Heterologous expression and biochemical characterization of recombinant alpha phosphoglucomutase from Mycobacterium tuberculosis H37Rv

Protein Expression and Purification 85 (2012) 117–124

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Heterologous expression and biochemical characterization of recombinant alpha phosphoglucomutase from Mycobacterium tuberculosis H37Rv Gagan Chhabra a, Divya Mathur a, Aparna Dixit b,⇑, Lalit C. Garg a,⇑ a b

Gene Regulation Laboratory, National Institute of Immunology, Aruna Asaf Ali Marg, New Delhi 110067, India Gene Regulation Laboratory, School of Biotechnology, Jawaharlal Nehru University, New Delhi 110067, India

a r t i c l e

i n f o

Article history: Received 17 May 2012 and in revised form 5 July 2012 Available online 15 July 2012 Keywords: Mycobacterium tuberculosis Phosphoglucomutase Molecular modeling

a b s t r a c t Phosphoglucomutase (PGM) plays an important role in polysaccharide capsule formation and virulence in a number of bacterial pathogens. However, the enzyme has not yet been characterized from Mycobacterium tuberculosis (Mtb). Here, we report the biochemical properties of recombinant Mtb-PGM as well as the in silico structural analysis from Mtb H37Rv. The purified recombinant enzyme was enzymatically active with a specific activity of 67.5 U/mg and experimental kcat of 70.31 s1 for the substrate glucose-1-phosphate. The enzyme was stable in pH range 6.5–7.4 and exhibited temperature optima range between 30 and 40 °C. Various kinetic parameters and constants of the rPGM were determined. A structural comparison of Modeller generated 3D Mtb-PGM structure with rabbit muscle PGM revealed that the two enzymes share the same overall heart shape and four-domain architecture, despite having only 17% sequence identity. However, certain interesting differences between the two have been identified, which provide an opportunity for designing new drugs to specifically target the Mtb-PGM. Also, in the absence of the crystal structure of the Mtb-PGM, the modeled structure could be further explored for in silico docking studies with suitable inhibitors. Ó 2012 Elsevier Inc. All rights reserved.

Introduction Tuberculosis (TB) is one of the leading causes of death globally and accounts for 2 million deaths annually. According to a World Health Organization (WHO) estimate, approximately 8–10 million new TB cases are reported every year [1]. Further, enhanced susceptibility to TB in HIV-infected patients, and the emergence of Multi- and Extensive drug-resistant TB (MDR-TB and XDR-TB) emphasize the importance of the development of new antitubercule drugs and protocols for effective control of Mycobacerium tuberculosis (Mtb) infection. The only TB vaccine currently available is the attenuated Mycobacterium bovis strain Bacillus Calmette– Guerin, which has been reported to have a variable protective efficacy ranging from 0% to 85% in different studies [2]. Mtb has evolved with an ability to survive in hostile environments. The enzymes and pathways required for the growth and survival of Mtb under nutritionally restrictive conditions of the phagosome are therefore attractive alternative targets for new anti-TB therapies. These can also target latent infection of the bacterium. A significant increase in the expression of glycolytic enzymes, meeting 70% of the energy requirement, allows the bacterium to adapt to hypoxic conditions when shifting from a strictly aerobic respiratory mode to the anaerobic mode during infection [3]. ⇑ Corresponding authors. Fax: +91 1126742125 (L.C. Garg), +91 1126742580 (A. Dixit). E-mail addresses: [email protected] (A. Dixit), [email protected], [email protected] (L.C. Garg). 1046-5928/$ - see front matter Ó 2012 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.pep.2012.07.004

Phosphoglucomutase (PGM; EC 5.4.2.2), a key enzyme that contributes to glycolysis by catalyzing the reversible transfer of a phosphoryl group from the 10 to the 60 position on a glucose monomer via glucose 1,6-diphosphate intermediate [4]. Glucose-1-phosphate, produced from glycogen by the action of glycogen phosphorylase, is converted to glucose-6-phosphate by PGM. Glucose-6-phosphate thus formed can enter either the glycolytic pathway or the pentose phosphate pathway. In the presence of higher glucose levels, the PGM-catalyzed reverse reaction results in the formation of glucose-1-phosphate, which is then converted to UDP-glucose, a precursor for polysaccharides synthesis. Thus, PGM plays an important role in carbon metabolism and directs its flow either towards anabolic or catabolic pathways. The involvement of PGM in the formation of lipopolysaccharide and other virulence factors, as well as its role in imparting resistance to bacteria against antimicrobial peptides have also been reported [5,6]. Since PGM contributes to glycolysis, which is crucial for the organism’s survival in macrophages, it would be of interest to characterize this enzyme from Mtb. Thus, the key roles that PGM plays in the biology of Mtb also make it a viable drug target. In the present study, we report the biochemical and molecular characterization as well as structural analysis of the recombinant Mtb-PGM, which will aid in effective therapeutic design.

