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Biochem. J. (2008) 415, 45–56 (Printed in Great Britain)
45
doi:10.1042/BJ20080242
Molecular basis of the substrate specificity and the catalytic mechanism
of citramalate synthase from Leptospira interrogans
Jun MA*‡1 , Peng ZHANG†‡1 , Zilong ZHANG*‡, Manwu ZHA†, Hai XU*2 , Guoping ZHAO*§3 and Jianping DING†3
Leptospira interrogans is the causative agent for leptospirosis, a
zoonotic disease of global importance. In contrast with most other
micro-organisms, L. interrogans employs a pyruvate pathway
to synthesize isoleucine and LiCMS (L. interrogans citramalate
synthase) catalyses the first reaction of the pathway which
converts pyruvate and acetyl-CoA into citramalate, thus making it
an attractive target for the development of antibacterial agents. We
report here the crystal structures of the catalytic domain of LiCMS
and its complexes with substrates, and kinetic and mutagenesis
studies of LiCMS, which together reveal the molecular basis of the
high substrate specificity and the catalytic mechanism of LiCMS.
The catalytic domain consists of a TIM barrel flanked by an
extended C-terminal region. It forms a homodimer in the crystal
structure, and the active site is located at the centre of the TIM
barrel near the C-terminal ends of the β-strands and is composed
of conserved residues of the β-strands of one subunit and the Cterminal region of the other. The substrate specificity of LiCMS
towards pyruvate against other α-oxo acids is dictated primarily
by residues Leu81 , Leu104 and Tyr144 , which form a hydrophobic
pocket to accommodate the C2 -methyl group of pyruvate. The
catalysis follows the typical aldol condensation reaction, in which
Glu146 functions as a catalytic base to activate the methyl group
of acetyl-CoA to form an enolated acetyl-CoA intermediate and
Arg16 as a general acid to stabilize the intermediate.
INTRODUCTION
into (R)-citramalate in the first reaction of this pathway. So far,
citramalate synthase has only been found in L. interrogans and
a few other micro-organisms, such as Methanococcus jannaschii
[13] and thus it could be an attractive target for the development of
antibacterial agents for both therapeutic and preventive purposes.
The biosynthesis of isoleucine in L. interrogans is very similar
to the biosynthesis of leucine in most micro-organisms, in which
α-isopropylmalate synthase catalyses the synthesis of α-isopropylmalate from α-Kiv (α-oxoisovalerate) and acetyl-CoA in
the first reaction, and the catalytic reaction is inhibited by the
end-product, leucine [14,15]. In addition, the biosynthesis of
isoleucine in L. interrogans employs several other enzymes that
are also utilized in the biosynthesis of leucine in most microorganisms, such as α-isopropylmalate isomerase (encoded by the
LeuC/D gene) and β-isopropylmalate dehydrogenase (encoded
by the LeuB gene) [12], suggesting that the two pathways
share some common features. The crystal structure of MtIPMS
(Mycobacterium tuberculosis α-isopropylmalate synthase) in
complex with its substrate α-Kiv has been solved, showing that
the enzyme functions as a homodimer and consists of a catalytic
domain and a regulatory domain connected together by two
subdomains and a disordered region [14]. However, the exact
mechanisms of the catalytic reaction and the feedback inhibition
Leptospirosis is a zoonotic disease which has emerged as a globally important infectious disease and has caused endemic
and epidemic severe pulmonary haemorrhage in recent years
(reviewed in [1,2]). It is more common in the tropics where
conditions for its transmission are favourable, but can also occur
in urban environments of industrialized and developing countries
and rural areas worldwide [3–7]. The pathogen of leptospirosis
is Leptospira interrogans, which belongs to the spirochetes, an
evolutionarily primitive species of bacteria [8]. It has long been
noticed that the metabolism of spirochetes differs substantially
from those of well-studied micro-organisms [9,10]. In particular,
most micro-organisms use the threonine pathway to synthesize
isoleucine, in which the first reaction, synthesis of α-Kb (α-oxobutyrate) from threonine, is catalysed by L-threonine dehydratase
(encoded by the ilvA gene). In our previous studies, we did not
find the ilvA gene in L. interrogans through analysis of the genome
sequence [11]; instead, we found that L. interrogans might use
a threonine-independent pathway, namely the Pyr (pyruvate)
pathway, to synthesize isoleucine [12]. Specifically, L. interrogans
uses LiCMS (L. interrogans citramalate synthase; encoded by the
CimA gene) to catalyse the conversion of Pyr and acetyl-CoA
Key words: aldol condensation, catalytic mechanism, citramalate
synthase, crystal structure, feedback inhibition, substrate
specificity.
Abbreviations used: Glx, glyoxylate; His6 , hexahistidine; HS-CoA, reduced CoA; ICP-MS, inductively coupled plasma MS; α-Kb, α-oxobutyrate; α-Kiv, αoxoisovalerate; Li CMS, Leptospira interrogans citramalate synthase; Li CMSC, C-terminal regulatory domain of Li CMS; Li CMSN, N-terminal catalytic domain
of Li CMS; Mal, malonate; MR, molecular replacement; Mt IPMS, Mycobacterium tuberculosis α-isopropylmalate synthase; Ni2+ -NTA, Ni2+ -nitrilotriacetate;
Pyr, pyruvate; RMSD, root mean square deviation; SAD, single-wavelength anomalous dispersion.
1
These authors contributed equally to this work.
2
Present address: State Key Laboratory of Microbial Technology, Shandong University, Jinan, Shandong 250104, China.
3
Correspondence may be addressed to either of these authors (email [email protected] or [email protected]).
The structural co-ordinates reported for the N-terminal catalytic domain of Leptospira interrogans citramalate synthase in complex with malonate, in
complex with pyruvate and in complex with pyruvate and acetyl-CoA will appear in the Protein Data Bank under accession codes 3BLE, 3BLF and 3BLI
respectively.
c The Authors Journal compilation c 2008 Biochemical Society
Biochemical Journal
*Laboratory of Microbial Molecular Physiology, Institute of Plant Physiology and Ecology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, 300 Feng-Lin
Road, Shanghai 200032, China, †State Key Laboratory of Molecular Biology and Research Center for Structural Biology, Institute of Biochemistry and Cell Biology, Shanghai Institutes
for Biological Sciences, Chinese Academy of Sciences, 320 Yue-Yang Road, Shanghai 200031, China, ‡Graduate School of Chinese Academy of Sciences, 320 Yue-Yang Road,
Shanghai 200031, China, and §Shanghai-MOST Key Laboratory for Health and Disease Genomics, Chinese National Human Genome Center, Shanghai 201203, China
46
J. Ma and others
are still unclear. LiCMS is 516 amino acids in length, with a
molecular mass of 56 kDa. On the basis of sequence homology
with MtIPMS and secondary-structure prediction, LiCMS appears
to be composed of an N-terminal catalytic domain (residues 1–
330) and a C-terminal regulatory domain (residues 390–516),
connected together by a linker region of approx. 60 amino-acid
residues. Although the catalytic reactions catalysed by LiCMS and
MtIPMS both belong to the aldol condensation reaction, the two
enzymes use different substrates: LiCMS has a high specificity
for Pyr, whereas MtIPMS prefers α-Kiv, which has an isopropyl
group at the C2 position instead of a methyl group as in Pyr.
To understand the catalytic mechanism and feedback-inhibition
mechanism of LiCMS and provide the molecular basis for
development of antibacterial agents to treat leptospirosis, we
carried out structural and biochemical studies of LiCMS.
