From the Department of Biochemistry and Biophysics,
Texas A & M University, College Station, Texas 77843-2128,
Department of Microbiology and Immunology, College of Veterinary
Medicine, Cornell University, Ithaca, New York 14853, and
¶ Department of Medical Microbiology, University of Szeged,
Szeged POB 8-6701, Hungary
Received for publication, September 9, 2002, and in revised form, October 17, 2002
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ABSTRACT |
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Establishment or maintenance of a
persistent infection by Mycobacterium tuberculosis requires
the glyoxylate pathway. This is a bypass of the tricarboxylic
acid cycle in which isocitrate lyase and malate synthase (GlcB)
catalyze the net incorporation of carbon during growth of
microorganisms on acetate or fatty acids as the primary carbon source.
The glcB gene from M. tuberculosis, which
encodes malate synthase, was cloned, and GlcB was expressed in
Escherichia coli. The influence of media conditions
on expression in M. tuberculosis indicated that this enzyme
is regulated differentially to isocitrate lyase. Purified GlcB had
Km values of 57 and 30 µM for its
substrates glyoxylate and acetyl coenzyme A, respectively, and was
inhibited by bromopyruvate, oxalate, and phosphoenolpyruvate. The GlcB
structure was solved to 2.1-Å resolution in the presence of glyoxylate
and magnesium. We also report the structure of GlcB in complex with the
products of the reaction, coenzyme A and malate, solved to 2.7-Å
resolution. Coenzyme A binds in a bent conformation, and the details of
its interactions are described, together with implications on the
enzyme mechanism.
In mycobacteria it has been found that the mechanism of persistent
infection is dependent on a switch of metabolism to the glyoxylate
bypass (1-3). Isocitrate lyase and malate synthase are the two enzymes
of this pathway, which has been described in various eubacteria, fungi,
and plants (see Ref. 4 for a review). In the glyoxylate bypass,
isocitrate lyase (ICL)1 and
malate synthase (GlcB) function sequentially to convert isocitrate to
succinate plus glyoxylate and glyoxylate plus acetyl-CoA to malate and
CoA, respectively. Together catalysis by ICL and GlcB ensures
the bypass of two oxidative steps of the tricarboxylic acid cycle,
permitting net incorporation of carbon during growth of most
microorganisms on acetate or fatty acids as the primary carbon source.
Thus, the glyoxylate bypass conserves carbon and ensures an adequate
supply of tricarboxylic acid cycle intermediates for biosynthetic
purposes when cells convert lipids to carbohydrates.
In Mycobacterium tuberculosis isocitrate lyase activity is
increased when the bacilli are in an environment of low oxygen tension
or in a transition from an actively replicating to a non-replicating state (5, 6). In addition, at least the first enzyme of the glyoxylate
bypass of M. tuberculosis is up-regulated when grown in a
medium containing either acetate or palmitate as the main carbon source
or upon infection of macrophages (3, 7). It has also been shown that a
A single malate synthase gene called glcB (also referred to
as aceB in M. tuberculosis CDC1551; MT1885; see
www.tigr.org) has been identified in M. tuberculosis
encoding a malate synthase G (MSG) (Rv1837c; see Ref. 12, and see
www.sanger.ac.uk/). GlcB is an 80-kDa monomeric protein with 741 amino acid residues, which is homologous to malate synthase (AceB) of
the Gram-positive bacterium Corynebacterium glutamicum (13)
and MSG of the Gram-negative Escherichia coli (14),
with ~60% identity. A second group of malate synthases called malate
synthase A (MSA) have been identified (15). Typically, MSAs have a
molecular mass of ~60 kDa and include MSA of E. coli K12 (16), Yersinia pestis (17), Vibrio
cholerae (18), yeast, and higher plants. The prokaryotic MSAs tend
to be monomeric whereas eukaryotic enzymes of this group are
homomultimers. There is high sequence identity within the MSA class
(~65%) whereas the similarity between the MSA- and MSG-type malate
synthases is much lower at 18-20%, with MSA and MSG from E. coli K12 showing 18% identity. Sequence alignments of
representatives from the MSA and MSG classes of malate synthase show 47 conserved residues among M. tuberculosis GlcB, M. leprae GlcB, E. coli K12 GlcB, C. glutamicum
GlcB, E. coli AceB, and Y. pestis GlcB (see Fig. 1a).
Long term persistence is one of the hallmarks of an infection by
M. tuberculosis and one of the main obstacles to the global control of the disease. TB remains a leading cause of mortality with ~3 million deaths per year in the 1990s (19). Anti-tubercular drugs that are currently available preferentially target bacteria during active growth and replication, requiring long treatment times to
successfully clear a TB infection. The reliance on enzymes of the
glyoxylate bypass for intracellular survival and persistent infection
of M. tuberculosis (3) and the absence of this pathway in
mammals has led to interest in these enzymes as attractive novel
targets for drug development. It is believed that drugs that are
effective against persistent bacteria will clear an infection more
quickly, reducing chemotherapy times and so decreasing the risk of
development of multi-drug-resistant TB. In this work we investigate the
expression of GlcB in M. tuberculosis under varying media conditions. We also purify recombinant malate synthase and present biochemical characterization of the enzyme together with its
three-dimensional structure in a substrate-bound form and product-bound form.