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Materials and methods Cloning of the Mtb pgmA gene The pgmA gene was amplified using genomic DNA isolated from BAC genomic library of M. tuberculosis (kindly provided by Prof. Stewart Cole of Pasteur Research Institute) and gene specific primers (Forward: 50 -GGACCATATGGTGGCCAACCCACGAGC-30 and reverse: 50 -AATTCTCGAGTCACCCGATGACCCGATC-30 ) were designed on the basis of sequence information of Mtb-pgmA (Rv3068c). The NdeI and XhoI sites were introduced in the forward and reverse primers respectively, for convenient cloning in the expression vector. PCR was performed for 30 cycles, each involving denaturation at 94 °C for 1 min, annealing at 55 °C for 1 min, and extension at 72 °C for 1 min, with a final extension at 72 °C for 7 min in GeneAmp PCR system (Perkin-Elmer, USA). The amplified product digested with NdeI and XhoI (NEB, USA) was cloned into pET28a(+) (Novagen, USA), digested with the same enzymes, and transformed into Escherichia coli BL21(DE3) cells. Putative recombinants were analyzed by restriction digestion and confirmed by automated DNA sequencing (Applied Biosystem Model 393A). The recombinant clone thus generated was designated as pET28.Mtbpgm. Expression of histidine-tagged recombinant PGM (6His–rPGM) in E. coli E. coli BL21 (DE3) cells harboring the pET28.Mtbpgm were cultured at 37 °C in Luria–Bertini medium in the presence of kanamycin (50 lg/ml). For the expression of rPGM, the cells were induced with 0.25 mM isopropyl-b-D-thiogalacto-pyranoside (IPTG). The induced cells harvested at 8 h post-induction were processed as described by Mathur et al. [7]. Soluble and insoluble fractions from the cell pellet were prepared as described earlier [7]. The pellet was resuspended and solubilized in 10 mM Tris–HCl (pH 7.4) containing 8 M Urea.

spectra were acquired using Flexcontrol 2.2 software with ion source voltage 25 kV and accelerating voltage 23.2 kV in the positive linear mode of operation and processed using Flexanalysis™ 2.2 software. Time to mass conversion was achieved by external calibration using protein standards obtained from Bruker, Germany. Protein identification was performed by searching for bacteria in the NCBI non-redundant database using the Mascot search engine (http://www.matrixscience.com/). Circular dichroism (CD) spectroscopy Far UV (200–250 nm) CD spectra of the 6His-rPGM (0.2 mg/ml in 10 mM Tris–HCl, pH 7.4) at 30 °C were recorded using a spectropolarimeter (JASCO J-815; path length, 0.1 cm; scan speed, 50 nm/ min). Ten successive spectra were accumulated and averaged followed by baseline correction. Mean residue weight ellipticities are expressed as degree  cm2  dmol1. Secondary structure contents of the refolded protein from the CD measurements were calculated using the K2D2 program (http://www.ogic.ca/projects/ k2d2). Determination of enzyme activity Phosphoglucomutase activity was determined as described previously [8]. The assay mixture consisted of 0.1 mM Tris–HCl (pH 7.4), 0.25 mM MgCl2, 15 lM BSA, 0.5 mM bNADP+, 2 mM glucose1-phosphate, 8 lM glucose-1,6-bisphosphate, and 1.25 U glucose6-phosphate dehydrogenase in a final reaction volume of 1 ml. The reaction mixture was incubated at 30 °C for 5 min, and the reaction was initiated by the addition of an aliquot of the purified recombinant enzyme. The activity was measured by monitoring the change in the absorbance at 340 nm for 5 min (Lambda25 Perkin-Elmer, USA). One unit of enzyme activity is defined as the amount of enzyme that catalyzes the conversion of 1 lmol of glucose-1-phosphate to glucose-6-phosphate per minute under the assay conditions.