We report here the crystal structures of LiCMSN (N-terminal
catalytic domain of LiCMS) in complex with Mal (malonate) at
2.0 Å (1 Å = 0.1 nm) resolution, in complex with Pyr at 2.6 Å
resolution and in complex with Pyr and acetyl-CoA at
2.5 Å resolution. Analyses of these structures, together with
mutagenesis and kinetic studies, identified the key residues
involved in the recognition and binding of the substrates and
the catalytic reaction. The structural and biochemical results
together provide the molecular basis for the substrate specificity
of LiCMS and reveal the catalytic mechanism of LiCMS. The
crystal structure of LiCMSC (C-terminal regulatory domain of
LiCMS) and the mechanism of feedback inhibition of LiCMS
will be described elsewhere (P. Zhang, J. Ma, Z. Zhang, M. Zha,
H. Xu, G. Zhao and J. Ding, unpublished work).
MATERIALS AND METHODS
Cloning, expression, and purification of Li CMS
The LiCMS gene (CimA) used for clone construction and
protein expression was amplified by PCR from the genomic
DNA of L. interrogans (serogroup Icterohaemorrhagiae, serovar
lai, type strain 56601). The following primers were used:
5 -CATATGGGACGTTCTCAAAAGGTATC-3 (NdeI restriction
site underlined), 5 -AAGCTTATGCCGGTTGTGAACATATT-3
(HindIII restriction site underlined). The gene fragment was digested by NdeI/HindIII and inserted into the pET-28b expression
plasmid resulting in N-terminal His6 (hexahistidine)-tagged pET28b-LiCMS. The plasmid was transformed into Escherichia coli
BL21(DE3) strain (Novagen) and the transformed bacterial cells
were cultured at 37 ◦C in TB (Terrific Broth) medium containing
50 μg/ml kanamycin. Protein expression was induced by adding
IPTG (isopropyl β-D-thiogalactoside) into the medium to a final
concentration of 1 mM. The cells were harvested by centrifugation at 5000 g for 10 min at 4 ◦C, resuspended in a lysis buffer
[50 mM Tris/HCl (pH 8.0), 300 mM NaCl and 1 mM PMSF], and
then disrupted using a French Press (Thermo Scientific).
The recombinant protein was purified with affinity chromatography using a Ni2+ -NTA (Ni2+ -nitrilotriacetate) Superflow
column (Qiagen) pre-equilibrated with buffer A [50 mM Tris/HCl
(pH 8.0) and 300 mM NaCl] and then washed with buffer B (buffer
A supplemented with 40 mM imidazole) to remove proteins which
have bound non-specifically. The target protein was eluted with
buffer C (buffer A supplemented with 300 mM imidazole), and the
eluted fractions were dialysed against buffer D [20 mM Tris/HCl
(pH 8.4) and 50 mM NaCl] and then digested by thrombin to
remove the N-terminal His6 -tag. The protein sample was further
purified using an anion-exchange Q-column (Pharmacia). After
the two-step purification, the target protein was of sufficient
purity (above 95 %) as shown by SDS/PAGE analysis (14 %
c The Authors Journal compilation c 2008 Biochemical Society
gels) and was then concentrated to approx. 20 mg/ml in buffer
D by ultrafiltration for further structural and biochemical studies.
Selenomethionine-substituted LiCMS protein suitable for
structure determination was prepared following a method
described previously [16]. Purification of the selenomethionine
LiCMS protein was performed using the same methods as for
the native protein. Constructs of mutant LiCMS containing point
mutations were generated using the QuikChange® site-directed
mutagenesis kit (Stratagene), and all of these clones were verified
by DNA sequencing. The plasmid pET-28b-LiCMSN containing
the gene fragment corresponding to LiCMSN (residues 1–330)
was also constructed using a method similar to that used to clone
the full-length gene. Expression and purification of the LiCMS
mutants and the LiCMSN truncation mutant were the same as for
the wild-type full-length enzyme as described above.
Crystallization and diffraction data collection
The full-length LiCMS protein was used for the crystallization
experiments, which were performed at room temperature (20 ◦C)
using the hanging-drop vapour-diffusion method. Two types of
crystals with different morphologies were grown in the same
drops containing equal volumes (2 μl) of the protein solution
(approx. 20 mg/ml LiCMS) and the reservoir solution [0.1 M
Hepes (pH 7.5) and 2 M sodium malonate]. Crystals of one
type are tetragonal bipyramids and the other type were hexagonal
plates. SDS/PAGE analyses (14 % gels) of the crystallization
solution and the dissolved crystals of both types showed that fulllength LiCMS had been hydrolysed into two fragments during
crystallization (Figure 1A). Addition of the protease inhibitor
PMSF could slow down, but could not prevent, this hydrolysis.
MS analyses indicated that the crystals of the bipyramidal shape
contain the larger fragment with a molecular mass of approx.
33 kDa, which corresponds to LiCMSN, and the crystals of the
plate shape contain the smaller fragment with a molecular mass of
approx. 14 kDa, which corresponds to LiCMSC. The two types of
crystals were used to determine the crystal structures of LiCMSN
and LiCMSC respectively. Attempts to crystallize the
LiCMSN truncation mutant in the absence or presence of Pyr
and/or acetyl-CoA have been unsuccessful in producing any
crystals so far.
For diffraction data collection, the LiCMSN crystals were first
cryo-protected using paratone oil (Hampton Research) and then
flash-cooled in liquid nitrogen. Selenium SAD (single-wavelength anomalous dispersion) diffraction data of LiCMSN were
collected to a resolution of 2.6 Å from a flash-cooled crystal at
100 K at the Photon Factory (Ibaraki, Japan), beamline BL17,
and the native diffraction data collected to a resolution of 2.0 Å at
beamline BL6A. The diffraction data were processed, integrated
and scaled together using the HKL2000 suite [17]. The crystals
of native LiCMSN belong to space group P31 21, with unitcell dimensions a = b = 85.2 Å and c = 112.9 Å, containing one
LiCMSN molecule in the asymmetric unit with a solvent content
of 60 %. Subsequent structure refinement has revealed that a Mal
molecule is bound at the active site, mimicking the substrate Pyr.
Therefore this structure should be considered as the structure of
LiCMSN in complex with Mal (LiCMSC–Mal). The crystals
of LiCMSN in complex with Pyr (LiCMSN–Pyr) were prepared
by soaking the crystals of the LiCMSN–Mal complex in the
crystallization drop supplemented with 50 mM Pyr for 24 h.
The crystals of LiCMSN in complex with Pyr and acetyl-CoA
(LiCMSN–Pyr–CoA) were prepared by soaking crystals of the
LiCMSN–Mal complex in the crystallization drop supplemented
with 50 mM Pyr and 40 mM acetyl-CoA for 24 h. The diffraction
data of the LiCMSN–Pyr complex were collected to 2.6 Å
Substrate specificity and catalytic mechanism of Li CMS
Figure 1
47
Characterization of Li CMS
(A) SDS/PAGE analysis of the Li CMS protein samples. Full-length Li CMS was hydrolysed during crystallization into two fragments: a larger fragment (corresponding to Li CMSN) and a smaller
fragment (corresponding to Li CMSC). Dissolved crystals of Li CMSN and Li CMSC, freshly purified full-length Li CMS and the crystallization solution after 1 month (Drop) were analysed.
Molecular-mass markers are shown in the lane between Li CMSC and Li CMS (in kDa). (B) The enzymatic activity of Li CMS in the absence and presence of different metal ions. Li CMS has very little
of the enzymatic activity of Li CMS by Mn2+ .
activity in the absence of bivalent metal ions and shows the highest activity in the presence of Mn2+ . Results are means +
− S.D., n = 2 (C) The activation
2+
+
n
=
2
(D)
The
enzymatic
activity
of
Li
CMS
in
the
presence
of
different
univalent
metal
ions
in
addition
to
0.8
mM
Mn
.
