Biochemical and structural characterization reveals Rv3400 codes for β‐phosphoglucomutase in Mycobacterium tuberculosis

Abstract

Mycobacterium tuberculosis (Mtb) adapt to various host environments and utilize a variety of sugars and lipids as carbon sources. Among these sugars, maltose and trehalose, also play crucial role in bacterial physiology and virulence. However, some key enzymes involved in trehalose and maltose metabolism in Mtb are not yet known. Here we structurally and functionally characterized a conserved hypothetical gene Rv3400. We determined the crystal structure of Rv3400 at 1.7 Å resolution. The crystal structure revealed that Rv3400 adopts Rossmann fold and shares high structural similarity with haloacid dehalogenase family of proteins. Our comparative structural analysis suggested that Rv3400 could perform either phosphatase or pyrophosphatase or β‐phosphoglucomutase (β‐PGM) activity. Using biochemical studies, we further confirmed that Rv3400 performs β‐PGM activity and hence, Rv3400 encodes for β‐PGM in Mtb. Our data also confirm that Mtb β‐PGM is a metal dependent enzyme having broad specificity for divalent metal ions. β‐PGM converts β‐D‐glucose‐1‐phosphate to β‐D‐glucose‐6‐phosphate which is required for the generation of ATP and NADPH through glycolysis and pentose phosphate pathway, respectively. Using site directed mutagenesis followed by biochemical studies, we show that two Asp residues in the highly conserved DxD motif, D29 and D31, are crucial for enzyme activity. While D29A, D31A, D29E, D31E and D29N mutants lost complete activity, D31N mutant retained about 30% activity. This study further helps in understanding the role of β‐PGM in the physiology of Mtb.

1. INTRODUCTION

Mycobacterium tuberculosis (Mtb) is the pathogen responsible for causing tuberculosis which includes pulmonary tuberculosis (TB), gastrointestinal TB, liver TB, TB peritonitis, and so forth. It accounts for the deaths of 1–1.5 million people every year making it the second largest infectious cause of death worldwide (World Health Organization, 2021). Current TB treatment involves a combination of antibiotics that specifically target vital bacterial functions like translation, transcription, replication, and so forth. Unfortunately, the current treatments for TB are becoming less effective worldwide, leading to the emergence of highly resistant strains known as MDR and XDR. Finding new antimicrobial therapeutic targets against Mtb with great potency and specificity is therefore urgently required. The genes that allow Mtb to thrive and persist in the host may be considered as potential drug targets. Ideally, these genes should only be essential for the pathogen and not to the host. The libraries of random transposon mutant and completely saturated transposon library experiments have identified the proteins and genes that are essential for the survival of Mtb (Chao et al., 2016; DeJesus et al., ²⁰¹⁷). The whole genome of the Mtb H37Rv strain has been sequenced to obtain a better understanding of its virulence and immunity. A significant fraction of the Mtb proteome consists of hypothetical proteins (HPs, a term often used in genomics and bioinformatics to describe a protein with unknown function), which make up around 25% of the approximately 4000 genes in the Mtb genome and are predicted to be expressed from an open reading frame (Hawkins & Kihara, 2007; Nimrod et al., ²⁰⁰⁸; Yang et al., ²⁰¹⁹). The annotation of HPs from Mtb will aid in the discovery of novel structures and functions and also serve as pharmacological indicators and targets for drug discovery, design, and screening (Galperin & Koonin, 2004; Lubec et al., ²⁰⁰⁵).

We selected one such conserved hypothetical protein, Rv3400 for its detailed structural and functional characterization. Rv3400 is shown to be non‐essential for in vitro growth of H37Rv (DeJesus et al., 2017; Minato et al., ²⁰¹⁹; Sassetti & Rubin, ²⁰⁰³) but important for the growth in C57BL/6J mouse spleen (Sassetti & Rubin, 2003). Mycobacterial cell entry (mce) loci in Mtb have been identified as important factors in promoting the uptake of bacteria into nonphagocytic cells and allows bacteria to evade the immune system and establish infections within host tissues (Joshi et al., 2006). Using genetic interaction mapping strategy Joshi et al have reported interaction of several Mtb genes including Rv3400 with mce4, indicating its potential involvement in the virulence (Joshi et al., 2006).

Rv3400 is annotated as a probable hydrolase in Mycobrowser (Kapopoulou et al., 2011) and the similarity search using its amino acid sequence in Basic Local Alignment Search Tool (BLAST), shows top hits with members of haloacid dehalogenase (HAD) family proteins. One of the sub‐category of phosphatases that belongs to the HAD superfamily is Phosphoglucomutase (PGM). The PGM catalyzes the transfer of phosphate group from the 1st position to the 6th position on the glucose molecule, or vice versa, that is, converting D‐glucose 1‐phosphate (G1P) to D‐glucose 6‐phosphate (G6P) in carbohydrate metabolism. PGM is the connecting link between the glycolysis and gluconeogenesis pathways. The phosphoglucomutase is divided into two classes: α‐phosphoglucomutase (α‐PGM, EC 5.4.2.2), which is found in all prokaryotes and eukaryotes and β‐phosphoglucomutase (β‐PGM, EC 5.4.2.6), which is known to present only in bacteria and protists (Bras et al., 2018; Koonin & Tatusov, ¹⁹⁹⁴; Neves et al., ²⁰⁰⁶). These two groups are distinguished by their substrate specificity and overall protein fold. The α‐PGM belongs to phosphohexomutase superfamily, while β‐PGM belongs to HAD superfamily (Shackelford et al., 2004). The primary cellular function of β‐PGM is to catalyze the isomerization of β‐D‐glucose 1‐phosphate (G1P) to β‐D‐glucose 6‐phosphate (G6P) with β‐D‐glucose 1,6‐bisphosphate (β‐G1,6bisP) as an intermediate (Figure 1). G6P is a universal source of cellular energy and leads to the formation of ATP and NADPH via glycolysis and pentose phosphate pathway respectively (Barrozo et al., 2018).

FIGURE 1.

Flowchart showing metabolism of maltose and trehalose as a carbon source in bacterial cell.

Trehalose is a peculiar non‐mammalian disaccharide abundantly present in mycobacteria (Elbein et al., 2003). Trehalose‐containing glycolipids play important roles in the virulence and metabolism of Mtb. These glycolipids are major components of the mycobacterial cell membrane and have been found to contribute significantly to the pathogen's ability to cause disease (Kapopoulou et al., 2011; Thanna & Sucheck, ²⁰¹⁶). Likewise, another sugar, maltose, plays a vital role in the physiology of Mtb. It is an essential component for the formation of α‐glucan that constitute the capsule. The capsule is the outermost layer of the Mtb cell envelope and plays an essential role in the pathogenesis of tuberculosis (Kalscheuer et al., 2019). Besides the role of these sugars in the physiology of Mtb, they can also be used as carbon source during stress or glucose starvation (Figure 1) (Jankute et al., 2015). However, the metabolism of trehalose and maltose in Mtb has not been completely understood.