Bacterial Strains and Growth Conditions--
Middlebrook 7H9
medium (Difco) containing 10% (v/v) OADC enrichment and 0.05%
Tween 80 was used. Either modified Dubos medium was used as a defined
minimal medium (2 g of asparagine, 1 g of KH2PO4, 2.5 g of
Na2HPO4, 10 mg of MgSO4·7
H2O, 50 mg of ferric ammonium citrate, 0.5 mg of
CaCl2, 0.1 mg of ZnSO4, 0.1 mg of CuSO4, 0.05% Tween 80, 0.5 g of Casitone (Difco), and
0.1 g of bovine serum albumin per liter), or unmodified Dubos
medium was used (5 g of bovine serum albumin and 7.5 g of glucose
per liter). Carbon sources were added to 10 mM with the
exception of palmitate, which was added to 0.1% (v/v), and glycolate
and acetate, which were used at 3 mM final concentration.
Non-aerated cultures were grown as 55-ml cultures in flasks with
25-cm2 growth area, without stirring. Aeration of cultures
was achieved with a 50-mm Teflon-coated magnetic stirring bar in a
500-ml Erlenmeyer flask containing 100-ml medium at a stir rate of 80 rpm. The M. tuberculosis strain CDC1551 was used. E. coli strains were grown in LB
(Luria-Bertani) medium with ampicillin (50 µg/ml) and kanamycin (50 µg/ml).
Preparation of Mycobacterial Cell-free Extracts--
Cells were
harvested, washed three times with PBST (PBS plus 0.05%
Tween 80) and resuspended in Tricine buffer (20 mM Tricine, pH 7.5, 5 mM MgCl2, 1 mM EDTA) supplemented with protease inhibitors
(tosyl-L-lysine chloromethyl ketone, 100 µg/ml; pepstatin
A, 50 µg/ml; leupeptin, 50 µg/ml; trans-epoxysuccinyl-L-leucylamido (4-guanidino)-butane, 50 µg/ml). The cells were disrupted with a "bead-beater"
(Biospec, Bartlesville, OK), and the lysate was centrifuged for 30 min
at 300,000 × g.
Cloning of glcB and Expression of the GlcB Protein--
A 2.3-kb
DNA fragment containing the glcB gene was amplified by PCR
using the following oligonucleotide primers: 5'-CAG TAC ATA TGA CAG ATC
GCG TGT C-3' and 5'-ATA TTG GAT CCC GCA AGC GGG CGG T-3' and M. tuberculosis CDC1551 DNA as the template. The amplified DNA was
digested with NdeI and BamHI and ligated into p6HisF-11d (kindly provided by Cheng-Ming Chiang, The Rockefeller University, New York). For overexpression, E. coli
HB101(pGP1-2) cells carrying the recombinant
p6HisF-11d(glcB) plasmid were grown to exponential phase at
30 °C in LB plus ampicillin. Expression of GlcB was induced by
shifting the temperature to 42 °C for 20 min, followed by a shift to
37 °C for an additional 90 min.
Purification of GlcB--
Cells were resuspended in Buffer 1 (50 mM NaH2PO4, 20 mM
Pipes, 100 mM NaCl, 10 mM imidazole, pH 8.0)
with protease inhibitors (as above) and lysed by sonication, and the
lysate was centrifuged at 300,000 × g for 30 min. The
cell extract was applied to metal affinity resin (TalonTM,
Clontech, Palo Alto, CA) equilibrated with Buffer 1 and then washed with Buffer 1. The His-tagged protein was eluted with
Buffer 2 (as Buffer 1, plus 5% glycerol, 1 mM
For the crystallization of the GlcB, p6HisF-11d(glcB)
plasmid was transformed into E. coli BL21(DE3) cells, and
expression was induced by adding
isopropyl-1-thio- Assay of Malate Synthase--
Malate synthase was assayed at
room temperature in 96-well plates. Typically, 50 µl of 20 mM Tricine-HCl, pH 7.5, containing 5 mM
MgCl2, 0.8 mM EDTA, 2 mM
glyoxylate, and 2 mM acetyl-CoA were mixed with 50 µl of
the purified protein (0.16 mg/ml) in Tricine-HCl, pH 7.5, 5 mM MgCl2, and 0.8 mM EDTA buffer
and incubated at room temperature for 30 min. Protein concentrations
were determined by the method of Bradford (20). The enzyme-catalyzed
reaction was stopped by adding DTNB to a final concentration of
2 mM in Tris-HCl, pH 8.0. The amount of CoA-SH
released was measured by titrating the free thiol groups with the DTNB
and measuring the change in absorbance at 412 nm. The
Km and Vmax values were
determined using a double-reciprocal Lineweaver-Burk or Hanes-Woolf plot. Ki values were determined from linear replots
of Lineweaver-Burk slopes versus inhibitor concentrations
(21). To determine the pH optimum for malate synthase activity, assays were performed using MES/NaOH at pH 5.5, 6, and 6.5, MOPS/NaOH at pH
6.5, 7, and 7.5, Tricine/HCl at pH 7.5, 8, and 8.5, and Tris/HCl at pH
8.5 and 9 in place of 20 mM Tricine-HCl, pH 7.5. The metal
ion dependence of malate synthase was investigated by preincubating the
enzyme with 5 mM MnCl2, CoCl2,
FeCl2, CaCl2, BaCl2,
NiCl2, ZnCl2, CuCl2, and
HgCl2 in place of MgCl2.