K
and
NH4 + can act as co-activators of Li CMS
Results are means +
S.D.,
−
in the presence of Mn2+ . (E) The co-activation of the enzymatic activity of Li CMS by K+ in the presence of Mn2+ .
resolution from a flash-cooled crystal at 100 K at the Beijing
Synchrotron Radiation Facility (Beijing, China) and processed
and scaled using HKL2000. The diffraction data of the LiCMSN–
Pyr–CoA complex were collected to 2.5 Å resolution from a flashcooled crystal at 100 K using an in-house Rigaku R-AXIS IV++
diffractometer and processed and scaled using the CrystalClear
suite [18]. The statistics of the diffraction data are summarized in
Table 1.
Structure determination and refinement
The structure of the LiCMSN–Mal complex was solved using the
SAD method implemented in the program SOLVE [19]. The SAD
phases were improved by statistical-density modification, including solvent flattening and histogram matching, using the program
RESOLVE [20], increasing the overall figure-of-merit from
0.39 to 0.77 at 2.6 Å resolution. The resultant electron density
c The Authors Journal compilation c 2008 Biochemical Society
48
Table 1
J. Ma and others
Summary of diffraction data (a) and structure refinement (b) statistics
For the resolution range, the numbers in parentheses refer to the highest resolution shell. Rmerge = hkl i |I i (hkl )i − I (hkl )|/ hkl i I i (hkl ). R factor = ||Fo |-|Fc ||/|Fo |.
(a)
Wavelength (Å)
Resolution range (Å)
Space group
Cell parameters a = b /c (Å)
Unique reflections [I/σ (I) > 0]
Average I/σ (I)
Redundancy
Completeness (%)
Rmerge (%)
Selenomethionine Li CMSN
Li CMSN–Mal
Li CMSN–Pyr
Li CMSN–Pyr–CoA
0.9795
50.0–2.6
(2.69–2.6)
P 31 21
85.5/113.8
15263
55.6 (15.4)
21.8 (22.2)
100 (100)
8.1 (43.1)
1.0000
50.0–2.0
(2.08–2.0)
P 31 21
85.2/112.9
32925
26.3 (5.0)
7.7 (7.8)
100 (100)
6.8 (42.7)
1.0000
50.0–2.6
(2.69–2.6)
P 31 21
85.1/116.1
15521
33.8 (5.6)
8.8 (8.8)
100 (99.6)
6.0 (45.6)
1.5418
20.0–2.5
(2.59–2.5)
P 31 21
85.0/115.7
17171
5.3 (1.9)
8.4 (8.7)
96.6 (96.6)
10.4 (35.9)
Li CMSN–Mal
Li CMSN–Pyr
Li CMSN–Pyr–CoA
22.3
25.4
307
154
22.4
25.9
306
79
22.7
27.7
311
82
39.1
38.1
–
35.9
44.8
0.006
1.4
57.7
59.9
–
46.3
51.7
0.010
1.7
90.8
8.4
0.7
87.5
12.1
0.4
(b)
R factor (%)
Free R factor (%)
Number of residues
Number of water molecules
Average B factor (Å2 )
Protein
Substrate (Mal or Pyr)
Acetyl-CoA
Zn2+
Water molecules
RMSD bond lengths (Å)
RMSD bond angles (◦)
Ramachandran plot (%)
Most favoured regions
Allowed regions
Generously allowed regions
map was of high quality and RESOLVE automatically built
60 % of the polyalanine model. The full-structure model was
built manually using the program Coot [21]. Structure refinement
was carried out against the 2.0 Å native data using the program
CNS with standard protocols [22]. The structures of the LiCMSN–
Pyr and LiCMSN–Pyr–CoA complexes were solved using
the MR (molecular-replacement) method using CNS, with the
structure of the LiCMSN–Mal complex used as the starting model.
In all three complexes, there was strong electron density at the
active site that matched the bound substrate (Mal or Pyr) very
well. However, the electron density for the bound acetyl-CoA
was poor and thus the occupancy of acetyl-CoA was set to 0.5 in
the structure refinement. In addition, there was a residual electron
density near the bound substrate that could be fitted with a bivalent
metal ion. The bound metal ion was identified as an Zn2+ ion copurified with the enzyme from the expression system (see the
Results and discussion section). The statistics of the structure
refinement and the quality of the structure models are summarized
in Table 1.
Enzymatic activity assay
The enzymatic activity of both wild-type and mutant LiCMS
and the LiCMSN truncation mutant was assayed by monitoring
the production of HS-CoA (reduced CoA) over time as described
previously [13]. Specifically, a typical reaction mixture consisted
of a varied concentration of Pyr (or other α-oxo acids in the
determination of substrate specificity), a varied concentration of
acetyl-CoA, 0.8 mM MnCl2 , 50 mM KCl and 15 nM LiCMS, in a
total volume of 50 μl using 0.1 M Hepes (pH 7.7). To determine
c The Authors Journal compilation c 2008 Biochemical Society
44.5
49.9
76.5
36.3
47.6
0.008
1.4
88.0
11.6
0.4
the effect of Pyr, the concentration of acetyl-CoA was fixed at
4 mM (four times K m ) and the concentration of Pyr was varied
from 30 μM to 800 μM. To determine the effect of acetyl-CoA,
the concentration of Pyr was fixed at 2 mM (30 times K m ) and the
concentration of acetyl-CoA was varied from 100 μM to 6 mM.
The reaction mixture was first equilibrated on ice for 10 min. The
reaction was performed in a water bath at 37 ◦C for 15 min and
then stopped by placing the reactant on ice for 10 min. For
measurement of the produced HS-CoA, 35 μl of 1 M Tris/HCl
(pH 8.0), 25 μl of 10 mM DTNB [5,5 -dithio-bis(2-nitrobenzoic
acid] in 0.1 M Tris/HCl (pH 8.0) and 390 μl of distilled H2 O
were added into the reactant to a total volume of 500 μl, and
the generated yellow 5-mercapto-2-nitrobenzoic acid was quantified at an absorbance (A) of 412 nm using a Beckman Du800
spectrometer and blanked against an identical incubation sample
without the substrate (either Pyr or other α-oxo acids). The
concentration of HS-CoA was calculated from a linear standard
curve generated with known concentrations (0 to 200 μM) of
2-mercaptoethanol. The production of HS-CoA was found to
be linear over the time period of the assay, and the product
formation was a linear function of the amount of the enzyme
added. All experiments were repeated at least twice under the
same conditions. All kinetic data were analysed using Prism 4.0
for Windows (GraphPad) and the kinetic parameters (V max , K m
and kcat ) were calculated from the Scatchard plots.
In the experiments to measure the effects of different metal
ions on the enzymatic activity of LiCMS, we first removed the
bound metal ions co-purified with the enzyme by using
the following procedure. The purified protein was first dialysed
six times for 30 min against 20 mM Tris/HCl (pH 8.0) containing
Substrate specificity and catalytic mechanism of Li CMS
50 mM KCl and 10 mM EDTA to remove the bivalent metal
ions, and then dialysed four times for 30 min against 20 mM
Tris/HCl (pH 8.0) containing 50 mM KCl to remove the EDTA.
The protein was further dialysed four times for 30 min against
20 mM Tris/HCl (pH 8.0) to remove univalent metal ions. This
sample was indeed metal-free as shown by ICP-MS (inductively
coupled plasma MS) analysis (see Supplementary Table S1
at http://www.BiochemJ.org/bj/415/bj4150045add.htm) and thus
was used for the activity assay of LiCMS in the presence of
different metal ions using the same method as described above.
RESULTS AND DISCUSSION
Enzymatic activity of Li CMS
The enzymes catalysing the adol condensation reaction require the
participation of bivalent metal ions for their activities. For
MtIPMS, Mg2+ and Mn2+ result in the highest activation and K+
can act as a co-activator, but Zn2+ and Cd2+ can inhibit enzymatic
activity [23]. To examine the effects of different bivalent and
univalent metal ions on the activity of LiCMS, we first removed
the metal ions co-purified with the enzyme with EDTA and then
measured the activity of the metal-free enzyme in the presence
of various metal ions (see the Materials and methods section).