Here, we have biochemically and structurally characterized Rv3400 and determined its crystal structure at 1.7 Å resolution. We present the comparative structural analysis and the biological function of this conserved hypothetical protein. Our data confirm that Rv3400 codes for β‐PGM enzyme and the DxD motif present in the active site of Rv3400 is important for its enzymatic function.

2. RESULTS

2.1. Bioinformatics analysis of Rv3400

The BLAST search using the amino acid sequence of Rv3400 revealed that it is conserved throughout the Mycobacterial species and shares about 66% sequence identity among them. Interestingly, a homolog of Rv3400 is also present in Mycobacterium leprae which has undergone extensive gene reduction and contains only about ~1600 functional genes (Cole et al., 2001; Gomez‐Valero et al., ²⁰⁰⁷). Presence of Rv3400 homolog (ML0393c) even in the minimalistic set of genes encoded in M. leprae emphasizes its importance in bacterial physiology/virulence. The BLAST search also reveals that the Rv3400 shares significant sequence identity with β‐phosphoglucomutase (75% sequence identity with 94% query coverage) and phosphatase (56% sequence identity with 93% query coverage) of other organisms, suggesting that Rv3400 could be a potential β‐phosphoglucomutase or phosphatase. Similarly, the BLAST search using the amino acid sequence of Rv3400 against PDB database shows sequence identity with the other members of HAD proteins like β‐phosphoglucomutase from Lactococcus lactis, (31% sequence identity with 81% query coverage), pyrophosphatase from Bacteroides thetaiotaomicron (24% sequence identity with 80% query coverage) and phosphatase from Thermococcus onnurineus (34% sequence identity with 44% query coverage). In addition, the Pfam (Protein family) database predicted that the Rv3400 is a member of Haloacid dehalogenase family (HAD) proteins. These HAD proteins are superimposable and have conserved structural motifs (motif I, II, III and IV) despite very low sequence identity (Figure 2a–c). To determine the exact function and to rule out the ambiguity of Rv3400 as a mutase, phosphatase or pyrophosphatase we carried out the detailed characterization of Rv3400 in this study.

FIGURE 2.

(a) Multiple sequence alignment of protein containing HAD‐domain. The alignment reveals conserved structural motifs labeled as I, II, III, and IV. Conserved residues are depicted as red on a white background, while identical residues are displayed as white on a red background. The amino acid residues conserved between the groups are boxed. Motifs that are conserved between the HAD members are marked and placed within the black box. Sequences are identified by the PDB ID; 1O03 (β‐phosphoglucomutase from Lactococcus lactis), 3QU2 (pyrophosphatase from Bacteroides thetaiotaomicron), 4YGQ (HAD phosphatase from Thermococcus onnurineus) and Rv3400 from Mycobacterium tuberculosis. Multiple sequence alignment was performed with the Multalign server (Corpet, 1988) and the figure was generated by ESpript 3.0 (Robert & Gouet, 2014). (b) Structural superposition of β‐phosphoglucomutase from L. lactis, PDB ID: 1O03 (orange), pyrophosphatase from B. thetaiotaomicron, PDB ID: 3QU2 (cyan) and phosphatase from T. onnurineus PDB ID: 4YGQ (magenta). (c) Catalytic motif representing the four conserved motif in pyrophosphatase from B. thetaiotaomicron.

2.2. Rv3400 encodes for β‐phosphoglucomutase

The gene encoding Rv3400 (789 base pairs) was amplified by PCR, cloned in pETDuetN vector and expressed in Rosetta (DE3) expression strain. The recombinantly expressed Rv3400 protein was purified further for its biochemical and structural characterization (described in the materials and methods section). The analytical size exclusion chromatography studies suggests that Rv3400 predominantly exists as a monomer in solution (Figure 3a).

FIGURE 3.

Purification and functional characterization of Rv3400. (a) Analytical Gel filtration profile of Rv3400. The inset shows the standard curve. Peak (i) in the graph corresponds to the void volume (7.7 mL) whereas peak (ii) is the elution volume of the protein Rv3400 (17.2 mL). The column used in the Size Exclusion Chromatography (SEC) experiment, has the bed volume of 24 mL. Testing Rv3400 for (b) phosphatase, (c) pyrophosphatase and (d) β‐PGM activities. Notably, the Rv3400^Δ16M2 protein also shows comparable β‐PGM activity to that of wild‐type. Negative control (NC) is the reaction mixture without Rv3400, Calf intestinal alkaline phosphatase (Thermo) and pyrophosphatase from S. cerevisiae were used as positive controls (PC) in phosphatase and pyrophosphatase assays, respectively. The enzyme concentration was kept 5 μM in all reactions. (e) The relative β‐PGM activity of Rv3400 in the presence of different divalent metal ions, NC represents the activity in the absence of any divalent metal ion. All the experiments were done in triplicates. (f) Michaelis–Menten plot showing the kinetic parameters for β‐PGM activity in Rv3400. For each concentration of the substrate, all readings were taken in triplicate. Error bars represents the standard deviation (SD).

The sequence analysis using bioinformatics tools suggested that Rv3400 may have a phosphatase or pyrophosphatase or β‐phosphoglucomutase activity. To determine the functional activity of Rv3400, we performed all three enzyme assays. For checking the phosphatase activity of Rv3400, a common substrate para‐NitroPhenyl Phosphate (pNPP), was used in the assay. Alkaline phosphatase was utilized as a positive control which shows an increase in absorbance at 405 nm due to the formation of p‐nitrophenol (pNP) owing to the phosphatase activity (Figure 3b). However, in the presence of Rv3400, no change in absorbance was observed at 405 nm suggesting that Rv3400 is unable to catalyze the dephosphorylation of pNPP and thus lacks the phosphatase activity.

The pyrophosphatase activity of Rv3400 was checked using a pyrophosphate assay kit (Sigma, CAT‐MAK168). For the assay, sodium pyrophosphate was used as a substrate while a known pyrophosphatase from Saccharomyces cerevisiae was used as a positive control. The substrate was hydrolyzed into phosphate ions by pyrophosphatase thereby decreasing the concentration of free pyrophosphate in the reaction. The concentration of free pyrophosphate in the sample was determined by the use of fluorogenic based pyrophosphate sensor (PPi sensor) which produces the fluorescent product (λ _ex = 316/λ _em = 456 nm) that is directly proportional to the pyrophosphate present. However, we did not observe any significant decrease in the fluorescence emission counts in the reaction containing Rv3400. These data suggest that Rv3400 was unable to convert pyrophosphate to orthophosphate and thus lacks pyrophosphatase activity (Figure 3c).

The β‐PGM activity of Rv3400 was checked spectrophotometrically by employing the method reported previously (G. Zhang et al., ²⁰⁰⁵). In the reaction assay, β‐G1P was used as a substrate and the formation of β‐G6P was monitored by the coupled reaction (wherein the increase in absorbance of at 340 nm due to formation of NADPH is directly proportional to the formation of β‐G6P). An increase in the absorbance at 340 nm confirms that Rv3400 possess β‐PGM activity (Figure 3d). Hence, here onwards we will be referring Rv3400 as Mtb β‐PGM.