Production of Polyclonal Antibodies to GlcB--
Antibodies
against two internal fragments of GlcB (QNTMKIGIMDEERRTT (MS1) and
YTEPILHRRRREFKAR (MS2)) were produced in New Zealand White rabbits
after coupling the peptide to ovalbumin. The polyclonal antibodies were
affinity-purified using the respective antigen coupled to
CNBr-activated Sepharose.
Flow Cytometry Analysis of GlcB::DsRedTM
Expression--
pGlcB::red (glcB::DsRedTM) was
derived from pMV262 (22) by substituting the glcB promoter
(227-bp 5' of the start codon of glcB) and the
glcB open reading frame fused at the 3' end to the DsRedTM
red fluorescent protein (Clontech). Flow cytometry
to quantify levels of GlcB::DsRed was carried out using a
FACScalibur cytometer (BD Biosciences). A 488-nm argon-ion laser
was used for excitation, and the emission detector (FL-2) was set to a
585-nm filter with a 42-nm bandpass. Analysis was performed with
CellQuest software (BD Biosciences). Bacterium-sized particles were
detected by their side- and forward-scatter profiles (23). FL-2,
forward-, and side-scatter data were collected using logarithmic
amplifiers. Data were collected on 104 bacterium-sized
particles per sample. Bacteria were pelleted (2,000 × g, 20 min), fixed (4% paraformaldehyde in PBS for 20 min),
washed once (PBS/0.05% Tween 80/0.1% bovine serum albumin), and
resuspended in PBS/Tween. The relative fluorescence is expressed as
geometric mean fluorescence above background.
Crystallization and Data Collection--
Crystals of malate
synthase were produced by the microbatch method in 3-µl drops
containing 3-5 mg/ml protein, 15% (w/v) PEG4000, 0.05 M
Tris-HCl, pH 8.5, and 0.05 M MgCl2 at 17 °C,
covered by paraffin oil. Crystals were observed after 2-3 days and
grew to the size of ~0.2 × 0.2 × 0.3 mm after 1-2 weeks.
The crystals belonged to the space group P43212 with unit cell
dimensions of a = b = 78.1 Å, c = 223.2 Å, contained
one molecule per asymmetric unit, and had an estimated solvent content
of ~42%. Crystals of selenomethionyl malate synthase were produced
as described above and were isomorphous with the native protein
crystals. Crystals containing the products malate and CoA were obtained
by hanging-drop vapor diffusion. The protein (~9 mg/ml) was
pre-incubated for 20 min with 2 mM acetyl-CoA and 2 mM glyoxylate in 20 mM Tris-HCl, pH 8.0, 10 mM MgCl2 at room temperature. 2.5 µl of the
protein was mixed with 2.5 µl of 1.6 M
(NH4)2SO4, 0.1 M MES,
pH 6.5, 10% Dioxane. These crystals belonged to the space group P41212 with unit cell dimensions of a = b = 120.78 Å, c = 232.8 Å, two molecules per asymmetric unit, and a solvent content of
~55%.
MAD diffraction data for a single selenomethionyl crystal were
collected at beamline 19ID at the Structural Biology Center (Advanced
Photon Source, Argonne National Laboratory, on a 3 × 3 mosaic CCD
area detector. 30% PEG400 was added to the precipitant solution as a
cryoprotectant, and crystals were flash-cooled in a liquid nitrogen
stream (120 K). Data sets at 4 wavelengths were collected on a single
crystal ( Structure Determination and Refinement--
Seventeen selenium
sites were automatically identified using the program SOLVE (26). SHARP
was used to refine the position of the selenium sites, and the phase
was calculated between 20 to 2.5 Å (27). Solvent flattening using DM
(CCP4) (28) and phase extension to 2.1 Å were used to improve the
electron density maps. With the initial experimental map, ~85% of
the sequence was assigned unambiguously and was built in the program O
(29). After simulated annealing refinement of the initial model in
CNS (30) and minimization, an additional 10% of the residues
were built into difference maps. A magnesium ion and a glyoxylate
molecule were built into the active site. 453 water molecules were
added using WATERPICK in CNS. Several rounds of positional minimization and B-factor refinement were carried out in CNS (30). The final model
contains 701 amino acids (>94%) consisting of residues 2-70, 77-301, 312-381, and 386-727. The missing residues were unable to be
built, because the electron density was too weak. 91.4% of the
residues were in the most favored region of the Ramachandran plot, and
none were in the disallowed region as verified using PROCHECK (31). The
crystallographic R value is 18.5%, and the Rfree is 23.1% (Table
I).
The structure of malate synthase complexed with malate and CoA was
solved by molecular replacement using AMoRe (CCP4) (32). The structure
of GlcB-glyoxylate, with all the non-protein atoms excluded, was used
as the search model. Two solutions were found after rigid-body
refinement with a correlation coefficient of 66.1 and an R
factor of 35.7. The model was refined by simulated annealing including
non-crystallographic symmetry averaging of the two molecules (30).
