The C-terminal regulatory domain is required for catalysis by Neisseria meningitidis α-isopropylmalate synthase

Article abstract of DOI:10.1016/j.bbrc.2010.01.114

α-Isopropylmalate synthase (α-IPMS) catalyses the first committed step in leucine biosynthesis in many pathogenic bacteria, including Neisseria meningitidis. This enzyme (NmeIPMS) has been purified, characterised, and compared to α-IPMS proteins from other bacteria. NmeIPMS is a homodimer which catalyses the condensation of α-ketoisovalerate (α-KIV) and acetyl coenzyme A (AcCoA), and is inhibited by leucine. NmeIPMS can use alternate α-ketoacids as substrates and, in contrast to α-IPMS from other sources, is activated by a range of metal ions including Cd²⁺ and Zn²⁺ that have previously been reported as inhibitory, since they suppress the dithiodipyridone assay system rather than the enzyme itself. Previous studies indicate that α-IPMS is a TIM barrel enzyme with an allosteric leucine-binding domain. To assess the importance of this domain, a truncated form of NmeIPMS was generated and characterised. Loss of the regulatory domain resulted in a loss of the ability to catalyse the aldol reaction, although the enzyme was still able to slowly hydrolyse AcCoA independently of α-KIV at a rate similar to that of the WT enzyme. This implies that the regulatory domain is not only required for control of enzymatic activity but may assist in the positioning of key residues in the catalytic TIM barrel. The importance of this domain to catalytic function may offer new strategies for inhibitor design.

Full text of DOI:10.1016/j.bbrc.2010.01.114

Biochemical and Biophysical Research Communications 393 (2010) 168–173
Contents lists available at ScienceDirect
Biochemical and Biophysical Research Communications
journal homepage: www.elsevier.com/locate/ybbrc
The C-terminal regulatory domain is required for catalysis
by Neisseria meningitidis a-isopropylmalate synthase
Frances H.A. Huisman a,b, Michael F.C. Hunter a,b, Sean R.A. Devenish a,c,1, Juliet A. Gerrard a,c
,
Emily J. Parker a,b,
*
a Biomolecular Interaction Centre, University of Canterbury, Christchurch, New Zealand
b Department of Chemistry, University of Canterbury, Christchurch, New Zealand
c School of Biological Sciences, University of Canterbury, Christchurch, New Zealand
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 25 January 2010
Available online 1 February 2010
a
-Isopropylmalate synthase (
a
-IPMS) catalyses the first committed step in leucine biosynthesis in many
pathogenic bacteria, including Neisseria meningitidis. This enzyme (NmeIPMS) has been purified, charac-
terised, and compared to -IPMS proteins from other bacteria. NmeIPMS is a homodimer which catalyses
the condensation of -ketoisovalerate ( -KIV) and acetyl coenzyme A (AcCoA), and is inhibited by leu-
cine. NmeIPMS can use alternate
a
a
a
Keywords:
a
-ketoacids as substrates and, in contrast to a-IPMS from other sources,
is activated by a range of metal ions including Cd2+ and Zn2+ that have previously been reported as inhib-
itory, since they suppress the dithiodipyridone assay system rather than the enzyme itself. Previous stud-
a-IPMS
Regulatory domain
Allosteric regulation
Leucine biosynthesis
Neisseria meningitidis
ies indicate that a-IPMS is a TIM barrel enzyme with an allosteric leucine-binding domain. To assess the
importance of this domain, a truncated form of NmeIPMS was generated and characterised. Loss of the
regulatory domain resulted in a loss of the ability to catalyse the aldol reaction, although the enzyme
was still able to slowly hydrolyse AcCoA independently of
a-KIV at a rate similar to that of the WT
enzyme. This implies that the regulatory domain is not only required for control of enzymatic activity
but may assist in the positioning of key residues in the catalytic TIM barrel. The importance of this
domain to catalytic function may offer new strategies for inhibitor design.
Ó 2010 Elsevier Inc. All rights reserved.
Introduction
tial, the branched-chain amino acid pathway is amongst the least
studied of the amino acid biosynthetic pathways.