2.3. Biochemical characterization of Mtb β‐PGM

We further performed biochemical characterization to know optimal conditions for the catalytic activity of Mtb β‐PGM. The β‐PGM requires divalent metal ions for its catalytic activity. Similarly, Mtb β‐PGM was not active in the absence of the Mg²⁺ ions. We performed enzyme activity in the presence of various divalent metal ions to determine metal ion preference for catalytic activity. The results suggested that Mtb β‐PGM possess a comparable catalytic activity in the presence of Mg²⁺, Mn²⁺, Co²⁺, and Ni²⁺ metal ions. The enzyme also shows ~60% activity in the presence of Fe²⁺ cofactor. However, the enzyme was not active in the presence of Ca²⁺, Ba²⁺, Zn²⁺ and Cd²⁺ metal ions (Figure 3e).

The kinetics parameters such as Km and specific activity were calculated for Mtb β‐PGM enzyme. The kinetic parameters for Mtb β‐PGM were determined using the non‐linear regression method to fit the data to the Michael‐Menten equation using ORIGIN 8.5 (Figure 3f). For each concentration of substrate, the readings were taken in triplicate. The calculated Km and specific activity values for Mtb β‐PGM are 608 ± 96 μM and 41 ± 3.4 nmol/min/mg, respectively. The value of Km is found to be comparable to L. lactis β‐PGM (Km 400 ± 40 μM) where the reaction was catalyzed without the reaction intermediate β‐G1,6bisP. However, in the presence of β‐G1,6bisP the Km of L. lactis β‐PGM reduced to 4 ± 5 μM (G. Zhang et al., ²⁰⁰⁵).

2.4. Crystallization, data collection, and refinement of Mtb β‐PGM

Although, we have successfully crystallized the full length Mtb β‐PGM (Rv3400), these crystals diffracted very poorly and therefore could not collect any x‐ray diffraction data from these crystals. Despite extensive optimization experiments, we could not improve the quality of the crystals. Therefore, to improve the quality of the crystal we have made a deletion construct Rv3400^Δ16 (wherein the first 16 amino acids from N‐terminal is deleted) which crystallized readily and yielded rectangular shaped diffraction quality crystals. However, the resolution of the data collected at our home source using these crystals was not good (~4 Å). Moreover, the BLAST (Altschul et al., 1997) search of the Rv3400 amino acid sequence against the Protein Data Bank (PDB) yielded no significant model that can be used for Molecular Replacement (MR) method (found only hits with maximum 31% identity with 81% query coverage). Therefore, we preferred to solve the crystal structure of Rv3400 by SAD phasing using selenium‐methionine (SeMet) labeled protein. As there were only two methionine residues were present in the native sequence of Rv3400, additional methionine residues were incorporated in the sequence to increase the chance of SAD phasing. The leucine residues at different positions were replaced to methionine yielding Rv3400^Δ16M1 and Rv3400^Δ16M2 constructs (detailed in method section).

The crystals of Rv3400^Δ16TM2 diffracted up to 1.7 Å and a complete x‐ray intensity data set was collected. The crystal belongs to orthorhombic space group C222₁ with unit cell parameters of a = 97.903 Å, b = 106.389 Å, c = 81.204 Å, α = β = γ = 90°. The structure was solved by SAD phasing. The electron density for one of the selected regions is shown in Figure 4a. The model building and refinement were carried out iteratively till R _cryst and R _free converged to 15.4% and 17.3%, respectively. The final model of Rv3400 consists of 273 amino acid residues, two Cl, one glycerol (GOL) and one EDO (1,2‐ethanediol) molecules. The Ramachandran plot analysis of the final model showed 98.78% of the residues are in the most favored region while 1.22% of the residues are in the allowed region. All the residues were traced in the electron density map except for the residues Asp 127, Asp 128, Asp 129 and His 186. These residues showed clear electron density for the back bone atoms, however, it is absent for their side chains. Therefore, the side chains for these residues were modeled based on the best rotamers. The data collection and refinement statistics for Rv3400^Δ16TM2 are shown in Table 1. Notably, an unintentional mutation of a His to Leu residue at position 123 was observed, probably due to PCR amplification while making the Rv3400^Δ16M2 construct. Nevertheless, the protein Rv3400^Δ16M2 showed comparable catalytic activity to that of Rv3400 indicating that the deletions, mutations introduced in the wild‐type did not have any effect on the function of the protein (Figure 3d).

FIGURE 4.

Crystal structure and comparative structural analysis of Mtb β‐PGM (a) Stereo view showing the 2Fo‐Fc Fourier map for Rv3400, contoured at 1σ. (b) Overall structure of Rv3400 showing α‐helices and 6 β‐stands. Secondary structures are labeled and represented in cyan (α‐helices), magenta (β‐stands) and light pink (loops). The cap domain is represented by the 4 α‐helical bundle while the core domain or the catalytic domain contains the α/β fold. Structural superposition of Mtb β‐PGM (Deep teal) with member of β‐PGM family from Lactococcus lactis, PDB ID: 5OLY (light pink), and Clostridioides difficile, PDB ID: 4GIB (TV orange) (c) a member of Pyrophosphatase family, from Bacteroides thetaiotaomicron, PDB ID: 3QU5 (Voilet) (d), and a member of phosphatase family from Escherichia coli, PDB ID: 1TE2, (yellow) (e).

TABLE 1.

Data collection and refinement statistics.

	SeMet‐Rv3400
Data collection	Elettra, Trieste Italy
PDB code	8H5S
Wavelength (Å)	0.97910
Resolution range (Å)	41.92–1.70 (1.73–1.70)
Space group	C2221
Unit cell parameters
a, b, c (Å)	97.90, 106.38, 81.20
α, β, γ (°)	90, 90, 90
Total number of reflections	498,700
No. of unique reflections	85,784
No. of unique reflections (non‐anomalous)	44,752
Average mosaicity (°)	0.09
Redundancy	11.1 (3.5) a
Average I/σI	30.3 (1.6)
Completeness (%)	95.4 (66.2)
R merge (%) b	5.7 (67.6)
CC1/2	1.00 (0.65)
Refinement statistics
Resolution range (Å)	33.34–1.70
No. of reflections used in the refinement	85,775
R cryst (%) c	15.4
R free (%) d	17.3
RMSD e
Bond lengths (Å)	0.006
Bond angles (°)	0.758
Ramachandran plot statistics
Most favored (%)	98.78
Allowed regions (%)	1.22
No. of protein atoms	1922
No. of solvent atoms	310
Others	12
Cl atoms	2
Glycerol atoms	6
1,2‐Ethanediol atoms	4
Wilson B‐factor (Å)	19.5
Average B‐factor (Å2)
Protein atoms	26.22
Solvent atoms	38.02
Others	35.46

^a Values for the last shell are in parentheses.

^b R _merge = ∑ _hkl ∑ _i |I _i (hkl) − I(hkl)|/∑ _hkl ∑ _i I _i (hkl) where I(hkl) is the intensity of reflection hkl.