Several rounds of manual correction of the model in O were carried out
followed by positional minimization and B-factor refinement to improve
the model. Coenzyme A and malate were observed in the active site in
an Fo Dependence of M. tuberculosis Malate Synthase Expression on Culture
Conditions--
Unlike ICL, whose activity increased ~4-fold, malate
synthase expression decreased ~2-fold under microaerophilic
conditions in rich medium (Middlebrook or Dubos) (3). This is in
agreement with Wayne and Lin (6) who did not detect an increase in
malate synthase activity upon adaptation to microaerophilic conditions, whereas isocitrate lyase activity increased significantly. Cultures grown in minimal medium supplemented with acetate, palmitate, or
glucose did not show any significant differences with respect to GlcB
expression, which is also in contrast to the ICL expression, which was
up-regulated 2- to 3-fold with growth on acetate or palmitate (7). In
M. tuberculosis, glycolate was the only carbon source that
induced a significantly higher expression of GlcB (2-fold) as measured
by flow cytometry and by Western blot analysis of cell lysates. In
E. coli, growth on glycolate has been shown to up-regulate
the expression of GlcB (14). These data suggest the GlcB of M. tuberculosis fulfills a role comparable with GlcB (MSG) of
E. coli.
Our results indicate that GlcB and ICL from M. tuberculosis
are regulated differentially and independently from each other. In
E. coli, the genes for isocitrate lyase, malate synthase,
and isocitrate dehydrogenase kinase/phosphatase are in an operon under the control of the same promoter and repressor elements (34). The
location of these genes are quite different in M. tuberculosis with ICL and GlcB being far apart on the chromosome.
In C. glutamicum, the aceA, encoding isocitrate
lyase, and aceB, encoding malate synthase, genes are
organized in an antiparallel way that predicts that both genes are
expressed independently from their own promoters (13). It is not known,
however, if the two genes in this organism are controlled by the
same regulatory mechanism or differentially. Wayne and Sohaskey
(35) suggest that the glyoxylate generated by ICL is converted into
glycine, consuming NADH in preparation for a metabolic downshift into
the persistent state. This contrasts with the traditional view that the
glyoxylate bypass generates malate to go on to gluconeogenesis.
However, the differential regulation of icl and
glcB observed in M. tuberculosis is more consistent with an uncoupled reaction, and more experimentation is
required to determine the routing of carbon through ICL and GlcB.
Properties of the Purified GlcB--
The specific activity of the
purified enzyme was 6 µmol/min/mg protein. The Km
of the recombinant protein was determined to be 57 µM for
glyoxylate and 30 µM for acetyl-CoA. These values are
similar to Km data reported for other malate
synthases. For example, malate synthase from C. glutamicum
displayed Km values of 30 µM and 12 µM for glyoxylate and acetyl-CoA, respectively (13).
The inhibition of GlcB activity by several compounds, known to be
effective against various malate synthases, was examined (36-39).
Oxalate, phosphoenolpyruvate, and bromopyruvate were the most
potent inhibitors with inhibition constants of 400, 200, and 60 µM, respectively. Malate was shown to inhibit the
activity to ~50% at 1 mM concentration.
3-Phosphoglycerate, 6-phosphogluconate, fructose-1,6-bisphosphate, and
malonic acid had no inhibitory effect at relevant concentrations.
Glycolate showed inhibition only at fairly high concentrations
(Ki of 900 µM), which is in contrast
to other malate synthases such as the enzyme from C. glutamicum, which has a Ki of 440 µM (4, 13, 37-39).
In the absence of divalent cations only negligible activity was
measured for the purified GlcB. Mg2+ at 5 mM
was found to be the most effective cation. Mn2+ was able to
replace Mg2+, yielding 40% of the activity obtained with
Mg2+. Co2+, Fe2+, Ca2+,
Ba2+, Ni2+, Cd2+, Zn2+,
Cu2+, and Hg2+ were not able to support
significant GlcB activity. The pH optimum for malate synthase activity
was found to be pH 7.5 using a Tricine buffer.
Structure of Malate Synthase Complexed with the Substrate
Glyoxylate--
The structure of GlcB from M. tuberculosis
was solved in complex with glyoxylate to 2.1-Å resolution by MAD
methods (40). The protein is a mixed
Although no structure for the lower molecular mass MSAs has
been reported, multiple sequence alignments and secondary structure predictions suggest that the TIM barrel and domain II are conserved. Biochemical data suggest that both MSAs and MSGs catalyze the condensation of glyoxylate and acetyl-CoA to malate and CoA with a
similar activity, and therefore catalytic residues are likely to be
contained within domains I and II. The active site is located at the
interface of the TIM barrel and domain II, and a loop, which consists
of residues 616-633, forms part of this interface. A glyoxylate
molecule that was not included in the crystallization condition was
found in the active site, as it was in E. coli GlcB (41). A
Mg2+ ion required for activity is bound in a near perfect
octahedral coordination by the carboxylate side chains of Glu-434, 2.1 Å away and OD1 of Asp-462 at 2.0 Å, one carboxylate oxygen (O3 2.1 Å away) and one aldehyde oxygen (O1 2.5 Å away) of the substrate glyoxylate, and two water molecules 2.1 Å and 2.2 Å away,
respectively (see Fig. 3a). Glyoxylate binds via the
aldehyde oxygen (O1) forming a hydrogen bond to NH1 of Arg-339, 3.1 Å away. O2 of glyoxylate is hydrogen bonded to the backbone NH group of
461 (2.9 Å), whereas O3 of the glyoxylate interacts with the backbone
NH of residue 462 at 3.0 Å. Both Glu-434 and Asp-462, important in
coordinating the Mg2+, and residue Arg-339, important in
binding glyoxylate, are found to be conserved in all known malate
synthase sequences of both the A and G types (Fig. 1a).