Purification of the 6His-rPGM Determination of temperature and pH optima The 6His-rPGM was purified from the solubilized inclusion bodies using Ni– NTA chromatography and refolded on column. The solubilized protein was allowed to bind to Ni–NTA resin (Qiagen, Germany) on a tube rotator for 1 h at room temperature. Subsequently, the protein-bound beads were collected by centrifugation at 1200g for 2 min. On column refolding was performed by washing the beads with 6, 4, 2, 1, and 0 M urea in 10 mM Tris–HCl, pH 7.4. After washing the column with 25 mM imidazole in 10 mM Tris–HCl, pH 7.4, the specifically bound refolded protein was eluted using 250 mM imidazole in 10 mM Tris–HCl, pH 7.4. The protein was concentrated using Amicon ultra-centrifugal filter device (Ultracel-30 k, Millipore, USA). MALDI-TOF mass-fingerprinting The matrix-assisted laser desorption/ionization time-of flight (MALDI-TOF) service was carried out using a Bruker Daltonic Ultraflex TOF/TOF system calibrated using Calibration std.II Bruker Daltonics (Bruker Daltonik GmbH, Germany) at the NxGenBio Life Sciences, New Delhi, India. The purified recombinant protein was electrophoresed on SDS–PAGE and the protein band was carefully excised out of the SDS–PAGE gel, cut into small pieces, washed with 25 mM ammonium bicarbonate (pH 8.0) containing 50% acetonitrile for 15 min. Before tryptic digestion gel pieces were dehydrated with 100 lL acetonitrile and finally dried with a Speed-Vac. MALDI-TOF analysis was performed using HCCA matrix. The

The optimum temperature for the 6His-rPGM activity was determined by performing the assay reaction at different temperatures (0–50 °C) in 10 mM Tris–HCl buffer, pH 7.4. For determination of optimum pH, the enzyme activity was measured in buffers of different pH (Imidazole buffer, pH 5–7; Tris–HCl buffer, pH 7.4– 9.0). Determination of Km and Ki for the 6His-rPGM The Km for the substrate glucose-1-phosphate was determined using a Lineweaver–Burk plot. The Ki for the non-competitive inhibitor, fructose-2,6-diphosphate, was determined by a Dixon plot using three different concentrations of the inhibitor with different concentrations of the substrate, glucose-1-phosphate. The data represent the mean of at least three independent determinations. Phylogenetic analysis Multiple sequence alignment of the Mtb-PGM sequence obtained from the TB Structural Genomics Consortium (www.doe-mbi.ucla.edu/TB/) [9] with the PGM sequences of other species was performed using ClustalW multiple sequence alignment tool [10]. The evolutionary history was inferred using the Neighbor-Joining method [11]. Phylogenetic analyses were conducted in MEGA5 [12].

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Homology modeling A PDB blast search for the amino acid sequence of the Mtb-PGM (Accession No. NP_217584) was performed in order to identify the most suitable template. Multiple sequence alignment of the target and the template was performed using ClustalW multiple sequence alignment tool [10]. Sequence alignment revealed that the best template structure for modeling was that of Salmonella typhimurium phosphoglucomutase (PDB code 3NA5) [13]. A homology model of Mtb-PGM was built using the MODELLER program [14]. The quality of the model was evaluated using PROCHECK and Ramachandran plot [15,16]. The modeled structure, and its superposition onto the known structures of the template and rabbit PGM were visualized using Swiss-PDB Viewer software (http://www.expasy.org/spdbv).

Results Sequence analysis of Mtb-pgmA gene Sequence analysis of the cloned PCR-amplified pgmA fragment revealed it to be a full length pgmA gene of 1641 bp. A genome database search revealed varying degrees of similarity of the Mtb-PGM with the PGMs of other species, at both the nucleic acid and amino acid levels. BlastP of the amino acid sequence of Mtb-PGM showed that the percentage identities of the Mtb-PGM with the PGMs of E. coli, S. typhimurium, Saccharomyces cerevisiae, Drosophila, human, mouse, and rabbit were 58%, 57%, 28%, 27%, 22%, 22%, 17%, respectively. Multiple sequence alignment of the Mtb-PGM with the PGMs of other species revealed that although there are significant differences among the PGMs from different species, the signature sequences of the PGM super family – SerHis-Asn-Pro and Gly-Glu-Glu-Ser – remain conserved (Supplementary Fig. S1).