Whereas mammals utilise environmental supplies of the
branched-chain amino acids, leucine, isoleucine and valine, many
bacteria and plants have enzymes for de novo synthesis of these
compounds. Several studies have shown that Mycobacterium bovis
requires the leucine biosynthetic pathway for survival inside mice
and host macrophages [1]. In addition, Corynebacterium glutami-
cum, Salmonella typhimurium, and Neurospora crassa exhibited leu-
cine auxotrophy when this pathway was disrupted [2,3]. These
enzymes are therefore potential targets for inhibition, as inhibitors
may be antibacterial with limited host toxicity. Despite this poten-
a
-Isopropylmalate synthase (
first committed step in leucine biosynthesis (Fig. 1), the condensa-
tion reaction between -ketoisovalerate ( -KIV) and acetyl coen-
zyme A (AcCoA) to form (S)- -isopropylmalate ( -IPM). This
enzyme belongs to the Claisen-condensing family, along with ma-
late synthase, citrate synthase, and homocitrate synthase. -IPMS
a-IPMS, EC 2.3.3.13) catalyses the
a
a
a
a
a
is feedback inhibited by the end product of its pathway, leucine,
in organisms such as Mycobacterium tuberculosis [4] and S.
typhimurium [5].
Previous studies of a-IPMS have been limited to few organisms,
including S. typhimurium [6,7], C. glutamicum [2], and M. tuberculo-
sis [4,8–13]. The most in-depth biochemical studies and only
Abbreviations:
AcCoA, acetyl coenzyme A;
-IPMS; NmeIPMS, N. meningitidis
a
-IPMS,
a
a
-isopropylmalate synthase;
-IPM, (S)- -isopropylmalate; MtuIPMS, M. tuberculosis
-IPMS; (S)- -HIV, (S)- -hydroxyisovalerate;
a-KIV, a-ketoisovalerate;
known crystal structures are of the
a-IPMS from M. tuberculosis
a
(MtuIPMS) [9].
a
a
a
a
WT, wild-type; DTP, 4,40-dithiodipyridone; DSF, differential scanning fluorimetry;
BTP, bis–tris-propane
MtuIPMS is a homodimer of two 70 kDa monomers both in solu-
tion and in the crystalline form [9]. Other -IPMS variants such as
the enzyme from S. typhimurium have been reported to form tetra-
mers [6]. Each monomer of MtuIPMS is composed of a (b/ TIM
barrel and a regulatory domain of novel fold, separated by a flexi-
ble linker domain.
-KIV and a Zn2+ ion bind at the C-terminal end
a
* Corresponding author. Address: Department of Chemistry, University of Can-
terbury, Private Bag 4800, Christchurch, New Zealand. Fax: +64 3 364 2110.
E-mail address: emily.parker@canterbury.ac.nz (E.J. Parker).
a)
8
1
Present address: Department of Biochemistry, University of Cambridge, Cam-
a
bridge, UK.
0006-291X/$ - see front matter Ó 2010 Elsevier Inc. All rights reserved.
doi:10.1016/j.bbrc.2010.01.114

F.H.A. Huisman et al. / Biochemical and Biophysical Research Communications 393 (2010) 168–173
169
Fig. 1. Reaction catalysed by a-IPMS.
of the TIM barrel, and modelling studies imply AcCoA binds in a
large pocket on the Re face of -KIV. Leucine binds the C-terminal
Metal Affinity resin. The resin was washed with Buffer A, and pro-
tein eluted with Buffer B (Buffer A plus 150 mM imidazole, pH 8.0).
Protein-containing fractions were desalted, and DTT (1 mM) and
EDTA (0.5 mM) were added. The His-tag was cleaved by overnight
incubation at 4 °C with TEV protease [16]. TEV protease and
cleaved His-tag were removed from solution using Talon Superflow
Metal Affinity resin, and the protein-containing fractions pooled
and further purified by anion exchange chromatography on a Sour-
ceQ column using 25 mM bis–tris-propane (BTP) buffer (pH 7.0)
and a linear gradient of KCl. Enzyme-containing fractions were
identified by gel electrophoresis as a major band, which corre-
sponded well with the calculated molecular weights of 56 and
40 kDa for WT and E365Term, respectively, then pooled and stored
at ꢀ80 °C at 5–10 mg/mL.
a
regulatory domain [9]. Two residues from one monomer (His379
and Tyr410) protrude into the active site of the other, and may play
a key role in allosteric regulation. It has been shown recently that
mutation of Tyr410 to phenylalanine negates all leucine-induced
inhibition, without preventing leucine from binding in the regula-
tory domain [4]. Several point mutations and a deletion of part of
the regulatory domain of the Saccharomyces cerevisiae
a-IPMS re-
sults in insensitivity to leucine [14]. However, the role of the entire
regulatory domain has never been probed. This is intriguing, as
inspection of the sequence databases indicates that several anno-
tated
a-IPMS enzymes appear to be ‘naturally truncated’, lacking
a complete C-terminal regulatory domain, such as some forms
from Staphylococcus aureus and Francisella tularensis.