Structure of Malate Synthase Complexed with Products Malate and
Coenzyme A--
The overall structure of GlcB in complex with the
products malate and coenzyme A (GlcB-malate-CoA) was solved to 2.7 Å and is almost identical to that of GlcB-glyoxylate binary complex, with
root mean square differences for C
The protein ligands for Mg2+ in GlcB-malate-CoA are the
same as in the GlcB-glyoxylate structure, with OE1 of Glu434 and OD1 of
Asp-462 1.8 and 2.5 Å, respectively, away from the metal ion. There is
a water involved in Mg2+ coordination that is 1.9 Å away
from the metal ion. Malate coordinates Mg2+ via one
carboxylate oxygen (O4) at 2.1 Å and via the hydroxyl oxygen (O3) 1.7 Å away. One of the water molecules that was seen coordinating
Mg2+ in GlcB-glyoxylate is replaced by this hydroxyl of the
malate. The backbone NH of residue 461 is 3.2 Å from O1 carboxylate of the malate forming an interaction with the product.
From the magnesium binding site a channel extends ~15 Å to the
surface near the
The structures of enzyme-CoA complexes are now known to be of diverse
three-dimensional folds. No consensus structure or sequence motif has
been identified signifying coenzyme A binding. Coenzyme A adopts either
a bent or extended conformation, for example, acyl-CoA dehydrogenase
and succinyl-CoA synthetase bind CoA in an extended conformation (49,
50) whereas enoyl-CoA hydratase and citrate synthase interact with CoA
in a bent conformation (48, 51, 52). Of TIM barrel containing proteins
the structure of methyl-malonyl CoA mutase in complex with CoA has been
reported (53). Here CoA binding is quite different to that seen in
malate synthase, because it is in an extended conformation, and the
pantetheine arm sits within the barrel making contact with small
hydrophilic side chains.
The Catalytic Mechanism of Malate Synthase--
Malate synthase
catalyzes the Claisen condensation of glyoxylate and acetyl-CoA to form
a malyl-CoA intermediate, which is subsequently hydrolyzed to release
the products, malate and coenzyme A. The reaction can be dissected into
three steps as follows: (i) enolization, (ii) condensation, and (iii)
hydrolysis (41). (i) First, an enol(ate) is formed on acetyl-CoA.
Activation requires abstraction of a proton from the
Comparison of the active sites in the GlcB-glyoxylate structure and of
GlcB-malate-CoA helps clarify our understanding of the catalytic
mechanism of malate synthase. OD1 of the carboxylate side chain of the
proposed catalytic base, Asp-633, is 3.0 Å away from S1 of the
coenzyme A. The enol(ate) intermediate of the acetyl-CoA is then most
likely stabilized by the guanidinium group of Arg-339. Another
possibility discussed by Howard et al. (41) is that the
Mg2+ may stabilize the enol(ate). Though this is
unlikely based on current structural information, which shows that the
Mg2+ is more than 6 Å away from S1 of the coenzyme A, it
cannot currently be ruled out using glyoxylate-bound and product-bound
structures. Mg2+ is, however, essential in the polarization
of the glyoxylate for nucleophilic attack. In the GlcB-glyoxylate
structure the Mg2+ is positioned by the strictly conserved
residues, Glu-434 and Asp-462, as well as via waters to other conserved
residues (Glu-273 and Asp-274) and to the carboxylate oxygen (03) and
aldehyde oxygen (01) of the substrate. The importance of
Mg2+ in developing the positive charge on the C2 of the
carbonyl group and in positioning the glyoxylate in a suitable
orientation for reaction have been discussed in the case of malate
synthase G from E. coli (41). As in E. coli an
Arg, in this case Arg-339, hydrogen bonded to the aldehyde oxygen of
glyoxylate, also important in stabilizing the enol(ate), facilitates
orientating the two substrates for condensation, stabilizing the
oxyanion and yielding a malyl-CoA intermediate.
Hydrolysis of the thioester bond is the next step of the reaction. In
the product-bound structure, the hydroxyl of malate replaces one of the
waters involved in coordinating the Mg2+ in the
glyoxylate-bound structure (Fig. 3). OD1
of Asp-274 is 2.6 Å, and OE1 of Glu273 is 3.8 Å away from this water.
We propose that one or both of these residues are involved in
activation of this water toward hydrolysis of the malyl-CoA
intermediate. Glu-273 is totally conserved in all malate synthases
including members of both the MSA and MSG families. Asp-274 is also
highly conserved, being substituted by a Glu in the sequence of only one malate synthase from Streptomyces arenae (55).
Comparison of Malate Synthase and Citrate Synthase--
Citrate
synthase, which converts oxaloacetate and acetyl-CoA to citrate and
CoA, catalyzes the same Claisen-type condensation as malate synthase.