Phylogenetic analysis Twenty-two PGM amino acid sequences (corresponding to phosphoglucomuatse enzymes of different species) were selected. A phylogenetic tree obtained by using Neighbor-Joining method illustrated that each of the 22 PGM proteins fell into one of the five major lineages (Fig. 1) with three dominant clusters. The Mtb-PGM sequence was found to be most closely related to the PGM of Arthrobacter aurescences. The Mtb-PGM also shared high sequence similarity with the PGMs of enteric bacteria Vibrio cholerae, S. typhimurium and E. coli. As evident from the tree, the Mtb-PGM

Fig. 1. Phylogenetic tree inferred using the Neighbor-Joining method, showing the most likely relationship between representative PGM amino acid sequences of different species with the respective UniProtKB accession codes shown in parentheses. The number associated with each internal branch is the local bootstrap probability, an indicator of confidence. The bar (0.2) denotes pair-wise distance estimates of the expected number of amino acid replacements per site.

was found to be very distantly related to the human and rabbit PGMs.

Expression and purification of the 6His-rPGM The Mtb-pgmA fragment cloned into the pET28a(+) was expressed in E. coli BL21(DE3) cells (Fig. 2a). Upon induction with IPTG, a prominent band on SDS–PAGE corresponding to the expected size of 6His-rPGM (60 kDa) was observed, suggesting an efficient expression of pgmA (Fig. 2a, lane 2). Analysis of both the soluble and insoluble fractions of the induced cell lysates revealed that the 6His-rPGM was expressed as inclusion bodies (Fig. 2a, lane 4). The 6His-rPGM from solubilized inclusion bodies was refolded on column and purified to near homogeneity (Fig. 2a, lane 5). Western blot analysis using anti-Histidine HRPO-conjugated antibody confirmed the authenticity of the 6His-rPGM (Fig. 2b). The authenticity of the recombinant protein to be the PGM of Mtb was further confirmed by MALDI-

Fig. 2. Expression, purification, and CD spectrum of the 6His-rPGM. (a) SDS–PAGE (12%) analysis of recombinant 6His-rPGM expressed in E. coli strain BL21(DE3). M denotes the protein molecular markers. Lanes 1 and 2 Represent the uninduced and induced cell lysates, respectively. Lanes 3 and 4 are the soluble and insoluble fractions prepared from the induced cell lysates. The purified refolded 6His-rPGM is shown in lane 5. (b) Western blot analysis of the purified 6His-rPGM using anti-Histidine HRPOconjugate antibody. (c) Far-UV CD spectrum of the 6His-rPGM. CD values are expressed as [h], mean residue mass elipticity in units of degree  cm2 dmol1.

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Fig. 3. MALDI-TOF mass spectrum analysis of the tryptic digests of the recombinant Mtb-rPGM: The PMF analysis was carried out for the fragments obtained through the tryptic digestion of purified 6His-rPGM. (A) Mass spectrum analysis of recombinant His tagged-PGM of Mtb (N-terminal His tag Rv3068c) (B) The Mascot search results indicating the identified protein, intensity coverage, amino acid sequence coverage and the identified peptides are shown. The matched peptide ions in the phosphoglucomutase of M. tuberculosis amino acid sequence are shown in red. The boxes (grey and dark grey) underneath the amino acid sequence indicate the peptide ions generated by the tryptic digestion of the recombinant PGM that matched with the peptides of Mtb PGM in the database. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

TOF analysis of the purified recombinant protein. The monoisotopic masses obtained for the individual peptides were in the range of 725.405–2780.244. Search of the sequences obtained from the peptide mass fingerprinting (PMF) of the digested protein using Mascot search engine identified and confirmed the recombinant protein as phosphoglucomutase protein from M. tuberculosis with a score of 172 (Fig. 3).