Dithiodipyridone-coupled assay at 324 nm. Initial velocity data
were obtained using 4,40-dithiodipyridone (DTP) to detect the for-
The MtuIPMS enzyme shows Zn2+ bound near the active site,
coordinating the carbonyl functionalities of
a-KIV, thereby orient-
mation of CoASH product at 324 nm, (
e
of 1.98 ꢁ 104 L/mol cm) at
ing the substrate and polarising the ketone for reaction [9]. All
a
-
25 °C. A typical reaction involved an assay solution containing
IPMS enzymes require a divalent metal for activity and some en-
zymes also display dependency on a monovalent cation [10].
Neisseria meningitidis is a human pathogen, able to infect the
respiratory system or larynx, and can also infect the blood causing
fatal septicaemia. This organism is best known for causing bacte-
rial meningitis, leading to brain damage or even death. In this
study we describe the expression and purification of N. meningitidis
80 lM AcCoA, 500 lM a-KIV, 100 lM DTP, 20 mM KCl, and
20 mM MgCl2 in 1 mL of 50 mM HEPES (pH 7.5) in a quartz cuvette,
brought to temperature, and initiated by addition of purified WT
enzyme to a concentration of 10 nM. Assays investigating uncou-
pled hydrolysis used no
centration of 3.3 M. Assays using E365Term contained a higher
AcCoA concentration of 500 M, and were initiated with 2.5
a-KIV, and an increased WT protein con-
l
l
lM
a
-IPMS (NmeIPMS), and the characterisation of the enzyme with
enzyme. All kinetic measurements were performed in duplicate,
and typical error was less than 10%. Apparent Km data were deter-
mined by fitting to the Michaelis–Menten equation, and inhibition
data were fitted to either a competitive or mixed inhibition model
using Grafit [17].
Direct assay at 232 nm. Direct assays were carried out as de-
scribed above for the DTP assay, substituting 50 mM BTP for HEPES
and omitting DTP. Changing metal concentration and pH caused
significant variation in abs(AcCoA)–abs(CoASH), so every assay
solution was calibrated using known concentrations (of the order
respect to metal dependency, leucine inhibition, and substrate
specificity. A truncated mutant, lacking the regulatory domain,
was also generated and found to be catalytically compromised.
This study highlights several common features between NmeIPMS
and MtuIPMS, and also illuminates some significant differences in
their inhibition by leucine.
Materials and methods
of 16 lM) of CoASH and AcCoA.
Cloning. The leuA gene was amplified from N. meningitidis MC58
(serogroup B) genomic DNA (ATCC) (primer information in Supple-
mentary Material). A TOPO-cloning kit (Invitrogen) was used to li-
gate the resulting 1.5 kbp PCR product into a pET-151 vector.
Plasmid was transformed into chemically competent Escherichia
coli OneShot TOP10 cells, plasmid DNA was purified, and the se-
quence was verified.
Quikchange site-directed mutagenesis was used to insert a stop
codon at residue Glu365, chosen as it is upstream of the regulatory
domain, not highly conserved and is located in undefined loop of
the MtuIPMS structure. Plasmids containing the leuA gene and
the truncated version were transformed into chemically compe-
tent E. coli BL21(DE3)Star cells for expression.
Purification. WT and E365Term NmeIPMS were expressed and
purified using identical protocols. E. coli BL21(DE3)Star cells con-
taining the desired plasmid were grown overnight in ZYM-5052
auto-inducing media at 37 °C [15]. Cells were harvested by centri-
fugation at 4700g for 30 min, and resuspended in Buffer A (50 mM
potassium phosphate, 300 mM KCl, pH 8.0). Cells were lysed by
sonication. The soluble fraction was separated by centrifugation
at 12,000g for 30 min and passed through 5 mL Talon Superflow
Gel filtration chromatography. The multimeric states of WT and
E365Term proteins were determined by gel filtration chromatogra-
phy using a Superdex 200 10/300 GL column (GE Healthcare). Sam-
ples (500
Tris–HCl (pH 7.5) with 100 mM KCl. WT enzyme was also run in
the presence of 200 M leucine.
lL, 2 mg/mL) and standards (Sigma) were run in 50 mM
l
Differential scanning fluorimetry. Differential scanning fluorime-
try (DSF) was carried out using a BioRad iCycler iQ5 Multicolour
Real-Time PCR Detection System. SYBROrange dye was added to
a protein and ligand mixture in a 96-well plate. This sealed plate
was subjected to a thermal melt program from 20 to 95 °C in
0.2 °C increments over 4 h. Each sample was measured in triplicate
and compared to a control containing ligand solution and dye, but
no protein. Melting temperatures were determined as the temper-
ature at which the greatest increase in fluorescence was measured.