The results showed that LiCMS has no activity in the absence of
metal ions; Mn2+ is the most effective activator, followed by Co2+ ,
Ca2+ , Mg2+ and Ni2+ ; and Cu2+ and Zn2+ can inhibit the activity
of LiCMS (Figure 1B). The activation of the activity of LiCMS
by Mn2+ occurs in a concentration-dependent manner, with a K act
of 75 μM (Figure 1C). K+ alone has a very minor effect on the
activity of LiCMS, but K+ and NH4 + could act as co-activators
in the presence of Mn2+ (Figures 1B and 1D). The co-activation
by K+ in the presence of Mn2+ also occurs in a concentrationdependent manner, with a K act of 1.2 mM (Figure 1E). Therefore
Mn2+ was chosen as the activator and K+ as the co-activator, and
the enzymatic activity of LiCMS was assayed at 0.8 mM Mn2+
(approx. 10 times K act ) and 50 mM K+ (approx. 40 times K act ) as
the saturated concentration (protein/Mn2+ /K+ ≈ 1:2800:10 000).
Under this condition, the enzymatic activity of full-length LiCMS
was determined to be 9.4 μmol/min per mg, and the K m value to be
60 μM for Pyr and 1.1 mM for acetyl-CoA, which is higher than
that of MtIPMS for acetyl-CoA (12 μM) [24]. We also measured
the enzymatic activity of the LiCMSN truncation mutant and the
results show that LiCMSN has weaker binding affinities for both
Pyr and acetyl-CoA (K m being increased by 2.5- and 5-fold
respectively), but has very little enzymatic activity (kcat being
decreased by 2280- and 940-fold respectively) (Table 2).
Overall structure of Li CMSN
The structure of the LiCMSN–Mal complex was solved using the
SAD method and refined to 2.0 Å resolution with an R factor of
22.3 % and a free R factor of 25.4 % respectively (Table 1). In this
complex, there was strong electron density at the active site that
matches a Mal molecule very well (see Supplementary Figure S1A
at http://www.BiochemJ.org/bj/415/bj4150045add.htm). Because
LiCMS has no enzymatic activity for Mal (results not shown),
the bound Mal is biologically irrelevant and appears to be an
artifact as a result of it being present at a high concentration
(2 M) in the crystallization solution, mimicking the substrate Pyr.
In addition, there was an electron density at the active site near
the bound ligand which closely matches a bivalent metal ion.
Since no bivalent metal ion was added in either the purification
or crystallization steps, the bound metal ion could be these ions
present at higher concentrations in the expression system (such as
49
Zn2+ , Mg2+ , Mn2+ , Ca2+ etc.) or Ni2+ stripped from the Ni2+ -NTA
column during purification.
To characterize the type and source of the bound metal ion,
we first performed ICP-MS analysis of the purified enzymes.
The results show that the purified protein contains both Zn2+ and
Ni2+ at high concentrations (Supplementary Table S1). The bound
Zn2+ was co-purified with the enzyme from the expression system,
whereas the concomitant Ni2+ was stripped from the Ni2+ -NTA
column during purification. These metal ions could be removed
to the background level by treatment of the purified enzyme with
EDTA. We then carried out fluorescence scans of the crystals of
the LiCMSN–Mal complex and the LiCMSN–Pyr–CoA complex
at a synchrotron and the results show that the crystals have a
strong anomalous signal at the wavelength of Zn2+ , but not other
metal ions. We further collected anomalous diffraction data at the
wavelengths of Zn2+ and Ni2+ respectively, and determined
the structures using MR. The results show that there is a strong
anomalous density peak (above 10σ contour level) for a Zn2+
ion at the active site (see Supplementary Figure S2 at http://
www.BiochemJ.org/bj/415/bj4150045add.htm), but a very weak
anomalous density peak (approx. 2σ contour level) for a Ni2+
ion. All of these results together indicate that the bound metal ion
at the active site is Zn2+ , which is co-purified with the enzyme
from the expression system.
The crystals of the LiCMSN–Pyr complex were obtained by
soaking the crystals of the LiCMSN–Mal complex in the crystallization solution supplemented with excess Pyr. The structure of
the LiCMSN–Pyr complex was solved using the MR method and
refined to 2.6 Å resolution (Table 1). At the active site, there
was very good electron density correlating to a Pyr molecule
(Supplementary Figure S1B) and a Zn2+ ion. Attempts to grow
crystals of LiMCS in complex with both Pyr and acetyl-CoA using
co-crystallization have been unsuccessful so far. Thus we prepared
crystals of LiCMSN–Pyr–CoA complex by soaking the crystals
of the LiCMSN–Mal complex in the crystallization solution
supplemented with excess Pyr and acetyl-CoA. The structure
of the LiCMSN–Pyr–CoA complex was solved using the MR
method and refined to 2.5 Å resolution (Table 1). At the active
site, there was very good electron density for a Pyr molecule
and a Zn2+ ion. In addition, there was poor electron density in
a deep surface groove near the bound Pyr corresponding to an
acetyl-CoA molecule (Supplementary Figure S1C). Although the
electron density for the adenosine moiety and thioacetyl moiety of
acetyl-CoA was fairly defined and consistent in several diffraction
datasets, the electron density for the middle part of acetylCoA was invisible, and anomalous diffraction data collected at
the wavelength of sulfur did not reveal an evident anomalous
density peak at the active site for the sulfur atom of acetylCoA. This is probably due to the low binding affinity of
acetyl-CoA with LiCMSN and a partial occupancy resulting
from the soaking experiment. Regardless, the electron density
for acetyl-CoA is sufficient for us to define its position at the
active site, which provides some valuable information about
the catalytic mechanism. Since this is the only surface groove
near the active site of LiCMS where the coenzyme could bind, we
believe that it is the binding site of acetyl-CoA. In the structure of
the MtIPMS–α-KIV complex, a similar surface groove adjacent
to the bound α-Kiv is also suggested to be the binding site of
acetyl-CoA [14].
The overall structures of LiCMSN in all three complexes are
very similar, with a RMSD (root mean square deviation) of
approx. 0.3 Å, indicating that the binding of Pyr and/or acetylCoA does not cause a significant conformational change to the
overall structure and to the active site. The structure model of
LiCMSN consists of amino-acid residues 7–325, with residues
c The Authors Journal compilation c 2008 Biochemical Society
50
J. Ma and others
c The Authors Journal compilation c 2008 Biochemical Society
Table 2
Kinetic data for the catalytic reaction of the active site (a), the C-terminal region of Li CMSN (b) and the substrate-binding site (c) of wild-type and mutant Li CMSa
All kinetic parameters are the means of duplicate determinations. N.D., not detectable; where the enzymatic activity is too low to be detected.
(a)
Li CMS
Effect on the binding
of acetyl-CoA
Wild-type
Li CMSN
E146D
E146Q
R16K/R16Q
F83A
Effect on the binding
of Pyr
Wild-type
Li CMSN
D17N∗
D17A∗
T179A
K m (μM)
k cat (s−1 )
k cat /K m (M−1 · s−1 )
1118 +
− 49
5273 +
− 2457
1511 +
− 584
2391 +
− 358
N.D.
5186 +
− 1807
10.3 +
− 0.5
−2
1.1 +
− 0.2 × 10−2
2.1 +
0.3 × 10
−
−2
2.4 +
− 0.4 × 10
N.D.
−2
8.5 +
− 1.6 × 10
3
9.2 +
− 0.6 × 10
2.2 +
1.1
−
1
1.4 +
− 0.6 × 101
1.0 +
− 0.2 × 10
N.D.