The catalytic mechanism of citrate synthase has been investigated
extensively by both high resolution structural and biochemical studies
(52, 56-61). Despite the similarity between the chemistry of the
reactions catalyzed by the two enzymes, they differ greatly in their
overall structure and the details of their active sites. Citrate
synthase is an Comparison of Malate Synthase and the Enolase Superfamily--
The
enolase superfamily is comprised of a large number of enzymes
catalyzing abstraction of an Because of the importance of the glyoxylate bypass in persistence
of M. tuberculosis, attention has been focused on the two enzymes of this pathway (3). A high resolution structure of isocitrate
lyase from this organism has been solved (65), and structure-based drug
design is underway, with the prospect of yielding new drugs that are
active against a persistent infection. We have now focused our
attention on the second enzyme in this shunt, malate synthase,
presenting here purification, biochemical characterization of the
enzyme, regulation of expression, and high resolution crystal
structures of both the glyoxylate-bound and malate-coenzyme A-bound
forms of the enzyme from M. tuberculosis. Further work is
clearly needed before we understand the routing and control of carbon
flux in M. tuberculosis via ICL and GlcB. One of the future
goals is to understand the role of malate synthase in the persistent
phase of a M. tuberculosis infection. Like ICL, malate synthase will be regarded as a promising target for the development of new inhibitors. Novel anti-tuberculars, particularly targeting persistence pathways are imperative in the fight for the
global eradication of TB.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
CONCLUSIONS
REFERENCES
icl mutant of M. tuberculosis is unable to
maintain a persistent infection in a mouse model (3), emphasizing the
importance of this pathway to the bacteria in sustaining a chronic
infection. The pathway also appears to be critical in the virulence of
other intracellular pathogens. Both genes of the glyoxylate bypass,
ICL1 and MLS1, encoding isocitrate lyase and malate synthase,
respectively, were induced upon phagocytosis of Candida
albicans by macrophages (8). A mutant of C. albicans lacking ICL1 was less virulent in mice than wild-type (8). The
glyoxylate pathway has also been implicated in the pathogenesis of
Brucella abortus (9) and Rhodococcus equi (10),
as well as in the virulence of the plant pathogen Rhodococcus
fascians (11).
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
CONCLUSIONS
REFERENCES
-mercaptoethanol, 300 mM imidazole). Fractions
containing malate synthase activity that were greater than 90% pure
were pooled.
-D-galactopyranoside to 1 mM in LB media. For the production of selenomethionyl
malate synthase, the plasmid was transformed into E. coli
B834(DE3) cells, a methionine auxotrophic strain, and the protein was
overexpressed in a minimal media containing selenomethionine.
Malate synthase was purified by Ni2+-chelating affinity
chromatography (HiTrap metal-chelating column; Amersham
Biosciences). The His6 tag was removed using the
protease thrombin, and the enzyme without the tag was further purified by Ni2+-chelating affinity chromatography. The protein was
concentrated to ~10 mg/ml and stored in 20 mM Tris-HCl,
pH 8.0, 1 mM dithiothreitol at
80 °C prior to crystallization.
1 = 0.9792 Å (edge, f'),
2 = 0.9794 Å (peak,
f"),
3 = 0.9421 Å (high energy remote), and
4 = 1.0197 Å (low energy remote)). The data were processed using the HKL2000
package (24, 25). Data on the product-bound malate synthase crystal
were collected at beamline 14BMC (BioCars; Advanced Photon Source,
Argonne National Laboratory) at 1.0 Å using paratone-N as the
cryoprotectant. The data were integrated, scaled, and reduced using
DENZO and SCALEPACK (24).
Data collection, phasing statistics, and refinement statistics
Fc difference map. The final
rounds of refinement were carried out in REFMAC5 using TLS
restrained refinement (33). Non-crystallographic symmetry averaging was
not implemented in the final stages of refinement, once the
R factor was ~22%, and the Rfree
was ~30%. At this stage, the statistics were improved without using
non-crystallographic symmetry restraints. The final model contains 1434 residues (96%). Monomer A contains residues 5-303 and 309-727, a
magnesium ion, two malate molecules, and a coenzyme A molecule. Monomer
B contains residues 3-151, 156-303, and 309-727, a magnesium ion, a
glyoxylate ion, and a malate molecule. Analysis of the protein crystals
by SDS-PAGE showed no indication of proteolysis of the 80-kDa protein. 640 water molecules were added by inspection of an Fo
Fc map. The final refined structure has an
R factor of 19.0% and Rfree of
28.7% (Table I) with more than 88% of the residues in the most
favored region of the Ramachandran plot (31).
RESULTS AND DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
CONCLUSIONS
REFERENCES
/
structure consisting of
three domains (Fig. 1b).
Domain I is an 8
/8
TIM barrel consisting of residues 115-134 and 266-557. Domain II at the C terminus (residues 591-727) is mostly helical, and the
-strand rich domain III is inserted between
1 and
2 of the TIM barrel (residues 135-265). In
addition, the first 90 amino acids at the N terminus form two strands
and three helices that lie close to domain III (Fig. 1b).
The overall structure of GlcB from M. tuberculosis is very
similar to GlcB from E. coli, which was solved in complex
with glyoxylate and Mg2+ to 2.0 Å (41). The root mean
square difference between the C
of the two GlcBs is 0.7 Å,
and the sequence homology between the two proteins is high at 56%
identity and 71% similarity. Similarity searches in DALI (42) show
highest scores for TIM
/
barrel proteins of the superfamily
phosphoenolpyruvate/pyruvate domain (Z = 12-13). A large number
of hits with Z scores in the range of 7 to 8 correspond to other TIM
barrel-containing proteins. With an estimated 10% of enzymes
containing such a TIM barrel, this is a very common fold that adopts a
wide range of functions (43-45). The SCOP protein structure database
currently distinguishes 24 superfamilies of TIM
/
barrels. Malate
synthase has been placed in its own superfamily called malate synthase
G (46, 47) (scop.mrc-lmb.cam.ac.uk/scop/). To date no other members of
this fold specifically contain an insert of an all
domain in the
barrel with an all
helical C-terminal domain. The closest fold is
that of the phosphoenolpyruvate/pyruvate superfamily containing pyruvate kinase, which has an all
domain inserted at
loop 3 and a mixed
/
C-terminal domain (scop.mrc-lmb.cam.ac.
uk/scop/).