Metal dependency. Residual metal from purification was re-
moved by dialysing 2 mL of 2 mg/mL enzyme at 4 °C against
250 mL of 50 mM BTP (pH 7.5), 300 mM KCl, and 250 mM EDTA
for 4 h, then against 250 mL of 50 mM BTP (pH 7.5), 300 mM KCl,
and 100
lM EDTA for 4 h, with the buffer being changed at 2 h.
Metal reactivation was measured using the DTP assay with MgCl2

170
F.H.A. Huisman et al. / Biochemical and Biophysical Research Communications 393 (2010) 168–173
replaced by various concentrations of the relevant metal as the
dichloride. Rates in the presence of Cd2+ and Zn2+ were measured
using the 232 nm assay.
Stability
Differential scanning fluorimetry (DSF) was used to investigate
Substrate analogue testing. Activity was measured according to
standard assay conditions with the substrate analogue replacing
the thermal stability of NmeIPMS in combination with various
additives (Fig. 2). WT protein denatured at 44.5 0.4 °C, and this
increased to 59 3 °C in the presence of leucine. This stabilisation
was not observed with valine or glycine. WT protein was also sta-
bilised by a-KIV, an effect that is cumulative with leucine stabilisa-
tion. Interestingly, E365Term showed similar stability to WT
a-KIV. Compounds inactive as substrates had their inhibitory
parameters determined using standard assay conditions modified
to contain varying concentrations of inhibitor and substrate. Reac-
tions were initiated with enzyme.
CoASH inhibition. CoASH inhibition was tested using a standard
232 nm assay, containing varying concentrations of CoASH and ini-
tiated with AcCoA instead of enzyme after 2 min of incubation
NmeIPMS, but exhibited no increased stability in the presence of
AcCoA,
a-KIV or leucine. Circular dichroism spectroscopy con-
firmed the melting temperature of WT NmeIPMS, and that
E365Term NmeIPMS adopted a similar secondary structure to that
of WT enzyme (Figs. S2 and S3, Supplementary Material).
(80 lM AcCoA). Time-dependence of CoASH inhibition was mea-
sured by determining the activity of preincubated NmeIPMS
(0.2 mg/mL in 50 mM BTP (pH 7.5) and 30 mM KCl) with concen-
trations of up to 1.2 mM CoASH for up to 20 min.
Kinetic characterisation
Leucine inhibition. Leucine inhibition was examined using the
standard DTP assay, with 250
concentrations from 0 to 200
bition was measured for WT enzyme at 232 nm, using 100
CoA and 250 -KIV. Assays were carried out in BTP buffer at pH
l
l
M AcCoA and
M. pH dependence of leucine inhi-
M Ac-
a
-KIV and leucine
Kinetic assays were carried out using a DTP-coupled assay,
monitoring absorbance at 324 nm to follow the generation of a
thio-pyridone by reaction of DTP with the enzyme reaction product
CoASH [10]. However, this assay method was not suitable for all ki-
netic determinations as certain conditions cause the CoASH + DTP
reaction to become rate limiting, such as high pH (>8.0) or the
presence of Zn2+, Cd2+ or CoASH. In these cases the enzyme-cata-
lysed reaction was followed by monitoring the loss of AcCoA at
232 nm.
l
lM a
6.5, 7.5, or 8.5 with leucine concentrations of 0–2 mM.
Results
Cloning and isolation
Using the DTP-coupled assay, Km values for WT NmeIPMS were
determined to be 30 2 and 35
respectively. These values are somewhat different from those re-
ported for MtuIPMS (12 M for -KIV and 136 M for Ac-
3 lM for a-KIV and AcCoA,
The sequence of the cloned WT gene was consistent with that
encoding -IPMS from N. meningitidis (NCBI Gene ID: 903489). A
a
1
l
a
5 l
mutant lacking the regulatory domain, E365Term, was generated
using site-directed mutagenesis to introduce a stop codon in place
of residue Glu365, upstream of the C-terminal regulatory domain.
This mutation was confirmed by sequencing and by the reduced
size of the expressed protein.
WT and mutant proteins were expressed and purified to homo-
geneity by a combination of immobilised metal affinity and anion
exchange chromatography. The tag was cleaved, leaving the pro-
tein with an N-terminal GIDPFT extension. The calculated molecu-
lar masses of 56,027 and 39,931 Da, for the purified WT and
E365Term proteins, respectively, corresponded well with the
masses seen by SDS–PAGE (Supplementary Material, Fig. S1).