^c R _cryst = ∑ _hkl ║F _obs | − |F _calc ║/∑|F _obs |.

^d R _free is the cross‐validated R‐factor computed for the test set of 5% of unique reflections.

^e Root mean square deviation.

The overall structure of Mtb β‐PGM is made up of 262 amino acid residues and contains 13 α‐helices and 6 β‐strands. The Mtb β‐PGM shows a Rossmann fold which is a three‐layered α/β sandwich with five parallel strands that forms the central β‐sheet. The Mtb β‐PGM structure is composed of a helical cap domain (residues 37–129) and the α/β core domain (residues 17–36 and 129–262) (Figure 4b). The β‐PGM proteins have four widely spaced motifs (Burroughs et al., 2006) which are conserved in Mtb β‐PGM as well. Motif I, present at the N‐terminus has the DxD signature residues which is usually a metal binding motif. Motif II contains a highly conserved Thr (or Ser) whereas motif III includes a Lys residue. Motif IV is composed of conserved acidic residues Glu and Asp. In addition to the four conserved motifs, Mtb β‐PGM exhibit C1 type of cap module as it contains four α‐helices in the middle of the flap motif (2 β‐sheets) which is important to determine the substrate specificity (Burroughs et al., 2006; Park et al., ²⁰¹⁵).

2.5. Comparison of Mtb β‐PGM with other structural homologs

We searched for structural homologs of Mtb β‐PGM using PDBefold server (Krissinel & Henrick, 2004; Velankar et al., ²⁰¹⁰) and the top hits include β‐PGM from L. lactis (PDB ID: 5OLY) with RMSD of 1.2 Å and β‐PGM from Clostridioides difficile (PDB ID 4GIB) with RMSD of 1.3 Å (Figure 4c). Other homologs include pyrophosphatase from Bacteroides thetaiotaomicron (PDB ID 3Qu5) with RMSD of 1.4 Å (Figure 4d), hydrolase from Bacillus cereus (PDB ID 1SWV) with RMSD of 1.5 Å, phosphatase from Escherichia coli K‐12 (PDB ID‐1TE2) with rmsd of 1.8 Å (Figure 4e). The β‐PGM from L. lactis (PDB ID: 5OLY) shares 30% sequence identity with Mtb β‐PGM (Rv3400). Similarly, the pyrophosphatase from Bacteroides thetaiotaomicron (PDB ID 3Qu5) shares 23% sequence identity with Mtb β‐PGM. The superimposition of crystal structure of β‐PGM, pyrophosphatase and phosphatase from various organisms with the Mtb β‐PGM demonstrates that the cap and the core domain of the proteins are quite conserved among these members of HAD family. However, an extended helix was observed in the cap domain of Mtb β‐PGM. This comparative structural analysis suggests that Rv3400 shares high structural similarity with phosphatase, pyrophosphatase and β‐PGM enzymes. Hence, it was difficult to assign functional role based on the structure alone.

2.6. Catalytic site of Mtb β‐PGM

The active site of Mtb β‐PGM resides at the core domain that is comprised of β‐sheets sandwiched between α‐helices and also one residue from the cap domain aids in catalysis. The side chain of Thr37 contributed by the cap domain and side chains of Asp29, Asp31, Val33, Thr37, Ser155, Lys190, Glu214, and Asp215 contributed by the core domain comprised the active site of Mtb β‐PGM (Figure 5a,b). Asp29, Asp31, Lys190, Glu214, and Asp215 are conserved among all HAD phosphotransferases. Crystal structure of β‐PGM L. lactis (PDB ID 1LVH) revealed that Asp8 (Asp29 in Rv3400) is phosphorylated (Lahiri et al., 2002). Also, an Mg²⁺ ion is coordinated with octahedral geometry by the three carboxylate side chains of Asp8 (Asp29), Glu169 (Glu214), and Asp 170 (Asp 215), the backbone carbonyl oxygen of Asp 10 (Asp 31) and a water molecule. These structurally equivalent residues in Mtb β‐PGM are Asp29, Glu214, and Asp 215, and Asp 31, respectively. However, we did not observe any electron density for the phosphate or Mg²⁺ ion(s) in the Mtb β‐PGM crystal structure, hence the structure we obtained corresponds to the apo form of the enzyme.

FIGURE 5.

(a) Active site superimposition of Mtb β‐PGM (Purple) and Lactococcus lactis (light blue) showing the residues involved in the catalysis and in the coordination of Mg²⁺. (b) Multiple sequence alignment of β‐PGM from different bacteria including Rv3400 from Mtb showing the conserved active site residues. The secondary structure elements and numbering of alignment corresponds to Rv3400. Conserved residues appear in red on a white background, whereas identical residues appear in white on a red background. The conserved amino acid residues between the groups are boxed. Amino acid residues that are involved in the formation of active site are shown in star mark. Multiple sequence alignment was performed with the Multalign server (Corpet, 1988) and the figure was generated by ESpript 3.0 on web server (Robert & Gouet, 2014). (c) CD spectra of Mtb β‐PGM, Mtb β‐PGM^Δ16 and active site mutant proteins. CD spectra show the well folded Mtb β‐PGM active site mutants comparable to that of wild type Rv3400. (d) Catalytic activity of Mtb β‐PGM mutants. Effect of active site mutants on the β‐PGM activity of Rv3400 which has shown loss in the activity except for D29A. The construct Mtb β‐PGM^Δ16 was also employed to check its β‐PGM activity. All readings to check the catalytic activity were taken in triplicate. Error bars represents the standard deviation (SD).

2.7. Mutations of DxD motif

To catalyze the β‐PGM reaction, catalytically active Asp residue in the highly conserved DxD motif undergoes phosphorylation (Hisano et al., 1996; Peisach et al., ²⁰⁰⁴). Like other β‐PGMs (Lahiri et al., 2002; Morais et al., ²⁰⁰⁰), Mtb β‐PGM is a divalent metal ion dependent (Mg²⁺) enzyme as reported in the previous section. The DxD, a conserved motif of HAD family and also a metal binding domain, has crucial role to play in the catalysis. So, to understand the role of DxD motif in the catalysis of β‐PGMs, Asp29 and Asp31 were individually mutated to Ala, Glu, Asn by site directed mutagenesis. These six point variants were purified to homogeneity. To test whether these mutations affect the folding of point variants, circular dichroism (CD) experiments were performed for all the mutant proteins. The CD spectrum of wild type and mutant proteins was similar suggesting there were no gross changes in the secondary structural contents. Also, the CD spectrum of Mtb β‐PGM^Δ16 was comparable to the wild type protein suggesting no gross changes in the secondary structural contents (Figure 5c). We performed β‐PGM enzyme assays for all the point mutants. Data presented here suggest that point mutants of DxD motif (D29A, D29E, D29N, D31A, D31E and D31N) of Mtb β‐PGM were catalytically inactive, however D31N retained ~30% activity (Figure 5d). These data suggest that D29 plays a crucial role in catalysis while D31 can tolerate substitution with Asn, however, it reduces the catalytic activity which is in agreement with the study already reported for L. lactis β‐PGM (Johnson et al., 2018).