1
1.6 +
− 0.7 × 10
60 +
−4
150 +
− 26
+
263 − 42
2036 +
− 787
979 +
− 76
9.13 +
− 0.72
−2
0.4 +
− 0.1 × 10
+
1.9 − 0.4 × 10−2
−2
2.9 +
− 1.2 × 10−2
4.9 +
− 0.5 × 10
5
1.5 +
− 0.2 × 10
25 +
7
−
1
7.3 +
− 1.8 × 101
1.4 +
0.8 × 10
−
1
5.0 +
− 0.6 × 10
(b)
Acetyl-CoA
Pyr
Li CMS
K m (μM)
k cat (s−1 )
k cat /K m (M−1 · s−1 )
K m (μM)
k cat (s−1 )
k cat /K m (M−1 · s−1 )
Wild-type
H302A/H302N
D304A
N310A
L311A
Y312A
1118 +
− 49
N.D.
5851 +
− 1132
2409 +
− 87
8921 +
− 1814
N.D.
10.3 +
− 0.5
N.D.
0.62 +
− 0.07
5.9 +
− 0.2
1.7 +
− 0.4
N.D.
3
9.2 +
− 0.6 × 10
N.D.
2
1.1 +
− 0.2 × 103
2.5 +
0.1 × 10
−
+
1.9 − 0.6 × 102
N.D.
60 +
−4
N.D.
153 +
− 16
170 +
− 22
1272 +
− 476
N.D.
9.1 +
− 0.7
N.D.
0.32 +
− 0.01
3.8 +
− 0.2
1.7 +
− 0.4
N.D.
5
1.5 +
− 0.2 × 10
N.D.
3
2.1 +
− 0.2 × 104
2.2 +
0.3 × 10
−
+
1.4 − 0.6 × 103
N.D.
(c)
Pyr
Li CMS
K m (μM)
k cat (s−1 )
Wild-type
L104V
Y144L
Y144V
L81A
L81V
60 +
−4
105 +
− 18
15529 +
− 2057
6859 +
− 491
282 +
− 22
198 +
− 22
9.1 +
− 0.7
2.7 +
− 0.6
0.12 +
− 0.01
1.7 +
− 0.1
0.58 +
− 0.05
0.9 +
− 0.1
5
1.5 +
− 0.2 × 104
2.5 +
0.7 × 10
−
7.7 +
− 1.3
2
2.4 +
− 0.3 × 103
2.1 +
0.3 × 10
−
3
4.5 +
− 0.8 × 10
∗
α-Kb
Glx
k cat /K m
(M−1 · s−1 )
K m (μM)
1470 +
− 133
2898 +
− 564
3498 +
1491
−
1023 +
− 243
+
10605 − 637
7225 +
− 412
α-Kiv
k cat (s−1 )
k cat /K m
(M−1 · s−1 )
K m (μM)
k cat (s−1 )
k cat /K m
(M−1 · s−1 )
0.51 +
− 0.05
0.6 +
− 0.1
0.005 +
0.001
−
0.03 +
− 0.01
+
0.19 − 0.01
0.21 +
− 0.01
2
3.5 +
− 0.5 × 102
1.9 +
0.6 × 10
−
1.7 +
− 0.8
1
3.4 +
− 1.3 × 10
+
1.8 − 0.2 × 101
1
3.0 +
− 0.3 × 10
695 +
− 65
471 +
− 130
12606 +
− 2995
6519 +
− 601
+
465 − 112
2924 +
− 418
0.09 +
− 0.01
0.03 +
− 0.01
0.005 +
− 0.002
0.62 +
− 0.06
+
0.05 − 0.01
0.014 +
− 0.002
2
1.3 +
− 0.2 × 101
6.8 +
2.9 × 10
−
0.5 +
− 0.2
1
9.6 +
− 1.3 × 102
+
1.0 − 0.4 × 10
4.8 +
− 1.0
Acetyl-CoA
K m (μM)
k cat (s−1 )
k cat /K m
(M−1 · s−1 )
N.D.
253 +
− 52
151 +
−6
52854 +
296
−
+
98 − 6
N.D.
N.D.
0.03 +
− 0.01
0.023 +
− 0.001
0.062 +
0.001
−
+
0.018 − 0.002
N.D.
N.D.
2
1.3 +
− 0.4 × 102
1.5 +
0.1 × 10
−
1.17 +
− 0.01
2
1.9 +
− 0.2 × 10
N.D.
These mutants have relatively low enzymatic activity. In order to produce the most activated enzyme, the concentration of Mn2+ was increased from 0.8 mM (for other mutants) to 5 mM.
K m (μM)
k cat (s−1 )
k cat /K m
(M−1 · s−1 )
1118 +
− 49
3028 +
− 386
1904 +
− 536
1691 +
438
−
1137 +
− 162
1214 +
− 208
10.3 +
− 0.5
3.9 +
− 0.6
0.017 +
− 0.002
0.6 +
0.2
−
0.7 +
− 0.1
1.1 +
0.2
−
3
9.2 +
− 0.6 × 103
1.3 +
0.2 × 10
−
1
1.0 +
− 0.3 × 102
3.4 +
1.3
×
10
−
2
5.8 +
− 1.3 × 102
9.2 +
− 2.4 × 10
Substrate specificity and catalytic mechanism of Li CMS
Figure 2
51
Structure of the catalytic domain of Li CMS
(A) Overall structure of Li CMSN in complexes with Pyr and acetyl-CoA. The bound Pyr and acetyl-CoA are shown as ball-and-stick models in pink and yellow respectively, and Zn2+ is shown
as a gold sphere. (B) Structure of the homodimeric Li CMSN. The two monomers (in magenta and grey respectively) are related by a two-fold crystallographic symmetry. The bound Pyr and
acetyl-CoA are shown with ball-and-stick models in the same colors as in (A). (C) Superposition of Li CMSN (grey) and Mt IPMSN (N-terminal catalytic domain of Mt IPMS; red). Although the
catalytic domains of the two enzymes share very low sequence homology, their structures are very similar. (D) Structure-based sequence alignment of Li CMSN and Mt IPMSN. The secondary
structures of the two enzymes are shown above and below the alignment respectively. Strictly conserved residues are highlighted in red shaded boxes and conserved residues in white boxes with blue
outlines.
298–309 disordered. The structure of LiCMSN assumes a TIM
barrel fold flanked by an extended C-terminal region (Figure 2A).
The TIM barrel is mainly composed of eight β-strands (β1–β8)
and eight long α-helices (α1–α8) arranged as eight β/α repeats
with repeats β1/α1, β4/α4 and β5/α5 inserted by short helices
α1 , α4 and α5 respectively. The inside and outside diameter of
the barrel is about 10 Å and 45 Å respectively. The C-terminal
region forms a long α-helix (α9) and an extended loop (aminoacid residues 285–325) which flank helices α1 and α8 of the
TIM barrel. In the crystal structures of LiCMSN in complexes
with the ligands, two LiCMSN molecules related by the two-fold
crystallographic symmetry form a homodimer (Figure 2B). The
dimer interface involves extensive hydrophilic and hydrophobic
interactions between helices α7 and α8 and turns β6/α6 and β7/α7
c The Authors Journal compilation c 2008 Biochemical Society
52
J. Ma and others
of each monomer. In addition, the C-terminal region of monomer
A extends to and covers the top of the active site of monomer
B, forming interactions with residues of the β6/α6, β7/α7 and
β8/α8 turns of the TIM barrel of monomer B (Figure 2B). Dimer
formation buries 2465 Å2 (or 18.0 %) of the solvent-accessible
surface area of each monomer.
Sequence and structural comparisons indicate that although
LiCMS and MtIPMS have a low amino-acid sequence homology
(overall 16.2 % identity and 30.7 % similarity, and 18.3 % identity and 32.5 % similarity for the catalytic domain), the catalytic
domains of the two enzymes show high structural similarity
with an RMSD of 1.8 Å for 281 Cα atom pairs (Figures 2C
and 2D). The β-strands of the TIM barrel core of the two catalytic domains can be superimposed closely. However, the outside
α-helices have some conformational differences. In particular,
helices α2, α4, α5, α8 and α9 of LiCMSN are slightly twisted
compared with those of MtIPMS. The C-terminal region of
LiCMSN is equivalent to part of subdomain I (residues 365–424)
of MtIPMS. This region is involved in dimer formation and forms
part of the active site in both MtIPMS and LiCMS (see below).