Size exclusion chromatography indicated that both WT and
E365Term proteins are homodimeric. The presence of leucine
had no effect on oligomeric state.
CoA [11]). The kcat for WT NmeIPMS was found to be 13 0.3 sꢀ1
,
compared with kcat values of 3.5 0.1 and 2.1 0.1 sꢀ1 reported
for MtuIPMS [11]. WT NmeIPMS also catalysed uncoupled hydroly-
sis of AcCoA in the absence of a-KIV, with a Km of 250 30 lM and
a kcat of 0.011 0.001 sꢀ1. WT NmeIPMS activity was shown to vary
with pH, with maximal activity observed at pH 8.5 (Supplementary
Material, Fig. S4), and the thermal optimum of the enzyme was
found to be 40 °C (Supplementary Material, Fig. S5).
The rate of the NmeIPMS-catalysed reaction was greatly re-
duced in the truncated mutant, to the extent that the rate of
uncoupled AcCoA hydrolysis obscured any condensation reaction.
AcCoA had a Km of 150 20
kcat of 0.010 0.001 sꢀ1. This rate increased by 40% in the presence
of 500 -KIV.
lM for uncoupled hydrolysis, with a
lM a
Fig. 2. Denaturation temperature of WT (black) and E365Term (grey) NmeIPMS in the presence of substrates and allosteric inhibitor. All solutions contained 25 mM BTP
buffer (pH 7.0) and 0.16 mg/mL protein.

F.H.A. Huisman et al. / Biochemical and Biophysical Research Communications 393 (2010) 168–173
171
Metal dependence
ity before dialysis, suggesting some activity is lost during the dial-
ysis process.
Dialysis against EDTA led to a 30-fold drop in activity compared
to freshly purified NmeIPMS, indicating a strong divalent metal
dependency. In the presence of Zn2+ or Cd2+, addition of CoASH
to DTP failed to give an increase in absorbance at 324 nm in control
experiments, so the direct assay at 232 nm was used in the pres-
ence of these metals (Fig. 3).
Substrate analogue testing
A variety of ketoacids were tested as substrates for WT
NmeIPMS (Table 1). An unbranched side chain, as in
a-ketobuty-
rate and -ketopropanoate, causes a higher Km but comparable kcat
a
,
With the exception of Mg2+, all metals have optimum activating
concentrations below 0.2 mM and show significant loss of activity
at higher concentrations. Mn2+ and Co2+ show strong activation,
whereas Ni2+, Cd2+, and Zn2+ are all weakly activating. Mg2+ is dis-
tinctive in that it requires much higher concentrations to activate
the enzyme, but also shows no reduced effect at concentrations
up to 20 mM. Also, unlike the other metals tested, except Cd2+, it
does not cause a large increase in stability of NmeIPMS as mea-
sured by DSF (Table S1, Supplementary Material). The metal
dependency profile for NmeIPMS differs considerably from that ob-
served for MtuIPMS, for which Zn2+ and Cd2+ were characterised as
inhibitors, and Mn2+, Co2+, and Ni2+ all showed activation curves
whereas shortening it, as in pyruvate and 3-fluoropyruvate, raises
Km and lowers kcat. All of these are substrates for MtuIPMS other
than 3-fluoropyruvate (which has not been tested for this enzyme)
[11,18].
Analogues that were not substrates were tested for inhibition.
Several analogues were weak inhibitors, whilst 3-bromopyruvate,
3,3-dibromopyruvate and (S)-
exhibited reversible, competitive inhibition with respect to
(Table 2). (S)- -HIV can be understood as a transition state analogue
a
-hydroxyisovalerate ((S)-
a
-HIV)
a-KIV
a
of the putative alkoxide intermediate, whereas the first two seem to
gain strong (but unreactive) binding affinity from their halogens.
similar to Mg2+ [10]. In contrast to some forms of
a-IPMS that
CoASH inhibition
are activated by monovalent ions, NmeIPMS activity was not en-
hanced by either KCl or NaCl (up to 20 mM). It should be noted that
even the best specific activity attained was not as high as the activ-
Using the direct assay, product CoASH was determined not to be
an inhibitor of WT NmeIPMS. Time dependent inactivation by
Fig. 3. Steady state kinetics for specific activity of dialysed NmeIPMS by divalent metals provided as the dichloride. Specific activity is relative to dialysed NmeIPMS with no
divalent metal added. (A) Activation by Mg2+ (–j–), Mn2+ (–N–) and Co2+ (ꢂꢂdꢂꢂ). (B) Activation by Ni2+ (–h–), Cd2+ (–4–) and Zn2+ (ꢂꢂsꢂꢂ).