3. DISCUSSION

Mtb can use lipids and various sugars as carbon sources depending on the conditions it encounters during infection. It can metabolize glucose, trehalose, maltose, fructose, mannose, galactose, xylose, and Arabinose (de Carvalho et al., ²⁰¹⁰; Youmans & Youmans, ¹⁹⁵³). One of the most prevalent sugar in Mtb is trehalose. It is a non‐reducing disaccharide present in free or glycoconjugate form in the cytosol as well as in the cell wall of Mtb (Elbein et al., 2003). Trehalose plays a crucial role in the composition of the mycobacterial cell envelope, particularly trehalose monomycolate (TMM) and trehalose dimycolate (TDM) that are found on cell surfaces (Jankute et al., 2015). Trehalose serves multiple functions in Mtb physiology. It can be utilized as a carbon and energy source, plays an important role in Mtb virulence, protects Mtb against osmotic stress desiccation and freezing (Harland et al., 2008). Mtb can use exogenous trehalose as well as synthesize it de novo by three pathways (Figure 6). Endogenously, Mtb can produce trehalose from glucose 6‐phosphate and UDP‐glucose, from glycogen‐like α (1 → 4)‐linked glucose, and from maltose (De Smet et al., 2000). Maltose is an important intermediate during conversion of trehalose to glucan or its utilization as a carbon source. Trehalose synthase (TreS) catalyzes the conversion of trehalose to maltose and vice versa (Boyer, 1960; R. Zhang et al., ²⁰¹¹). TreS mediated interconversion of trehalose and maltose in Mtb provides metabolic flexibility in response to changing environmental conditions and nutrient availability. Maltose also plays an important role in Mtb physiology however enzymes involved in utilizing maltose as carbon source are less studied. Unlike trehalose, maltose is a reducing disaccharide which is involved in the formation of capsular glucan, a protective layer outside the Mtb cell wall. Besides the role of these sugars in the physiology of Mtb, they can also be used as carbon source during stress or glucose starvation. Both trehalose and maltose in the presence of their respective phosphorylase yields β‐glucose and β‐glucose‐1‐phosphate (β‐G1P). Glucokinase phosphorylates β‐glucose to yield β‐glucose‐6‐phosphate (β‐G6P) while β‐G1P is converted into β‐G6P by phosphoglucomutase. β‐G6P can then enter glycolysis or pentose phosphate pathway to provide energy.

FIGURE 6.

Trehalose metabolism and proposed role of Mtb β‐PGM in the physiology of Mtb. GL, glycolysis; PPP, pentose phosphate pathway.

The role of β‐phosphoglucomutase in the physiology of B. subtilis and L. lactis is well studied; they play a role in carbohydrate metabolism. Insertional mutagenesis of the β‐phosphoglucomutase enzyme resulted in the growth deficiency of B. subtilis on minimal medium supplemented with starch or maltodextrins (maltose to maltoheptaose) (Mesak & Dahl, 2000). In Lactococcus, both maltose and trehalose are catabolized by the sequential action of phosphorylase and β‐PGM (Andersson & Rådström, 2002; Looijesteijn et al., ²⁰⁰¹). L. lactis β‐PGM‐deficient has shown impaired or no growth when trehalose or maltose is the sole carbon source (Levander et al., 2001). However, enzymes performing β‐PGM activity in Mtb have not yet been reported. Here, we show biochemically and structurally that the uncharacterized protein Rv3400 from Mtb encodes for β‐PGM.

The most prevalent pathway for producing trehalose, known as the OtsAB pathway, starts with UDP‐glucose and glucose‐6‐phosphate that yields trehalose‐6‐phosphate, which is then dephosphorylated to produce free trehalose. The enzymes trehalose‐6‐phosphate synthase (OtsA) and trehalose‐6‐phosphate phosphatase (OtsB), respectively, are known to catalyze the reaction. The OtsB gene in Mtb is encoded by two distinct open reading frames (ORFs): Rv2006 for OtsB1 and Rv3372 for OtsB2. Though the activity of Rv2006 is unknown, Rv3372 has trehalose phosphate phosphatase activity (Murphy et al., 2005; Shan et al., ²⁰¹⁶). Rv2006 is a multifunctional enzyme encoded as a single polypeptide; its N‐terminal domain is comparable to ORF Rv3400 (35% identity over 238 aa), whereas the extra C‐terminal region shares a sequence with a protein having phosphorylase activity that is encoded by Rv3401 (27% identity over 777 aa), and its core domain shows homology with the otsB2 gene (De Smet et al., 2000). Since Rv3400 shows homology to the N‐terminal domain of otsB1, it is interesting to find its role in trehalose metabolism through this pathway and also the significance of having its link to the otsB1 enzyme and with Rv3401. Notably, Rv3401 shows sequence homology with the phosphorylase of other organisms that converts trehalose to glucose and β‐G1P (Andersson et al., 2001; Inoue et al., ²⁰⁰²; Murphy et al., ²⁰⁰⁵), although, a discrepancy in gene annotations between different bioinformatics resources were observed wherein Rv3401 is annotated as probable trehalose/maltose/kojibiose phosphorylase (https://orca2.tamu.edu/U19/pages/Rv3401.html). Nevertheless, the structural and functional insights provided in this study reveals that Rv3400 encodes for β‐PGM that converts β‐G1P to β‐G6P that ultimately enters glycolytic pathway. The organization of Rv3400 and Rv3401 in an operon suggests these genes may have a role in catabolizing trehalose during starvation and persister formation directly or via maltose metabolism in Mtb.

Based on available literature, gene annotation and data presented in the present study, we propose the following model for trehalose and maltose metabolism in Mtb. Trehalose acts as energy storage and the synthesis of cell wall components (TMM and TDM), and α‐glucan for capsule formation, thus involving its catabolism and biosynthesis through various pathways. Biosynthesis generally occurs through three pathways: OtsAB or TPS/Tpp, TreS pathway, and TreYZ pathway, but only the OtsAB pathway is essential for trehalose biosynthesis (De Smet et al., 2000). Trehalose is utilized by the enzymes Pks13 and CmrA to produce TMM, which is a mycolyl carrier. In the periplasm, TMM is transported from the cytoplasm to the periplasm for biosynthesis of TDM releasing free trehalose in pseudoperiplasmic space (Gavalda et al., 2014; Li et al., ²⁰¹⁴). Trehalose synthase (TreS) is the primary enzyme in the TreS pathway, converting trehalose to maltose and vice versa. The enzyme Pep2, a maltokinase that converts maltose to maltose‐1‐phosphate, links the TresS and GleE pathways. GlgE, the maltosyl‐transferase enzyme, extends glucan chains from maltose‐1‐phosphate, whereas the enzyme GlgB introduces α 1,6‐linked branches to linear glucans, which then form capsular glucan (Kalscheuer & Jacobs, 2010; Leiba et al., ²⁰¹³).