Physical and kinetic properties of the 6His-rPGM The two local minima, at 208 and 220 nm, in the CD spectrum of the purified refolded 6His-rPGM indicate the predominant presence of a-helical regions (Fig. 2c). Analysis of the spectrum of the refolded 6His-rPGM using K2D2 also confirmed the presence of a-helices predominantly (50.16%), with 7.41% as b sheets, and the remainder as random coil.

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Fig. 4. Biochemical characterization of the 6His-rPGM. (a) Determination of the temperature optimum for the 6His-rPGM. The activity of the recombinant enzyme was estimated at different temperatures (0–50 °C) using the 6His-rPGM (6.66 nM) in 10 mM Tris–HCl buffer, pH 7.4. (b) Determination of pH optimum for the 6His-rPGM. Enzyme activity was measured in the pH range of 5–9 in different buffers (pH 6.0–7.0, imidazole buffer; pH 7.4–9.0, Tris–HCl buffer) using the 6His-rPGM (6.66 nM) at 37 °C. (c) Determination of the Km of the 6His-rPGM. The Michaelis–Menten plot was plotted using different concentrations of glucose-1-phosphate and 6.66 nM of 6HisrPGM. The inset shows the Lineweaver–Burk plot. (d) Dixon plot for determination of Ki of the 6His-rPGM for non-competitive inhibitor, fructose-2,6-diphosphate. The Ki was calculated by using three different concentrations (0.1, 0.5 and 2 mM) of the inhibitor with three different concentrations (0.5, 1 and 2 mM) of the substrate, glucose-1phosphate.

Mtb-PGM, like rabbit muscle PGM, is made up of a single polypeptide chain containing an active site, and a metal-binding site. However, unlike rabbit PGM with five cysteines, Mtb-PGM has only two cysteines – Cys279 & Cys447 [17]. The Mtb-PGM with 547 amino acid residues has a molecular mass of 58.2 kDa, which falls within the range of molecular masses of a-PGMs (alpha phosphoglucomutases) from other species, which differ from the bacterial b-PGM of approximately half the molecular mass [18–20]. The refolded 6His-rPGM was enzymatically active, indicating efficient refolding of the recombinant protein. On determination of the enzyme activity at different pH, Mtb 6His-rPGM exhibited comparable activity, within the measurable range of the assay, between pH 6.5 and 7.4 (Fig. 4a). Maximum activity of the enzyme was obtained between 35 and 40 °C. The enzyme was significantly less active at temperatures lower than 30 °C and higher than 45 °C, and was completely inactive at temperatures greater than 50 °C (Fig. 4b). The effect of glucose-1-phosphate concentration on enzyme activity was determined. The enzyme followed Michaelis– Menten kinetics (Fig. 4c). The Km of the 6His-rPGM, using Lineweaver–Burk plot, for glucose-1-phosphate and glucose-1,6-biphosphate were determined to be 0.12 mM and 2.0 lM, respectively. The catalytic constant for G1P (kcat) for 6His-rPGM was determined to be 70.31 s1. Catalytic efficiency [kcat/Km (G1P)] of the 6His-rPGM was determined to be 5.84  105 M1 s1, which is much lower than that of rabbit muscle PGM (9.0  107 M1 s1) [21]. The specific activity of the recombinant 6His-rPGM was determined to be 67.5 lmol/min/mg. The recovery and yield of

the recombinant active protein during refolding process are given in Table 1. Fructose-2,6-bisphosphate inhibits phosphoglucomutase noncompetitively with respect to the cofactor glucose 1,6-bisphosphate, an intermediate formed during the PGM catalyzed reaction [22]. Inhibition of the 6His-rPGM by fructose-2,6diphosphate was examined and the inhibition constant (Ki) for fructose-2,6-diphosphate was calculated to be 2.0 mM (Fig. 4d). Structure modeling and evaluation of the phosphoglucomutase model Amino acid sequence alignment of Mtb-PGM with the PGM sequences of other organisms available from the PDB showed that Mtb-PGM shared highest sequence identity (57%) with S. typhimurium PGM. A homology model for Mtb-PGM was built using the Modeller program, based on the S. typhimurium PGM chain A (PDB code: 3NA5) as a template (Fig. 5a). PROCHECK analysis confirmed the quality of the model to be good. The Ramachandran plot revealed that the modeled Mtb-PGM has 93.4% residues in most favorable regions, and 6.3% residues occur in allowed regions. Only one residue-Ala200 was found in the disallowed regions (data not shown). Thus, the modeled structure fulfills the parameters expected from a good quality model. The superposition of the modeled structure of Mtb-PGM onto the structure of the 3NA5 monomer (Chain A of PDB entry 3NA5) matches 532 Ca atoms with RMSD of 0.22 Å. Additionally, the core of the phosphoglucomutase is highly conserved in both the struc-