Table 1
Activities of alternate substrates. Assays performed in 50 mM HEPES (pH 7.5), 20 mM MgCl2, 80 lM AcCoA, and 0.6 mM KCl at 25 °C. Dialysed enzyme was used.
Substrate
Km (mM)
kcat (sꢀ1
)
Substrate
O
Km (mM)
kcat (sꢀ1
)
0.035 0.002
5.4 0.1
0.28 0.03
0.027 0.001
O
-O2 C
-O2C
0.62 0.07
1.36 0.06
3.4 0.1
3.3 0.4
1.5 0.3
105 20
0.63 0.04
0.3 0.1
O
O
-O2 C
O
-O2 C
O
F
-O2 C
-O2 C

172
F.H.A. Huisman et al. / Biochemical and Biophysical Research Communications 393 (2010) 168–173
lent metals. In contrast, only Mg2+ shows simple saturation kinet-
ics for NmeIPMS, whilst other divalent metals have distinct activity
Table 2
Inhibition of NmeIPMS by
of above 1 mM in the presence of 100
detected at 1 mM.
a-KIV analogues. ꢄ Indicates weak inhibition observed (IC50
lM a-KIV). ND indicates no inhibitory activity
maxima, and are less activating at concentrations greater than
200 l
M. Zn2+ and Cd2+ were characterised as strongly inhibitory
Inhibitor
Ki (mM)
Inhibitor
O
Ki (mM)
in previous studies [10,13]; however, these results could be arte-
factual, caused by these ions suppressing the DTP dependent assay.
Using the direct assay these metals are modest activators of
NmeIPMS. NmeIPMS shows no activation by monovalent cations
at any concentration, whereas a strong correlation between mono-
valent cation concentration and activity has been reported for
MtuIPMS, although MtuIPMS has no obvious binding site for such
a cation.
0.06 0.03
ꢄ
O
Br
Br
-O2C
H
-O2 C
O
0.03 0.02
ꢄ
OH
-O2C
-O2 C
Br
2.5
1
ꢄ
ꢄ
OH
O
O
-
CO2
Sequence alignments show that MtuIPMS and NmeIPMS share
-O2 C
-O2C
-O2C
all key residues associated with the binding of
unsurprising that they both tolerate similar changes to substrate
-KIV. Whilst pyruvate is a poor substrate, introduction of a 3-flu-
a-KIV. It is therefore
ND
ND
O
O
a
-O2C
-O2C
Ph
oro substituent increases Km 70-fold, and a 3-bromo substitution
converts pyruvate into a potent reversible inhibitor. This suggests
that bromine is a good fit for the side chain pocket of the enzyme,
unlike the significantly smaller fluorine. The three nearest residues
-
CO2
to the
possibly supporting the binding of modestly polar bromo substitu-
ent compared to the nonpolar isopropyl group of -KIV.
In common with many other enzymes that catalyse the first
committed step in a biosynthetic pathway, -IPMS is inhibited
by the pathway end product, leucine. For NmeIPMS this inhibition
pattern does not show the unusual time-dependency that was ob-
served for MtuIPMS, indicating that there is some variation in the
nature of allosteric regulation between enzymes from different
sources. The structure of MtuIPMS indicates that catalysis is med-
a-KIV side chain are the polar His99, Ser131, and Asn159,
CoASH, as observed for Saccharomyces cerevisiae
not observed for NmeIPMS.
a-IPMS [19] was
a
a
Allosteric inhibition
Allosteric inhibition of WT enzyme by leucine fits a mixed, non-
competitive model with respect to both -KIV and AcCoA (Table 3).
a
Inhibition of WT catalytic activity is specific to leucine; similar
amino acids (valine, glycine) had no effect. Uncoupled hydrolysis
of AcCoA was unaffected by leucine in both the WT and E365Term
enzymes.
iated by the (b/
a
)8 barrel domain and, as for many other (b/
a)8 bar-
rel containing enzymes,
a
-IPMS has an accessory domain that is
associated with regulation. Intriguingly, a truncated NmeIPMS,
lacking the allosteric domain, was unable to catalyse the aldol
reaction, indicating that catalytic and regulatory functions of this
enzyme cannot be readily uncoupled. In an earlier study, protease
treatment gave a MtuIPMS truncated at both the N-terminal and C-
terminal ends, reportedly giving an active barrel core, although it is
unclear whether uncoupled AcCoA hydrolysis was assessed [13]. In
contrast, our engineered NmeIPMS mutant catalysed only the
uncoupled hydrolysis of AcCoA, a reaction that was over 1000
times slower than the aldol reaction catalysed by the wild-type
enzyme.
de Carvalho et al. found that, in the presence of leucine, reac-
tions catalysed by MtuIPMS showed an initial burst of product for-
mation, followed by a slower, linear rate [8]. No such time-
dependent inhibition was observed for WT NmeIPMS (Fig. S6, Sup-
plementary Material).