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Fig. 1.
a, multiple sequence alignment
of malate synthases. Conserved residues are shown in green,
and similar residues are indicated in yellow. The
first four sequences are of malate synthase G from
M. tuberculosis GlcB (741 amino acids; Rv1837c),
Mycobacterium leprae GlcB (731 amino acids; ML2069),
E. coli K12 GlcB (723 amino acids; b2976), and C. glutamicum GlcB (739 amino acids; NC_003450; COG2225). The
lower two sequences are of malate synthase A from Y. pestis AceB (532 amino acids; YPO3726) and E. coli AceB
(533 amino acids; b4014). There is homology within the MSGs (~60%
identity) and within the MSAs (~65%); however a much lower sequence
identity of around 18-20% is seen between the two groups. Residues
proposed to be important in catalysis including Asp-633, Glu-434, and
Asp-462 (numbered according to GlcB from M. tuberculosis)
are conserved in all types of malate synthase. b, a
ribbon representation of the structure of malate synthase.
In domain I, the 8 /8
TIM barrel consisting of residues 115-134
and 266-557, is shown with
helices in cyan and
strands shown in blue. Domain II at the C terminus consists
of residues 591-727, is mostly helical, and is shown in
green. The
-rich domain III is inserted in the TIM barrel
between
1 and
2 and is shown in orange. The N-terminal
110 residues are shown in white. Glyoxylate is indicated in
ball-and-stick representation sitting at the C-terminal end
of the
-sheet. The Mg2+ ion sitting in the active site
is shown in magenta.
of <0.5 Å. Between residues 300 and 312, six more residues were built into electron density of the
GlcB-malate-CoA structure in both molecules of the asymmetric unit, forming two short anti-parallel
-strands. However,
residues 304-308 were unable to be assigned because of weak
density, suggesting that this loop is flexible in both the
glyoxylate-bound and product-bound structures. To obtain these
product-bound crystals, GlcB was incubated with the substrates
glyoxylate and acetyl-CoA and then set up under the crystallization
conditions. Electron density for CoA was clear in one of the molecules
in the asymmetric unit but not in the other molecule. Overall the
protein backbones are structurally conserved in the two molecules, and
therefore it is not clear why there is a preference for CoA binding to
only one of the molecules. There are some subtle differences in a loop
region (residues 310-318) between the two molecules at a crystal
lattice contact point, which may lead to a preference for
crystallization of the protein in this state. In the active site of the
second monomer in the asymmetric unit, a glyoxylate molecule is bound
in the same conformation as we report in the GlcB-glyoxylate structure.
-rich domain. An Fo
Fc electron density map revealed clear density
for a single molecule of CoA bound to the protein in this channel in a
bent conformation (Fig. 2a). The N6 of the adenine ring of CoA forms a hydrogen bond with the main
chain carbonyl of Pro-543. Phe-126 may also stabilize the ring via
-stacking interactions. Salt bridges are made between AP3 of the ADP
moiety and Arg-125 (NH2 being 2.6 and 2.7 Å away from A07 and A09
phosphate oxygens, respectively) and Arg-312 (NH1 and NH2 are 3.0 and
2.8 Å away from the phosphate oxygen A08) (Fig. 2b). The
nucleoside is in a 2'-endo conformation as is most commonly seen in
other protein-CoA complexes (48). Few interactions between the
diphosphate and the protein were observed, though NZ of Lys-621
is shifted 2.7 Å compared with its position in the GlcB-glyoxylate
structure, and NZ of Lys-621 is within 3.0 Å and is likely to be
important in stabilizing AP2. CoA binding is also stabilized by an
interaction between PN4 and OG of Ser-275 (3.4 Å). In this
conformation there is a bend at the pyrophosphate group allowing
interactions between the adenine ring and the pantetheine moiety,
specifically stabilized by a hydrogen bond between AN7 of the ring and
the PO10 hydroxyl group of the pantetheine 2.9 Å apart (Fig.
2b). The pantetheinyl moiety lies almost straight in the
channel down into the active site (Fig. 2c).
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Fig. 2.
a, simulated-annealing omitted
Fo Fc map of coenzyme
A. The map was contoured at 3 sigma. b, the interactions
between coenzyme A and GlcB. The coenzyme A is shown in
white, and the carbons of the interacting amino acids are in
gold. The malate and magnesium ion in the active site are
also shown. c, binding of coenzyme A to the active site of
GlcB. Surfaces were made around the protein atoms and colored according
to the electrostatic potential, red for acidic, and
blue for basic residues, and were made using the program
SPOCK (66). Mg2+ in the active site is shown as a
blue sphere.
