. Smerdon for suggesting AMP-
PNP as an alternative ATP analogue to be tested.
Work in L.P.S.C.’s laboratory is funded by the Medi-
cal Research Council (MC_UP_A253_1111).
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