MtIPMS contains a 64-residue insertion before strand β1, which
is involved in the dimer formation, and a 21-residue insertion in
the C-terminal region, which forms a long α-helix α10 and a 310
α-helix η5 [14].
Structure of the active site of Li CMS
The active site of LiCMS is located at the centre of the TIM barrel
near the C-terminal ends of the eight β-strands, and consists of
several conserved residues of the β-strands and the C-terminal region of the adjacent monomer. In both the LiCMSN–Pyr complex
and the LiCMSN–Pyr–CoA complex, there is a Pyr molecule and
a Zn2+ ion bound at the active site. The substrate Pyr forms a
hydrogen bond with the hydroxyl group of Thr179 (2.6 Å) through
the carboxyl oxygen and two co-ordination bonds with the Zn2+
ion through two carbonyl oxygens (2.3 Å and 2.4 Å respectively)
(Figure 3A). Mutagenesis results showed that mutation of Thr179
into an alanine residue resulted in a 16.4-fold increase in the
K m for Pyr and a 186-fold decrease in the kcat , confirming its
functional role in the binding of Pyr and the catalytic reaction
(Table 2a). The Zn2+ ion is bound near Pyr and has six ligands in
an octahedral geometry, including the two carbonyl oxygens of
Pyr, the side-chain Nε2 atoms of His207 and His209 (2.5 Å for both),
one carboxylate oxygen of Asp17 (2.2 Å) and a water molecule
(2.4 Å) (Figure 3A). Mutagenesis results showed that replacement
of Asp17 with an asparagine or an alanine residue caused a 4.4and 34-fold increase in the K m for Pyr, and a 480- and 315fold decrease in the kcat respectively (Table 2a). Moreover, the
concentration of Mn2+ in the enzymatic activity assay for those
mutants had to be increased from 0.8 mM (used in the standard
activity assay) to 5 mM, suggesting that these mutants have a
much weaker binding affinity for the metal ion. These results
indicate that the effects on the K m of Pyr and the enzymatic
activity of these mutations are through the destabilization of the
metal-ion binding. In the LiCMSN–Mal complex, there is a Mal
molecule and a Zn2+ ion bound at the active site, and the bound Mal
occupies the same position as Pyr and also forms a hydrogen bond
with the hydroxyl group of Thr179 (2.6 Å) through the carboxyl
oxygen, and two co-ordination bonds with the Zn2+ ion through
the two carbonyl oxygens (2.1 and 2.4 Å respectively), mimicking
the substrate binding (Figure 3B).
In the structure of the LiCMSN–Pyr complex, there is a deep
surface groove near the bound Pyr of approx. 21 Å in length
and 8 Å in width. This groove is formed by residues from
the α1 /α1 loop, β2/α2 turn and β3/α3 turn of one monomer
c The Authors Journal compilation c 2008 Biochemical Society
and the C-terminal region of the other. In the structure of the
LiCMSN–Pyr–CoA complex, acetyl-CoA is bound exactly in
the groove (Figure 3C). Acetyl-CoA can assume widely varying
conformations in different enzymes [25]. In our ternary complex,
acetyl-CoA assumes a U-shape conformation. The adenine moiety
of acetyl-CoA is stabilized through hydrophobic interactions with
Phe83 of β3, and mutation of Phe83 into an alanine residue results
in a 5-fold increase in the K m for acetyl-CoA and a 120-fold
decrease in the kcat (Table 2a). The thioacetyl moiety of acetylCoA is positioned near Pyr, with the carbonyl oxygen of the acetyl
group forming one hydrogen bond with the side-chain Nη2 of
Arg16 (2.9 Å) and the methyl group being sandwiched between the
side-chain Oε2 of Glu146 and the C2 atom of Pyr (distances of 3.2–
3.5 Å) (Figure 3A). The side chain of Arg16 is further stabilized
by forming a salt bridge with the side chain of Glu48 . These two
residues play very important roles in the catalytic reaction (see
below). Glu146 appears to function as a catalytic base to abstract
a proton from the methyl group of acetyl-CoA to form enolated
acetyl-CoA, and thus mutation of Glu146 to either Asp or Gln
has minor effects on the binding of acetyl-CoA, but can cause a
decrease in the kcat by more than 400-fold (Table 2a). Arg16 appears
to function as a general acid to stabilize the enolated acetyl-CoA
and thus mutation of Arg16 to either lysine or a glutamine residue
completely abolishes the enzymatic activity of LiCMS (Table 2a).
In the structure of the MtIPMS–α-Kiv complex, subdomain I
of MtIPMS forms part of the active site, and residues His379 and
Tyr410 of this region (corresponding to His302 and Tyr312 of LiCMS
respectively) are suggested to be involved in the binding of the
coenzyme [14].The C-terminal region of LiCMSN is equivalent to
part of subdomain I of MtIPMS. In the structure of the LiCNSN–
Pyr–CoA complex, the C-terminal region covers the top of the
active site of the adjacent monomer and, in particular, the disordered region of residues 298–309 appears to form part of the
binding site for acetyl-CoA. To verify this notion, we performed
mutagenesis studies of residues in the C-terminal region and
tested the effects of these mutations on the binding of acetyl-CoA
and Pyr and the enzymatic activity (Table 2b). The results show
that mutation of His302 to either an alanine or asparagine residue
completely disrupts the enzymatic activity of LiCMS. Similarly,
mutation of Tyr312 to an alanine residue abolishes the enzymatic
activity of LiCMS. Both residues are conserved in MtIPMS. In
addition, D304A and L311A mutations in LiCMS substantially
weaken the binding of both acetyl-CoA and Pyr and also decrease
the kcat . On the other hand, N310A mutation has a much smaller
effect on the binding of Pyr and acetyl-CoA and on the enzymatic
activity. It seems that the closer the residues are to the active site,
the more severe the effects of mutations are on the binding of
acetyl-CoA and Pyr and the enzymatic activity. The structural
and mutagenesis results together indicate that LiCMS functions
biologically as a homodimer and the active site is composed of
structural elements from both monomers. In particular, the Cterminal region of LiCMSN forms part of the active site and is
involved in the binding of the coenzyme and the substrate, and
thus plays an important role in the catalytic reaction.
On the other hand, it is intriguing to observe that although
LiCMSN can bind both Pyr and acetyl-CoA, it is enzymatically
inactive (Table 2). As the C-terminal region of LiCMSN is only
equivalent to part of subdomain I of MtIPMS, and residues 298–
309 are disordered in the structure of the LiCMSN–Pyr–CoA
complex, it is plausible that the flexible linker between LiCMSN
and LiCMSC might be involved in the proper positioning of the
C-terminal region of LiCMSN and/or the formation of the acetylCoA binding site. The inactivity of LiCMSN might be due to
the lack of the flexible linker because it might affect the binding
of acetyl-CoA and Pyr. This suggestion is consistent with the
Substrate specificity and catalytic mechanism of Li CMS
Figure 3
53
Structure of the active site of Li CMS
(A) The active site of Li CMS. The side chains of residues forming the active site are shown (in cyan). The bound Pyr (in pink) and acetyl-CoA (in yellow) are shown in ball-and-stick models, and the
Zn2+ ion is shown as a gold sphere. Water (Wat) is shown by a red sphere. The hydrogen-bonding interactions are shown with dashed lines. (B) Overlay of Mal in the Li CMSN–Mal complex (in
cyan) with Pyr in the Li CMSN–Pyr complex (in pink) based on the superposition of the two structures. Bond lengths are shown (in Å). (C) Electrostatic surface of Li CMSN showing the binding site
of the substrate and coenzyme. The bound Pyr and acetyl-CoA are shown as ball-and-stick models. (D) Structural comparison of the active sites of Li CMS and Mt IPMS. Residues forming the active
site of Li CMS, the bound substrate, coenzyme and Zn2+ are coloured as indicated in (A). The equivalent residues of Mt IPMS are coloured in grey. (E) Substrate specificity of Li CMS. Upper panel:
chemical structures of different α-oxo acids: Glx, Pyr, α-Kb and α-Kiv. Lower panel: Residues Leu81 , Leu104 and Tyr144 (grey) of Li CMS form a hydrophobic pocket to accommodate the C2 -methyl
group of Pyr (yellow). The van der Waals spheres of atoms are shown with dots. The C2 -methyl group of Pyr has favourable hydrophobic contacts with the three residues. When α-Kiv or α-Kb is
docked into the substrate-binding site, the bigger substituent at the C2 position of α-Kiv (isopropyl group) or α-Kb (ethyl group) would have steric conflicts with the surrounding residues. For
clarity, only the docked α-Kiv is shown (magenta).