WT NmeIPMS was far more sensitive to leucine at pH 6.5 than at
pH 8.5. Inhibitor at 200 lM caused a loss of 20% activity at pH 8.5,
54% loss at pH 7.5 and 89% loss at pH 6.5, whereas 2 mM inhibitor
reduced activity by 50%, 81%, and 95% at pH 8.5, 7.5, and 6.5,
respectively. pH dependence was also shown for
a-IPMS from S.
typhimurium [6], which was found to be 30-fold more sensitive
Uncoupling of regulatory and catalytic sites has been achieved
recently for MtuIPMS, where a point mutation Y410F created a leu-
cine insensitive variant [4]. The activity of this protein was consid-
erably compromised, however, giving a maximum catalytic rate
(ꢃ3% of wild-type enzyme) lower than the reported rate for the
maximally inhibited enzyme (10%). The equivalent residue
(Tyr313) and the structural element bearing it were deliberately
retained in the engineered NmeIPMS truncation. It appears likely
that the regulatory domain assists in optimal positioning of this
key residue and supports the active site structure for the uninhib-
ited enzyme. It would be interesting to resolve how the active site
to leucine inhibition at pH 6.5 than at pH 8.5.
Discussion
Active WT NmeIPMS was heterologously expressed and purified
from E. coli. In general, this enzyme exhibits similar properties to
other characterised
parameters, a tolerance for alternative
preference for alkaline pH for maximal activity, and a dimeric qua-
ternary structure. As with most other forms of -IPMS that have
a-IPMS enzymes, having comparable kinetic
a-ketoacid substrates, a
a
been characterised to date, NmeIPMS displays activation by diva-
is supported in a-IPMS enzymes that are ‘naturally truncated’. It is
also interesting to note that there are closely related enzymes cat-
alysing similar reaction chemistry that share a homologous barrel
structure and active site architecture, including the recently iden-
tified Re-citrate synthase from Clostridium kluyveri [20] and the
aldolase portion of the bifunctional enzyme 4-hydroxy-2-ketoval-
erate aldolase/acylating acetaldehyde dehydrogenase [21].
Although Re-citrate synthase is yet to be structurally characterised,
these enzymes clearly display significant variation in their extra
barrel regions. The observation that an intact regulatory domain
is critical to support catalytic function of NmeIPMS may enable
inhibitor design targeted specifically at this enzyme.
Table 3
Leucine inhibition of WT NmeIPMS with respect to AcCoA and
inhibition of MtuIPMS with respect to -KIV.
a
-KIV, compared with
a
Inhibition constants (lM)
Nme
Mtu [8]
AcCoA
a
-KIV
57 23
18
a-KIV
Ki
Ki0
8
1
22
8
2
1
52 17
2

F.H.A. Huisman et al. / Biochemical and Biophysical Research Communications 393 (2010) 168–173
173
[7] T.R. Leary, G.B. Kohlhaw, -Isopropylmalate synthase from Salmonella
a
Acknowledgments
typhimurium. Analysis of the quaternary structure and its relation to
function, J. Biol. Chem. 247 (1972) 1089–1095.
This work is supported by the Maurice Wilkins Centre for
Molecular Biodiscovery. S.R.A.D. thanks the Foundation for Re-
search, Science and Technology for postdoctoral funding (Contract
UOCX0603).
[8] L.P. de Carvalho, A. Argyrou, J.S. Blanchard, Slow-onset feedback inhibition:
inhibition of Mycobacterium tuberculosis
a-isopropylmalate synthase by L-
leucine, J. Am. Chem. Soc. 127 (2005) 10004–10005.
[9] N. Koon, C.J. Squire, E.N. Baker, Crystal structure of LeuA from Mycobacterium
tuberculosis, a key enzyme in leucine biosynthesis, Proc. Natl. Acad. Sci. USA
101 (2004) 8295–8300.
[10] L.P. de Carvalho, J.S. Blanchard, Kinetic analysis of the effects of monovalent
cations and divalent metals on the activity of Mycobacterium tuberculosis
isopropylmalate synthase, Arch. Biochem. Biophys. 451 (2006) 141–148.