During starvation conditions or persister formation, free trehalose is metabolized by different enzymes to yield glucose 6‐phosphate (β‐G6P), which generates ATP and NADPH through glycolysis or the pentose phosphate pathway for the survival of the cell. The reversible enzyme TreS converts trehalose to maltose, which can be further converted to β‐G1P and glucose by the action of maltose phosphorylase. Another pathway involves the enzyme trehalose phosphorylase (Trep), which in the presence of inorganic phosphate catalyzes the reversible hydrolysis of trehalose into glucose and β‐G1P. The β‐G1P formed is catalyzed by the β‐phosphoglucomutase to β‐G6P, whereas the glucose is phosphorylated by glucokinase. In both pathways, β‐G1P serves as a substrate for Rv3400 and is converted to β‐G6P. Glucose‐6‐phosphate thus formed can be used for the synthesis of ATP and NADPH via the glycolytic pathway or the pentose phosphate pathway (Figure 6).

To summarize, using structural and biochemical studies we established that the Rv3400 of Mtb encodes for β‐PGM enzyme. Based on the available literature and detailed study of β‐PGM in the physiology of B. subtilis and L. lactis, we propose that Rv3400 is one of the key enzymes involved in trehalose metabolism in Mtb. Our research represents the first comprehensive characterization of the β‐PGM enzyme from Mtb. Our study also highlights challenges in predicting functions using comparative structural and sequence‐based analyses. The findings presented here will further aid to study the maltose/trehalose metabolism in pathogenic mycobacterial species and their role in bacterial physiology and virulence.

4. EXPERIMENTAL PROCEDURES

4.1. Cloning of Rv3400 constructs

The DNA encoding Rv3400 (789 bp) was amplified by PCR using Mtb H37Rv genomic DNA as a template and designed primers (Table S1). The primers were synthesized by Sigma‐Aldrich, India. The amplified PCR product was purified using gel extraction kit (Thermo Fisher scientific Cat no‐K0702) and digested with the NheI and XhoI restriction enzymes at 37°C for 1 h. The digested PCR fragment was then ligated into pETDuet‐N vector (a modified pETDuet‐1 vector where BamHI restriction site was mutated to in‐frame NheI restriction site with N‐terminal His₆‐site) that was also digested with the same restriction enzymes. The ligation was done by T4 DNA ligase and the reaction was kept at 22°C for 2–4 h. The ligated product was transformed in E. coli TOP10 cells (Novagen) followed by plating on nutrient agar supplemented with ampicillin. The plates were incubated at 37°C for 16–18 h or until colonies appeared. The colonies on the plates were carefully marked and partially picked for checking the plasmid harboring Rv3400 gene by colony PCR. The colonies showing the positive amplification in the PCR were inoculated in 5 mL of LB broth with added ampicillin as a selection marker. The primary culture was kept in an incubator shaker overnight at 37°C at 200 RPM. Next day the cultures were harvested and plasmids were isolated using a commercial miniprep kit (GeneJet kit, Thermo Scientific, USA). The integration of the desired gene into the plasmid was further confirmed by double digestion using Nhe1 and Xho1 restriction enzymes for 20 min at 37°C. The digested products were run on 1% agarose gel to check the fallout of desired size. The correctness of the sequences were verified by automated DNA sequencing and the clone was named as pDuetN‐Rv3400.

In addition, several gene constructs were created to aid the purification and crystallization of Rv3400. The constructs Rv3400^Δ16 and Rv3400^Δ29 were made by deleting the disordered amino acids as predicted by PSIPRED server (McGuffin et al., 2000). For the construct Rv3400^Δ16, the first 16 amino acid residues and for Rv3400^Δ29, the first 29 residues were deleted. The rv3400 ^Δ16 and rv3400 ^Δ29 genes were cloned in pETDuet‐N vector using the same protocol as that of full‐length Rv3400. The primers used for these constructs are listed in Table S1.

4.2. Site directed mutagenesis

To assist the crystal structure determination of Rv3400 by single‐wavelength anomalous diffraction (SAD) method, additional methionine residues were introduced in the native protein. Two methionine mutant constructs of Rv3400^Δ16 were generated based on the combined results of Multiple sequence alignment (MSA) and the PSI‐blast based secondary structure prediction (PSIPRED) analysis (Figure S1). The amino acid leucine was selected as it is the “safe” residue substitution of methionine due to similar non‐polar side chain and hydrophobicity to methionine (Finney et al., 1980; Guy, ¹⁹⁸⁵). According to MSA analysis, the leucine residues where methionine was present in the Rv3400 homologs, were selected as the site of mutation. From the secondary‐structure prediction, mutated residues were selected in the helices as they can accumulate mutation without changing the structural stability of protein due to the higher numbers of inter‐residue contacts (Abrusan & Marsh, 2016). Three methionine residues were incorporated by replacing the leucine residue at 90th, 197th and 202th positions yielding construct Rv3400^{Δ16 L90M,L197M,L202M} whereas, the leucine residue at 126th, 197th and 202th positions were replaced by methionine yielding Rv3400^{Δ16 L126M,L197M,L202M}. These constructs thereafter named as Rv3400^Δ16M1 for Rv3400^{Δ16 L90M,L197M,L202M} and Rv3400^Δ16M2 for Rv3400^{Δ16 L126M,L197M,L202M}. The methionine mutants were made using site‐directed mutagenesis method (Zheng et al., 2004). The amplification was done by PCR using Phusion polymerase (Thermo Fischer Scientific, USA), a set of forward and reverse primers (Table S1) and pETDuetN‐Rv3400^Δ16 as template. The PCR product was purified by commercially available PCR cleanup kit (GeneJet kit, Thermo Scientific, USA). The amplified and purified PCR product was incubated with DpnI at 37°C for 3–4 h to destroy the methylated template plasmid. The unmethylated DNA was transformed into E. coli Top10 competent cells and plated on nutrient agar with added ampicillin. Incubation of the plate was done for 14–16 h and once colonies appear, a single colony is used to inoculate 5 mL LB broth with added ampicillin and allowed to grow overnight at 37°C for the isolation of the plasmid. Automated DNA sequencing was used to confirm mutations at the required sites.

In addition, the catalytic site mutants D29A, D29E, D29N, D31A, D31E and D31N of Rv3400, were also generated using the same protocol as mentioned above and the mutations at the required positions were verified by DNA sequencing.