biochemical results that the binding affinities of LiCMSN for
acetyl-CoA and Pyr were decreased by approx. 5- and 2.5-fold respectively compared with those of the full-length enzyme (Table 2).
It is also in agreement with the structural results that the electron
density for bound acetyl-CoA in the structure of the LiCMSN–
Pyr–CoA complex was poor, partly because of the weak binding of
the coenzyme in the absence of the linker. Nevertheless, since the
missing linker does not substantially affect the binding of acetylCoA and Pyr, it is unlikely to cause significant conformational
change of the active site and thus should not prejudice
the conclusions drawn from the structural and biochemical
results.
c The Authors Journal compilation c 2008 Biochemical Society
54
J. Ma and others
Although LiCMS and MtIPMS have a low sequence similarity,
the residues forming the active site are strictly conserved with
very similar conformations in the structures of both enzymes
(Figure 3D). The only difference occurs in the orientation of
the side chain of Gln20 (α1 ) of LiCMS, which forms hydrogen
bonds with the side chain Oε1 of Glu239 (2.6 Å), the side-chain
Nη of Arg16 (2.8 Å), and the carbonyl oxygen of the acetyl group
of acetyl-CoA (3.2 Å), whereas the equivalent residue Gln84 of
MtIPMS points its side chain away from the active site. Since the
catalytic domains of both LiCMS and MtIPMS have high structural similarity in the overall structure and at the active site, it is
possible that the two enzymes might share a common evolutionary
ancestor and a similar catalytic mechanism.
Substrate specificity of Li CMS
LiCMS shows high substrate specificity for Pyr, but has very
weak or no detectable activities for other α-oxo acids, such as Glx
(glyoxylate), α-Kb and α-Kiv (Table 2c) [12]. This high substrate
specificity is also seen for M. jannaschii citramalate synthase [13].
These α-oxo acids have different substituents at the C2 position
(Figure 3E). Structural analysis of LiCMSN reveals that three
hydrophobic residues, Leu81 of β3, Leu104 of β4 and Tyr144 of β5
at the active site, form a hydrophobic pocket to accommodate the
C2 -methyl group of Pyr through hydrophobic contacts (distances
of 3.6–4.4 Å) (Figure 3E). Modelling studies indicate that when
α-Kiv or α-Kb is docked into the substrate-binding site, the C2 isopropyl group of α-Kiv or C2 -ethyl group of α-Kb would have
steric conflicts with the surrounding residues (Figure 3E). In the
structure of the MtIPMS–α-Kiv complex, a similar hydrophobic
pocket is formed by residues Leu143 , Tyr169 and Ser216. This pocket
is slightly larger as a result of the change of Tyr144 (LiCMS) to
Ser216 (MtIPMS), and all three residues have hydrophobic contacts
with the C2 -isopropyl group of α-Kiv.
To investigate the functional roles of these residues in the determination of substrate specificity, we performed kinetic studies
with mutant LiCMS containing mutations L81A, L81V, L104V,
Y144L or Y144V. The kinetic results showed that these mutations
lead to weaker binding of Pyr (K m being increased by 2–260-fold),
with the Y144L mutation having the most serious effect and the
L104V mutation the smallest effect, but all of them have less pronounced effects on the kcat (decreased by 3–76-fold) (Table 2C).
These results can be explained structurally because those
mutations would lead to the formation of a bigger binding pocket
for the C2 -methyl group of Pyr and thus destabilize the binding
of the substrate, but have a less profound effect on the enzymatic
activity. On the other hand, LiCMS can also catalyse the conversion of some other α-oxo acids with much weaker enzymatic
activity, and mutations of these three residues have varied effects
on the binding of these substrates and the enzymatic activity
(Table 2C). For Glx, which has no substituent at the C2 position,
these mutations (in particular L81A and L81V) can cause
substantial increases in the K m . For α-Kiv, which has a bigger
substituent (isopropyl) at the C2 position, these mutations create a
bigger pocket for the substitution group and thus result in tighter
binding of the substratum, as shown by the moderate K m and
measurable enzymatic activity (Table 2C). For α-Kb, which has
an ethyl group at the C2 position, the L104V and L81A mutations
slightly increase the binding of the substrate, whereas Y144L,
Y144V and L81V mutations moderately decrease the binding
of the substrate. Taken together, the structural and biochemical
results demonstrate that the three hydrophobic residues at the
active site (Leu81 , Leu104 and Tyr144 ) determine the substrate specificity; in particular, Tyr144 plays an important role in the binding of
c The Authors Journal compilation c 2008 Biochemical Society
Pyr, α-Kiv and α-Kb, whereas Leu81 has a role in the binding
of Glx.
Catalytic mechanism of Li CMS
LiCMS catalyses the conversion of Pyr and acetyl-CoA into
citramalate and HS-CoA in an aldol condensation reaction. The
mechanism of the aldol condensation reaction catalysed by malate
synthase has been extensively studied, and the reaction takes place
in three steps: (i) enolization, (ii) condensation and (iii) hydrolysis [25–28]. Structural analysis of LiCMSN in complexes with
Pyr and acetyl-CoA has provided detailed information about the
interactions between the enzyme and the substrate and coenzyme.
In addition, mutagenesis and kinetic studies have confirmed the
functional roles of the key residues in the binding of the substrate
and coenzyme and the catalytic reaction. These structural and
biochemical results together enable us to propose the mechanism
of the aldol condensation reaction catalysed by LiCMS (Figure 4).
First, acetyl-CoA is activated to form an enolate, which is believed
to be the rate-limiting step in the catalytic reaction. The activation
requires abstraction of a proton from the methyl group of acetylCoA by a catalytic base and the enolate intermediate is stabilized
by a general acid. In the crystal structure of LiCMSN, the
methyl group of acetyl-CoA is sandwiched between the carboxylate group of Glu146 and the C2 atom of Pyr. In particular, the carboxylate Oε2 of Glu146 is positioned 2.8 Å away from the methyl
group of acetyl-CoA and appears to function as the catalytic base
to abstract a proton from the methyl group. This suggestion is supported by the mutagenesis results that mutation of Glu146 to either
an aspartic acid or a glutamine residue has a minor effect on the
binding of acetyl-CoA, but a significant impact on the enzymatic
activity of LiCMS. Arg16 forms a hydrogen bond with the carbonyl
oxygen of the acetyl group of acetyl-CoA (2.9 Å) and thus appears
to function as the general acid to stabilize the enolated acetyl-CoA.