-methyl group
of the thioester of the acetyl-CoA by a catalytic base. The enol(ate)
intermediate is stabilized by an active site general acid. Formation of
an enolic intermediate is believed to facilitate the removal of the
-proton of a carbon acid by overcoming the high
pKa (~20-30) of the
-proton (54). (ii) The
electrophilic substrate, glyoxylate, is polarized for nucleophilic
attack, a step in which magnesium is essential, leading to the
formation of the malyl-CoA intermediate. In this condensation step
another general acid is required for the protonation of the carbonyl
group of the glyoxylate. (iii) Finally, an activated water molecule
hydrolyzes the thioester of the malyl-CoA, leading to the formation of
malate and coenzyme A. Enolization is believed to be the rate-limiting
step in the reaction mechanism.
View larger version (24K):
[in a new window]
Fig. 3.
a, active site of the GlcB-glyoxylate
binary complex. Mg2+ is held in an octahedral coordination
by the carboxylate side chains of Glu-434 and Asp-462, one carboxylate
oxygen, one aldehyde oxygen of glyoxylate- and two water molecules.
b, active site of GlcB-malate-CoA ternary complex. A water
molecule that is seen coordinating Mg2+ in GlcB-glyoxylate
is replaced by the hydroxyl of malate.
-helical, dimeric protein that does not require a
divalent cation for catalysis (52, 56). The general acid-base catalysis
for the enolization and condensation in citrate synthase are carried
out by Asp-375, which acts as the general base to abstract the
-proton of the acetyl-CoA. The two histidines (His-274 and His-320)
are the general acids, stabilizing the enolic intermediate and
protonating the carbonyl group of oxaloacetate (58, 59).
Superimposition of the active sites of malate synthase from M. tuberculosis and that of citrate synthase from pig heart suggests
that the catalytic base, GlcB-Asp-633, functions in essentially the
same role as citrate synthase-Asp-375, catalyzing the deprotonation of
the terminal acetyl group on acetyl-CoA. In citrate synthase, Asp-375
acts in concert with His-274. Citrate synthase-His-320 interacts with
the carbonyl of oxaloacetate and withdraws electrons, activating it for
nucleophilic attack. In malate synthase the role of His-274 and His-320
appear to be fulfilled by Arg-339 and Mg2+. Therefore,
these two enzymes, which are very different in sequence and fold,
appear to have converged on a very similar catalytic mechanism for
their respective reactions.
-proton of a carboxylic acid to form
the enolic intermediate as the first step in their overall reaction
(62). Members include enzymes catalyzing reactions such as racemization
(e.g. mandelate racemase),
-elimination of water
(e.g. enolase), and
-elimination of ammonia
(e.g.
-methylaspartate ammonia lyase). All members of the
superfamily contain an 8
/8
TIM barrel and require at least one
divalent cation (Mg2+ or Mn2+) for catalysis.
In many respects malate synthase seems to be more closely related to
the enolases than to citrate synthase. Mandelate racemase, a member of
this superfamily, catalyzes the racemization of mandelic acid via a
concerted general acid-general base mechanism. In this case, a lysine
and Mg2+ act as the general base stabilizing the enolic
intermediate, and the side chain of a histidine acts as the general
acid (62). In the case of E. coli enolase, which catalyzes
the dehydration of 2-phosphoglycerate in a stepwise manner, two
Mg2+ per subunit are required for catalytic activity, as
well as a lysine and a glutamate, the proposed catalytic residues.
Mg2+ binds to the high affinity cation binding site
in an octahedral coordination (63, 64). The overall similarity of the
fold of the enolases, plus the requirement for a divalent cation and the initial abstraction of an
-proton leading to an enol(ate) intermediate, clearly has similarities to the reaction catalyzed by
malate synthase.
CONCLUSIONS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
CONCLUSIONS
REFERENCES
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ACKNOWLEDGEMENTS |
---|
Use of the Argonne National Laboratory Structural Biology Center beamlines at the Advanced Photon Source was supported by the United States Department of Energy, Office of Energy Research, under Contract W-31-109-ENG-38. We thank the staff at BioCARS Sector 14 and at the Structural Biology Center on beamline 19-ID at the Advanced Photon Source for help with data collection.
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FOOTNOTES |
---|
* This work was supported by the Robert A. Welch Foundation, National Institutes of Health Grant AI46392, and GlaxoSmithKline.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
The atomic coordinates and structure factors (codes 1N8I and 1N8W) have been deposited in the Protein Data Bank, Research Collaboratory for Structural Bioinformatics, Rutgers University, New Brunswick, NJ (http://www.rcsb.org/).
§ Present address: Laboratory of Molecular Biology, NIDDK, National Institutes of Health, Bethesda, MD 20892-0560.
** To whom correspondence may be addressed. Tel.: 607-253-3401; Fax: 607-253-4058; E-mail: dgr8@cornell.edu.
To whom correspondence may be addressed. Tel.: 979-862-7636;
Fax: 979-862-7638; E-mail: sacchett@tamu.edu.
§§ Present address: Tulane University School of Medicine, 1430 Tulane Ave., SL38, New Orleans, LA 70112.
Published, JBC Papers in Press, October 21, 2002, DOI 10.1074/jbc.M209248200
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ABBREVIATIONS |
---|
The abbreviations used are: ICL, isocitrate lyase; MS, malate synthase; PBS, phosphate-buffered saline; MES, 4-morpholineethanesulfonic acid; MOPS, 4-morpholinepropanesulfonic acid; Tricine, N-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycine; OADC, oleic acid/albumin/dextrose/catalase; DTNB, 5,5'-dithiobis(2-nitrobenzoic acid); MAD, multiwavelength anamolous dispersion; r.m.s., root mean square.
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