4.3. Protein expression and purification

For Rv3400 protein expression, the pDuetN‐Rv3400 plasmid was transformed into E. coli Rosetta DE3 (Novagen). The transformed cells were plated on the nutrient agar plates supplemented with ampicillin and chloramphenicol as selection markers and kept overnight at 37°C. A single colony was inoculated in 10 mL of LB media added with ampicillin and chloramphenicol and kept overnight at 37°C in an incubator shaker at 200 RPM. The 1% of overnight grown culture was used to inoculate 1 L of fresh LB media containing ampicillin and chloramphenicol as secondary culture in a 2 L flask. The secondary culture was allowed to grow at 37°C in an incubator shaker with constant shaking at 200 RPM till the absorbance at 600 nm reached a value ~0.6–0.8. The expression of Rv3400 was induced by adding 0.3 mM isopropyl β‐D‐1‐thiogalactopyranoside (IPTG) and incubated further for 3 h at 37°C. The cells were pelleted down by centrifugation at 8000g for 10 min at 4°C. The cell pellet was resuspended in a lysis buffer containing 50 mM Tris pH 8.0, 150 mM NaCl, 10% glycerol and 1 mM Dithiothreitol (DTT). Also, 1 mM phenylmethylsulfonyl fluoride (PMSF) was added along with one tablet of cocktail protease inhibitor (Roche, Applied Science, Mannheim, Germany). The lysis of the cells was done by sonication (Sonics, NewTown, CT USA) for 30–45 min at pulse rate of 8 s ON and 12 s OFF cycle with 25% amplitude. The centrifugation of the lysate was done at 12,000 g for 45 min at 4°C and the cell debris were separated from the supernatant containing the soluble Rv3400 protein. The N‐terminal His‐tagged Rv3400 protein was purified using His‐select Ni‐NTA beads (Merk‐Sigma Aldrich, USA). The supernatant was passed through the Ni‐NTA column which was pre‐equilibrated with the lysis buffer followed by washing the beads with the same buffer. The Rv3400 protein was eluted from the column with the lysis buffer containing 20–500 mM imidazole and each fraction of eluted protein was loaded on 15% SDS‐PAGE to check the expression of Rv3400 and its purity. An Amicon concentrator, (Millipore, USA) with a 10 kDa cut off membrane was used to concentrate the Rv3400 protein. The concentration of protein was estimated by Denovix nanodrop spectrophotometer (DS‐11 Series). Further, purification was done by using the gel filtration chromatography using Superdex™ 210/300 GL (GE Healthcare) column that was equilibrated with the buffer containing 50 mM Tris, pH 8.0, 150 mM NaCl, 10% glycerol and 1 mM DTT before loading the protein. The expression and purification of the deletion constructs Rv3400^Δ16 and Rv3400^Δ29 were done by using the same protocol as that of full length protein.

4.4. Preparation of selenomethionine‐labeled protein

The plasmids containing genes rv3400 ^Δ16M1 and rv3400 ^Δ16M2 were transformed in Rosetta (DE3) cells (Novagen). The transformed cells were plated on nutrient agar with added chloramphenicol and ampicillin and incubated at 37°C for 16 h. Only a single colony was picked to inoculate 10 mL LB broth with added antibiotics as primary culture and the cells were harvested in the log phase by centrifugation for 5 min at 4000 RPM. The pellet thus obtained was resuspended in 5 mL of M9 minimal media and used to inoculate the 1 L minimal media (secondary culture). The culture was incubated at 37°C with shaking at 200 RPM. When absorbance at 600 nm reached ~0.6, the amino acids—phenylalanine 100 mg, threonine 100 mg, valine 50 mg, lysine 100 mg, isoleucine 50 mg, and selenomethionine 60 mg were added. The culture was further incubated at 37°C with shaking at 200 RPM. After 15 min, the culture was induced with 0.3 mM IPTG and kept at 16°C for 18–20 h in an incubator shaker with 200 RPM. Harvesting of cells by centrifugation at 9000g for 10 min was done and protein was purified using Ni‐NTA chromatography and then by size exclusion chromatography similar to that of wild type Rv3400 protein.

4.5. Crystallization, structure determination, and refinement

The purified Rv3400 (10 mg/mL) protein in buffer containing 20 mM Tris pH 8.0, 1 mM DDT, 150 mM NaCl and 10% glycerol was used for setting up crystallization plates with commercially available cryoscreens from Molecular Dimensions, UK and Hampton Research, USA. Using an automated NT8 robotics system (Formulatrix Inc., USA), we set up 96‐well sitting‐drop crystallization trays. Each drop of the crystallization tray contained 150 nL of reservoir solution and 150 nL of protein sample (10 mg/mL). The total reservoir volume was kept of 40 μL in the Swissci 96‐well plate. The crystallization trays were kept at 20°C in a Rock Imager 1000 (Formulatrix) for storage with automatic pre‐scheduled imaging. In the initial crystal screening, hits were obtained within a day in conditions containing 0.05 M sodium acetate pH 4.5, 0.2 M ammonium sulfate, and 30% (w/v) polyethylene glycol (PEG) and 0.1 M sodium cacodylate pH 6.5, 0.2 M sodium acetate trihydrate, and 30% (w/v) polyethylene glycol (PEG) 8000. These initial conditions were further optimized by varying the pH, precipitant and molarity of the buffer used. Also, microbatch under oil plates (Hampton Research, USA) were used to improve the crystal quality.

The selenomethionine (SeMet) labeled crystals of Rv3400^Δ16M2 were obtained using the same protocol as for Rv3400 and were named thereafter as Rv3400^Δ16TM2. Since the protein yield was low, so the crystallization trials were set up at 4 mg/mL concentration. The rectangular shaped crystals appeared in almost all the conditions as mentioned above however, crystals took months to grow. The data were collected on the crystals obtained in a condition having 0.05 M sodium acetate pH 4.5, 0.2 M ammonium sulfate, and 30% PEG 8000 and harvested after 2 months. The SeMet derivative crystals were diffracted and data were collected at 11.2 C beamline at ELETTRA synchrotron radiation source, Trieste Italy. The complete data set for the SeMet labeled Rv3400^Δ16TM2 was collected at wavelength (0.97910 Å) using DECTRIS PILATUS 6 M detector. The data were collected at 100 K under cryogenic conditions. The data set of 360 images was collected by keeping the crystal to detector distance at 392.7 mm. The x‐ray diffraction data set was processed using XDS software package (Kabsch, 2010). The crystal structure of the protein was determined by SAD method using AutoSol module in the Phenix software suite (Liebschner et al., 2019). The model was improved by several iterative cycles of model building by Coot (Emsley & Cowtan, 2004) and refinement using PHENIX (Liebschner et al., 2019).

4.6. Other computational tools

The amino acid sequence of Rv3400 (Accession number: NC_000962.3) was retrieved from GenBank (Altschul et al., 1997). Basic Local Alignment Search Tool (BLAST) programs was used to obtain homologs of Rv3400 from different databases. The sequence alignment was done using multalign web server (Corpet, 1988). The alignment figures with superimposed structure features were generated using ESPRIPT (Robert & Gouet, 2014). PSI‐blast based secondary structure PREDiction (PSIPRED) was used to predict the secondary structure of proteins (McGuffin et al., 2000). PDBeFOLD was used to find structural homologs for the query protein. PyMOL (The PyMOL) Molecular Graphics System, Version 2.5, Schrödinger, LLC) was used to generate the molecular graphic figures and to calculate RMSD. The conservation of Rv3400 protein among the homologs was checked using the ConSurf server (Armon et al., 2001). The data analysis and graphical representation of activity assay results were performed using Origin software (OriginPro 2016). For figure assembly, we used adobe photoshop CS6.