The mutagenesis results show that mutation of Arg16 to either a
lysine or glutamine residue completely disrupts the enzymatic
activity of LiCMS. Secondly, the substrate Pyr is polarized by a
bivalent metal ion for nucleophilic attack by enolated acetyl-CoA,
leading to the formation of citramalyl-CoA. Previous biochemical
results have shown that Mg2+ is essential in the polarization of Glx
in malate synthase and α-Kiv in MtIPMS [23,26]. The catalytic
reaction of LiCMS is also dependent on bivalent metals, with
Mn2+ showing the highest activity, followed by Ca2+ , Mg2+ and
Ni2+ , suggesting that LiCMS might use Mn2+ instead of Mg2+ in
the catalytic reaction. In the crystal structure of LiCMSN, a Zn2+
ion is bound at the active site which appears to occupy the position
of the catalytic metal ion. The bound Zn2+ is co-ordinated with
the two carbonyl groups of Pyr, the conserved residues Asp17 ,
His207 , and His209 and a conserved water molecule, and is in a
proper position to polarize the C2 atom for nucleophilic attack
by acetyl-CoA. Finally, the thioester group of the citramalyl-CoA
intermediate is hydrolysed to form the products citramalate and
HS-CoA. The Zn2+ -bound water molecule is in a good position to
participate in the hydrolysis of the thioester bond of citramalylCoA.
In summary, the structural and biochemical results reveal the
molecular basis of the high substrate specificity and the catalytic
mechanism of LiCMS. The substrate specificity of LiCMS
towards Pyr against other α-oxo acids is dictated primarily by
residues Leu81 , Leu104 and Tyr144 , which form a hydrophobic
pocket to accommodate the C2 -methyl group of Pyr. The conversion of Pyr and acetyl-CoA into citramalate and HS-CoA
catalysed by LiCMS follows a typical aldol condensation reaction,
in which Glu146 functions as a catalytic base to activate the methyl
Substrate specificity and catalytic mechanism of Li CMS
Figure 4
55
A schematic diagram of the proposed catalytic mechanism of Li CMS
The aldol condensation reaction catalysed by Li CMS could take place in three steps: (i) enolization, (ii) condensation and (iii) hydrolysis. Glu146 appears to function as a catalytic base to abstract a
proton from the methyl group of acetyl-CoA, leading to the formation of enolated acetyl-CoA. Arg16 appears to function as a general acid to stabilize the enolated acetyl-CoA intermediate. A bivalent
metal ion (M2+ ) plays an essential role in polarizing the C2 atom of Pyr for nucleophilic attack by acetyl-CoA.
group of acetyl-CoA to form the enolated acetyl-CoA intermediate and Arg16 functions as a general acid to stabilize the
intermediate.
We thank Dr Xiaokui Guo (Department of Microbiology and Parasitology, Shanghai
Jiaotong University School of Medicine, Shanghai, China) for providing us with the
genomic DNA of L. interrogans (serogroup Icterohaemorrhagiae, serovar lai , strain
56601). We thank the staff members at the Photon Factory (Ibaraki, Japan) and the
Beijing Synchrotron Radiation Facility (Beijing, China) for technical support in diffraction
data collection, and other members of our groups for helpful discussion. This work
was supported by grants from the Ministry of Science and Technology of China
(2004CB720102, 2006AA02Z112, 2006AA02176 and 2007CB914302), the National
Natural Science Foundation of China (30570379, 30770480, 30730028, 30770111,
30670102 and 30470018), the Chinese Academy of Sciences (KSCX2-YW-R-107) and
the Science and Technology Commission of Shanghai Municipality (07XD14032).
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SUPPLEMENTARY ONLINE DATA
Molecular basis of the substrate specificity and the catalytic mechanism of
citramalate synthase from Leptospira interrogans
Jun MA*‡1 , Peng ZHANG†‡1 , Zilong ZHANG*‡, Manwu ZHA†, Hai XU*2 , Guoping ZHAO*§3 and Jianping DING†3
*Laboratory of Microbial Molecular Physiology, Institute of Plant Physiology and Ecology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, 300 Feng-Lin
Road, Shanghai 200032, China, †State Key Laboratory of Molecular Biology and Research Center for Structural Biology, Institute of Biochemistry and Cell Biology, Shanghai Institutes
for Biological Sciences, Chinese Academy of Sciences, 320 Yue-Yang Road, Shanghai 200031, China, ‡Graduate School of Chinese Academy of Sciences, 320 Yue-Yang Road,
Shanghai 200031, China, and §Shanghai-MOST Key Laboratory for Health and Disease Genomics, Chinese National Human Genome Center, Shanghai 201203, China
Figure S2
Figure S1
Electron density of the substrate and coenzyme
Composite-omit map (1σ ) for Mal in the structure of the Li CNSN–Mal complex (A), Pyr
in the structure of the Li CNSN–Pyr complex (B) and acetyl-CoA in the structure of the
Li CNSN–Pyr–CoA complex (C).
Anomalous electron density of the bound Zn2+ ion
(A) Crystal of the LiCMSN–Mal complex. (B) Crystal of the LiCMSN–Pyr–CoA complex soaked
with Zn2+ ion. These crystals were prepared by supplementing the crystallization drop with
50 mM Pyr, 40 mM acetyl-CoA and 10 mM Zn2+ for 48 h. In both structures, there is a strong
anomalous density peak (above the 10σ contour level) for the bound Zn2+ ion at the active site.
1
These authors contributed equally to this work.
Present address: State Key Laboratory of Microbial Technology, Shandong University, Jinan, Shandong 250104, China.
3
Correspondence may be addressed to either of these authors (email [email protected] or [email protected]).
The structural co-ordinates reported for the N-terminal catalytic domain of Leptospira interrogans citramalate synthase in complex with malonate, in
complex with pyruvate and in complex with pyruvate and acetyl-CoA will appear in the Protein Data Bank under accession codes 3BLE, 3BLF and 3BLI
respectively.
2
c The Authors Journal compilation c 2008 Biochemical Society
J. Ma and others
Table S1
ICP-MS analysis of different metal-ion concentrations
We performed ICP-MS analyses of different metal ion concentrations (μM) for the following three
protein samples. Sample 1, Li CMS purified with a Ni2+ -NTA column; sample 2, Li CMS purified
with a Ni2+ -NTA column, followed by treatment with EDTA; and sample 3, Li CMS purified using
a Zn2+ -affinity column. The concentrations of the metal ions in the different protein samples
were normalized to the concentration of Li CMS (175.8 μM). The results show that freshly
purified Li CMS contains both Zn2+ and Ni2+ at high concentrations; the EDTA-treated enzyme
contains very low traces of metal ions at the background level; and the enzyme purified with
the Zn2+ column contains only Zn2+ at a high concentration. These results indicate that: (1) the
bound Zn2+ ion in the purified protein was co-purified with the enzyme from the expression
system, (2) the concomitant Ni2+ ion in the purified protein was stripped from the Ni2+ -NTA
column during purification, and (3) treatment of the purified protein with EDTA can remove all
bound metal ions to the background concentration.
Ion
Sample 1
Sample 2
Sample 3
Mg2+
Mn2+
Ni2+
Cu2+
Zn2+
Ag2+
metal
− 1.5 +
− 0.4
1.5 +
− 0.2
103.1 +
− 1.5
12.9 +
− 0.3
92.4 +
− 4.6
0.6 +
− 0.1
208.9 +
− 4.9
7.9 +
− 0.5
0.1 +
− 0.0
5.6 +
− 0.2
0.5 +
− 0.1
7.2 +
− 0.3
0.8 +
− 0.0
22.1 +
− 0.6
2.4 +
− 0.2
0.9 +
− 0.0
11.3 +
− 0.2
17.5 +
− 0.4
119.9 +
− 2.8
0.1 +
− 0.0
152.1 +
− 2.8
Received 28 January 2008/19 May 2008; accepted 23 May 2008
Published as BJ Immediate Publication 23 May 2008, doi:10.1042/BJ20080242
c The Authors Journal compilation c 2008 Biochemical Society