[11] L.P. de Carvalho, J.S. Blanchard, Kinetic and chemical mechanism of
a
-
Appendix A. Supplementary data
a-
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bbrc.2010.01.114.
isopropylmalate synthase from Mycobacterium tuberculosis, Biochemistry 45
(2006) 8988–8999.
[12] W. Chanchaem, P. Palittapongarnpim, The significance and effect of tandem
repeats within the Mycobacterium tuberculosis leuA gene on a-isopropylmalate
synthase, FEMS Microbiol. Lett. 286 (2008) 166–170.
[13] K. Singh, V. Bhakuni, Cation induced differential effect on structural and
References
functional properties of Mycobacterium tuberculosis
synthase, BMC Struct. Biol. 7 (2007) 39.
a-isopropylmalate
[1] F.C. Bange, A.M. Brown, W.R. Jacobs, Leucine auxotrophy restricts growth of
Mycobacterium bovis BCG in macrophages, Infect. Immun. 64 (1996) 1794–
1799;
[14] D. Cavalieri, E. Casalone, B. Bendoni, G. Fia, M. Polsinelli, C. Barberio,
Trifluoroleucine resistance and regulation of alpha-isopropyl malate
synthase in Saccharomyces cerevisiae, Mol. Gen. Genet. 261 (1999) 152–160.
[15] F.W. Studier, Protein production by auto-induction in high density shaking
cultures, Protein Expr. Purif. 41 (2005) 207–234.
[16] R.B. Kapust, J. Tözsér, J.D. Fox, D.E. Anderson, S. Cherry, T.D. Copeland, D.S.
Waugh, Tobacco etch virus protease: mechanism of autolysis and rational
design of stable mutants with wild-type catalytic proficiency, Protein Eng. 14
(2001) 993–1000.
[17] R.J. Leatherbarrow, Grafit Version 6, Erithacus Software Ltd., Horly, United
Kingdom, 2007.
[18] K. Zhang, M.R. Sawaya, D.S. Eisenberg, J.C. Liao, Expanding metabolism for
biosynthesis of nonnatural alcohols, Proc. Natl. Acad. Sci. USA 105 (2008)
20653–20658.
R.A. McAdam, T.R. Weisbrod, J.L. Martin, J.D. Scuderi, A.M. Brown, J.D. Cirillo,
B.R. Bloom, W.R. Jacobs, In vivo growth characteristics of leucine and
methionine auxotrophic mutants of Mycobacterium bovis BCG generated by
transposon mutagenesis, Infect. Immun. 63 (1995) 1004–1012.
[2] M. Pátek, K. Krumbach, L. Eggeling, H. Sahm, Leucine synthesis in
Corynebacterium glutamicum: enzyme activities, structure of leuA, and effect
of leuA inactivation on lysine synthesis, Appl. Environ. Microbiol. 60 (1994)
133–140.
[3] C. Jungwirth, S.R. Gross, P. Margolin, H.E. Umbarger, The biosynthesis of
leucine. I. The accumulation of b-carboxy-b-hydroxyisocaproate by leucine
auxotrophs of Salmonella typhimurium and Neurospora crassa, Biochemistry 2
(1963) 1–6.
[4] L.P. de Carvalho, P. Frantom, A. Argyrou, J. Blanchard, Kinetic evidence for
[19] G.B. Kohlhaw,
a-Isopropylmalate synthase from yeast, Methods Enzymol.
inter-domain communication in the allosteric regulation of a-isopropylmalate
synthase from Mycobacterium tuberculosis, Biochemistry (2009).
(1988) 414–423.
[20] F. Li, C.H. Hagemeier, H. Seedorf, G. Gottschalk, R.K. Thauer, Re-citrate synthase
from Clostridium kluyveri is phylogenetically related to homocitrate synthase
and isopropylmalate synthase rather than to Si-citrate synthase, J. Bacteriol.
189 (2007) 4299–4304.
[21] B.A. Manjasetty, J. Powlowski, A. Vrielink, Crystal structure of a bifunctional
aldolase-dehydrogenase: sequestering a reactive and volatile intermediate,
Proc. Natl. Acad. Sci. USA 100 (2003) 6992–6997.
[5] E. Teng-Leary, G.B. Kohlhaw, Mechanism of feedback inhibition by leucine.
Binding of leucine to wild-type and feedback-resistant
a-isopropylmalate
synthases and its structural consequences, Biochemistry 12 (1973) 2980–
2986.
[6] G.B. Kohlhaw, T.R. Leary, H.E. Umbarger,
a-Isopropylmalate synthase from
Salmonella typhimurium. Purification and properties, J. Biol. Chem. 244 (1969)
2218–2225.