Table 1 Flowsheet of the recombinant Mtb-PGM (Rv3068c) refolding and purification. Steps

Amount of Protein (mg)

Total activity (U)

Amount of active protein (mg)

Specific activity (U mg1)

% of refolding

Inclusion bodies after washing Ni++–NTA bound protein Refolded protein After concentration

103.2 68.8 48.0 40.0

– – 2568 2700

– – 38.0 40.0

– – 53.5 67.5

– – 55.2 58.1

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Fig. 5. Homology modeling and structural analysis of the Mtb-PGM. (a) Ribbon diagram of the Mtb-PGM structure based on template 3NA5. A general view showing the Nand C- terminal as Swiss PDB viewer representation in secondary structure succession color scheme. (b) Structural superposition of homology model of Mtb-PGM with crystal structure of rabbit PGM. Color scheme Mtb-PGM vs. rabbit PGM domain 1- Blue, Red; domain 2- Green, Yellow; domain 3- Pink, Sky blue; domain 4- Dark blue, dark red. Nterminal of Mtb-PGM shown in blue and rabbit PGM in red. (c) Amino acid sequence alignment of the Mtb-PGM and rabbit PGM with structural features of the two shown on top (for the modeled Mtb-PGM) and bottom (for rabbit PGM, PDB ID 3PMG). The a helices and b sheets are shown as cylinders and arrows, respectively. The shaded residues represent conserved catalytic regions. The structural differences between the two structures are shown in the boxed regions. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

tures. The RMSD of <0.5 Å indicates a very small deviation between the modeled structure of the target and the template structure, suggesting the model is a feasible structural model (data not shown). The unavailability of human PGM 3D structure necessitated comparative structural analysis of the modeled structure of MtbPGM with the available structure of rabbit PGM (PDB: 3PMG) [23]. The superposition of the two showed a high RMSD of 1.5 Å, indicating that the two structures have many structural differences (Fig. 5b and c).

Discussion It has recently been discovered that PGM plays an important role in polysaccharide capsule formation and in the virulence of pathogenic bacteria [5,24–26]. Avirulent pgm mutants in Brucella abortus and Streptococcus iniae have been reported as potential candidates for live attenuated vaccine strains [27,28]. Important for the virulence of many pathogens, PGM can serve as a potential therapeutic target. The present study describes the biochemical and molecular characterization of the recombinant phosphoglucomutase encoded by pgmA gene from M. tuberculosis. As depicted in the phylogenetic analysis, Mtb-PGM is distantly related to human PGM and rabbit PGM, showing minimal identity at the amino acid level. Given the absence of a human PGM crystal structure, and the high sequence identity (96%) between human PGM and rabbit PGM, the predicted 3D structure of Mtb-PGM was compared with the crystal structure of the rabbit muscle PGM.

Mtb-PGM exhibits very low sequence identity with rabbit PGM, characterized earlier, except at the active site region [17]. Amino acid sequence comparisons between these two proteins clearly identified the conservation of important functional elements (80– 100%) as evident from Supplementary Fig. S1, despite a relatively low overall sequence identity (17%). The superposition of the modeled 3D structure of Mtb-PGM onto the rabbit PGM (PDB ID: 3PMG) reveals that although they share the same overall heart shape and four-domain architecture, there is a high degree of variability between the two structures, as is evident from the calculated RMSD (1.5 Å, with 303 Ca atoms) between the two superposed structures. Since rabbit PGM (562 residues) has 15 residues more than Mtb-PGM (547 residues), it has some additional secondary structural elements; these tend to fall on the periphery of the molecule, distant from the active site. The structural alignment demonstrates that critical residues in the active site, including the putative phosphorylation site (Ser-147) surrounded by the catalytic reaction center (Thr-Pro-Ser147-His-Asn-Pro), a metal binding site (Asp-Ala-Asp-Ala-Asp-Arg), and the glucose ringbinding site (Gly-Glu-Glu-Ser), are in very similar positions, except for a single amino acid difference in the glucose ring binding site, wherein the Mtb-PGM consists of a glycine in place of cystine in rabbit PGM [23]. The similarity in the active sites in these two proteins makes it difficult to design an effective drug targeting this site. However, other important differences between the two phosphoglucomutases might be exploited to achieve this purpose. The identification of such differences would enable the design of drugs/molecules