4.7. General phosphatase assay

To check the general phosphatase activity of Rv3400, para‐Nitrophenyl Phosphate (pNPP) assay was used. pNPP is a chromogenic non‐proteinaceous substrate commonly used to detect the activity of alkaline phosphatase and acid phosphatase enzymes. When pNPP is hydrolyzed by alkaline phosphatase or acid phosphatase, it produces p‐nitrophenol, which is a yellow‐colored compound that has a maximum absorbance at 405 nm. The intensity of the yellow color is directly proportional to the enzymatic activity of the phosphatase (J. Zhang et al., ²⁰¹⁷). pNPP (Thermofischer) was dissolved in a buffer containing 100 mM Tris (pH 7–9), 5 mM MgCl₂ and 100 mM NaCl. The Rv3400 protein at different concentrations (1–50 μM) was incubated with pNPP. The formation of yellow color was monitored spectrophotometrically at 405 nm.

4.8. Pyrophosphatase assay

Pyrophosphatase activity of the protein was checked using a sigma pyrophosphate assay kit (CAT‐MAK168). For the assay, pyrophosphate concentration in the sample is determined by the use of fluorogenic based pyrophosphate sensor (PPi sensor). The PPi sensor detects the presence of free pyrophosphate in the reaction and produces the fluorescent product (λ _ex = 316/λ _em = 456 nm) that is directly proportional to the pyrophosphate present. The reaction was carried out in 96 well flat bottom black ELISA plates. Sodium pyrophosphate was used as a substrate and different concentrations of Rv3400 (1 μM‐5 μM) was used. The reaction mixture consists of 100 mM NaCl, 100 mM Tris pH 9.5, 5 mM MgCl₂, and Rv3400. The reaction was kept at 37°C for 30 min. After that, 50 μL master mix (assay buffer + pyrophosphate sensor stock solution, provided in the kit) was mixed with 50 μL of reaction mixture and incubated further for 10–30 min at room temperature as per the manufacture's protocol. For each reaction, the pyrophosphate release was measured at 316 and 456 nm to check the pyrophosphatase activity of protein.

4.9. β‐Phosphoglucomutase (β‐PGM) assay

To check the β‐PGM activity, the Rv3400 was incubated in the reaction mixture containing substrate and buffers. The reaction mixture included 2 mM MgCl₂, 50 mM HEPES (pH 7.5), 450 μM β‐D‐Glucose 1‐phosphate (protocol for synthesis and confirmation in Figure S3), 0.4 mM NADP, 15 U of glucose 6‐phosphate dehydrogenase (G6PD) (HIMEDIA, India). The reaction was initiated by adding 50 μg of Rv3400. It was a coupled reaction in which product β‐G6P was catalyzed by the enzyme G6PD and led to the reduction of NADP. Thus, the increase in the absorbance of NADPH which is directly proportional to the formation of product β6P was monitored for 60 min at 340 nm using UV–Visual spectrophotometer (Perkin Elmer Lambda 25, USA).

The kinetic constants Vmax and Km were determined by monitoring the β‐PGM activity of Rv3400 at varying concentration of substrate β‐D‐Glucose 1‐phosphate (0–900 μM). The extinction coefficient for NADPH was used to compute the reaction rate (76.22 L/mmol/cm). The kinetic parameters were determined using program ORIGIN 8.5 by non‐linear regression method to fit the data to Michaelis–Menten equation. For each concentration of substrate, the experiments were done in triplicate.

4.10. Effect of different metal ions on catalytic activity of Rv3400

To find the effect of various metal ions on β‐PGM activity of Rv3400, enzymatic reactions were carried out using the same components as well as the same method as described above. The relative activity of the Rv3400 for β‐D‐Glucose 1‐phosphate was examined in presence of different divalent metal ions such as Mn²⁺(MnCl₂), Co²⁺(CoCl₂), Ni²⁺ (NiCl₂), Fe²⁺(FeCl₃), Ca²⁺(CaCl₂), Ba²⁺(BaCl₂), Zn²⁺(ZnCl₂) and Cd²⁺(CdCl₂).

4.11. Circular dichroism studies

The circular dichroism (CD) studies of Rv3400 was done to analyze its folding and secondary structure content. The experiments involved collecting far‐UV CD spectra using a Jasco‐810 CD spectropolarimeter, which was flushed with nitrogen at a flow rate of 9–12 L/min. A quartz cuvette with a path length of 1 mm was utilized for data acquisition, covering a wavelength range of 250 to 198 nm. Each spectrum was obtained as an average of five scans and recorded as raw ellipticity. Data collection for all protein samples was done using a concentration of 0.2 mg/mL. The mean residue ellipticity (MRE) was calculated by raw ellipticity according to the equation,

$${\left[\theta \right]}_{\mathrm{MRE}}=\left(\left[\theta \right]\times 100\times M_{\mathrm{r}}\right)/\left(c\times l\times N_{\mathrm{A}}\right),$$

where [θ]_MRE depicts the mean residue ellipticity, [θ] stands for the raw ellipticity in degrees, M _r is molecular weight of the protein (in Da), l is the path length of the cuvette (in cm), c is the concentration of the protein (in mg/mL), and the number of residues of the protein is depicted by N _A.

Supporting information: This article contains supporting information (Barrozo et al., 2018; Dai et al., ²⁰⁰⁹; Johnson et al., ²⁰¹⁸; Kvam et al., ¹⁹⁹⁷; Lahiri et al., ²⁰⁰²; Lahiri et al., ²⁰⁰³; Mesak & Dahl, ²⁰⁰⁰; Nilsson & Radstrom, ²⁰⁰¹; G. Zhang et al., ²⁰⁰⁵).

AUTHOR CONTRIBUTIONS

Krishan Gopal Thakur: Conceptualization; funding acquisition; methodology; validation; visualization; writing – review and editing; project administration; formal analysis; software; supervision; resources. Latika Singh: Conceptualization; methodology; investigation; writing – original draft; writing – review and editing; visualization; data curation; software; formal analysis; validation. Subramanian Karthikeyan: Conceptualization; funding acquisition; methodology; validation; visualization; writing – review and editing; software; formal analysis; project administration; supervision; resources.

FUNDING INFORMATION

This study was supported by funding and grant to S.K. and K.G.T. by Council of Scientific and Industrial Research (CSIR), India. L.S. is a recipient of senior research fellowship from the Department of Biotechnology, India.

CONFLICT OF INTEREST STATEMENT

The authors declare no conflicts of interest.

Supporting information

Table S1. List of primers and their nucleotide sequence used in the study.

Figure S1. Proposed detailed activation and catalytic mechanism for β‐PGM of Mtb.

Figure S2. Multiple sequence alignment of homologs of Rv3400.

Figure S3. Synthesis of β‐D‐glucose 1‐phosphate (β‐G1P).

Singh L, Karthikeyan S, Thakur KG. Biochemical and structural characterization reveals Rv3400 codes for β‐phosphoglucomutase in Mycobacterium tuberculosis . Protein Science. 2024;33(4):e4943. 10.1002/pro.4943

DATA AVAILABILITY STATEMENT

The atomic coordinates and structure factors for the reported crystal structure generated during this study are available at the Protein Data Bank with accession code 8H5S.