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G. Chhabra et al. / Protein Expression and Purification 85 (2012) 117–124 Table 2 Comparison of kinetic properties of the recombinant Mtb-PGM with the phosphoglucomutases of other bacterial species. Organism

Kinetic parameters kcat (s

Mycobacterium tuberculosis Escherichia coli Pseudomonas aeruginosa Acetobacter xylinum Sphingobium chungbukense(SP-1) Toxoplasma gondii (TgPGM-I)

1

70.31 54 22.5 88.4 126.8 398

)

Reference Km (G1P) (mM)

kcat/Km (G1P) (M

0.12 0.05 0.022 0.29 0.22 0.19

5.8  105 10.8  105 10.2  105 3.0  105 5.7  105 20.0  105

that would target the Mtb-PGM specifically but exclude the host enzyme. This approach has recently been reported to be highly beneficial in developing effective inhibitors against Mtb proteasomes [29]. Since the crystal structure of Mtb-PGM is not known, the modeled structure reported in the present study can aid in the development of successful therapeutic agents. Like the native PGMs from other species [13,23], the predominant presence of a helices (50%) with relatively lower percentage of b sheets together with rest of the structure as random coil in the refolded 6His-rPGM can be seen in the CD analysis. The crystal structures of the template PGM of S. typhimurium [13] used for the homology modeling in the present study and of other species [23] also show similar pattern. The refolded 6His-rPGM was also biologically active indicating that the protein folded correctly. Unlike human PGM which shows pH optima between a very narrow range of pH 7.4–7.6 [30], the recombinant 6His-rPGM of Mtb, showed a broad pH optima for the (6.5–7.4). Such a broad pH optima range have also been reported for PGM from other organisms such as Fusarium oxysporum (pH 6–8), Cassia corymbosa (pH 6.8–8.3) and Sphingobium chungbukense (pH 7–9) [30 and references there in]. Under the assay conditions, the recombinant Mtb-PGM was active between 35 and 40 °C after which 50% loss in the activity was found when assayed at 45 °C. The loss in activity at higher temperatures could be due to loss of tertiary structure, thus affecting ligand binding and subsequently catalysis. The rPGMs from other species have also been shown to be stable at 40 °C [30–32]. Interestingly, The PGMs from warm-adapted Mediterranean mussel Mytilus galloprovincialis and the cold-adapted Mytilus trossulus from North Pacific Ocean showed similar temperature sensitivity and kinetic properties at 40 °C in vitro, suggesting that the cellular environment in the two might be playing a role in adaptation of the enzyme to cold vs. high temperatures [32]. Thus, the native PGM of M. tuberculosis may also have different temperature sensitivity in vivo. Michaelis–Menten parameters, Km and kcat, for PGM are known to vary considerably across species. The Km (G1P) values have been reported to range from 1 lM for PGM from F. oxysporum to 220 lM for S. chungbukense PGM [4,30]. Km (G1P) for PGM from some of the thermophilic organisms such as Thermococcus kodakarensis has been reported to be as high as 3 mM [30]. The calculated Km value of 120 lM for the recombinant Mtb-PGM falls within the range of the reported Km values for the corresponding PGM from some of other bacteria listed in Table 2. Similarly, the refolded recombinant Mtb-PGM exhibits kcat (70 s1) comparable to that of E. coli [18]. However, it is relatively lower kcat (70 s1) when compared to that of Toxoplasma gondii (398 s1) and rabbit muscle PGM (730 s1) [30,33]. The data clearly show that the kinetic parameters of the refolded protein are comparable with those reported for other bacterial PGMs [34–37] indicating efficient refolding. This is expected as the catalytic domain of PGM has remained conserved amongst various species. Understanding the molecular and kinetic characteristics of the Mtb-PGM is a significant step towards gaining better insight into this member of the glycolytic pathway.

1

s

1

) Present study [18] [34] [35] [36] [37]

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