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Introduction

Although tuberculosis (TB) is an ancient disease resulting from infection with Mycobacterium tuberculosis (M. tuberculosis), it remains a great threat to both individual and public health throughout the world. It is reported that approximately one-third of the world’s population has been latently infected (1). The prevalence of human immunodeficiency virus has enhanced the spread of multi-drug resistant and extensively drug resistant tuberculosis strains, and the morbidity and mortality of TB have been rising yearly without much curative success using existing anti-TB drugs (2–4). Therefore, it is a matter of urgency to discover targets for new anti-TB drugs. Serine acetyltransferase (CysE) is involved in the biosynthesis of cysteine, which catalyzes the conversion of acetyl-CoA (AcCoA) and L-serine (L-Ser) to CoA and O-acetyl-L-serine (OAS) (5,6). This reaction is the first step in the two-step biosynthesis of L-cysteine in microorganisms and plants (7,8). Because of the differing pathways for cysteine anabolism in humans and microorganisms (9), serine acetyltransferase exists only in microorganisms. An ideal drug target should be unique to the pathogen, thus M. tuberculosis serine acetyltransferase is regarded as a potential drug target (10,11).

The CysE protein has been purified and characterized from certain bacteria, such as Escherichia coli (6,12,13), Salmonella typhimurium (5,14) and Haemophilus influenzae (15). Bioinformatic analyses have shown that M. tuberculosis Rv2335 is homologous to E. coli CysE, S. typhimurium CysE and H. influenzae CysE. Therefore, M. tuberculosis Rv2335 (GenBank accession no. CAB06152.1) could be a cysE gene that encodes the CysE protein.

In this study, we cloned and expressed the M. tuberculosis cysE (Rv2335) gene in E. coli and characterized the purified M. tuberculosis CysE protein. The kinetic studies on M. tuberculosis CysE allow for the screening of its inhibitors in the development of anti-TB drugs.

Materials and methods

Microorganisms and plasmids

E. coli NovaBlue and E. coli BL21 (DE3) (Novagen) were maintained as the hosts for cloning and expression, respectively. The cloning plasmid pMD18-T (Takara) with the ampicillin resistance gene was utilized to clone and sequence the target gene or DNA fragment. The expression vector pET29b (Novagen) carrying the kanamycin resistance gene was used for gene expression in E. coli. M. tuberculosis H37Rv genomic DNA was supplied by Colorado State University via an NIH contract.

Cloning the cysE (Rv2335) gene from M. tuberculosis H37Rv genomic DNA

The M. tuberculosis cysE gene was amplified from M. tuberculosis H37Rv genomic DNA using the following set of primers: cysE forward, 5′-AACATATGCT GACGGCCATGCGGG-3′ (underlined sequence is the NdeI site) and cysE reverse primer, 5′-AACTCGAGGATCGAG AAGTCCTCGCCG-3′ (underlined sequence is the XhoI site). The amplified PCR product was ligated into pMD18-T to generate the plasmid pMD18-cysE, which was transformed into E. coli NovaBlue. The positive recombinant plasmid pMD18-cysE was confirmed by digestion with restriction endonucleases (EcoRI) and subsequently sequenced. The cysE gene was subcloned into the NdeI and XhoI sites of pET29b, yielding the expression vector pET29b-cysE.

Expression, purification and identification of CysE protein

The plasmid pET29b-cysE was transformed into E. coli BL21 (DE3). BL21 (DE3)/pET29b-cysE culture was induced with 1 mM IPTG at 37°C for 3 h. The cells were harvested and suspended in lysis buffer (20 mM Tris-HCl pH 8.0, 100 mM NaCl, 25 mM MgCl2, 5% (v/v) glycerol, 1 mM EDTA, 1 mM β-mercaptoethanol and 1 mM PMSF). The cells were homogenized by sonication and the cell lysate was centrifuged at 20,000 × g for 20 min. The supernatant was then loaded onto a 1-ml Ni-NTA agarose column (Qiagen). The column was then washed with 20 ml of wash buffer (20 mM Tris-HCl pH 8.0, 500 mM NaCl, 20% glycerol, 60 mM imidazole and 1 mM PMSF), and the CysE protein with a His-tag at its C-terminus was eluted with 10 ml of elution buffer (20 mM Tris-HCl pH 8.0, 500 mM NaCl, 20% glycerol, 300 mM imidazole and 1 mM PMSF) and examined by SDS-PAGE and western blotting. The purified CysE protein was further confirmed by matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF-MS) (BIG, China).

Enzyme assays

The serine acetyltransferase activity of the CysE protein was determined by monitoring the increase in the absorbance of Ellman’s reagent (DTNB) due to its reaction with CoA (16,17). Briefly, a 50-μl reaction mixture (50 mM Tris-HCl pH 7.5, 5 mM MgCl2, 0.4 mM AcCoA, 2 mM L-Ser and 0.037 μg purified CysE protein) in a 96-well microtiter plate was incubated at 37°C for 20 min. A blank control without L-Ser and AcCoA, and a positive control containing standard CoA (0.2 mM) only were included. The reaction was terminated with 50 μl of stop solution (50 mM Tris-HCl pH 7.5, 6 M guanidine hydrochloride). Fifty microliters of Ellman’s reagent (50 mM Tris-HCl pH 7.5, 0.2 mM DTNB and 1 mM EDTA) was added to the reaction mixture. The mixture was incubated at room temperature for 10 min. The absorbance values were obtained using a microplate reader (Multiskan Ascent; Thermo Scientific) at a wavelength of 405 nm (18). One unit of specific enzyme activity was defined as 1 μmol of CoA-SH produced by 1 mg protein/min under specific conditions.

Characterization of M. tuberculosis CysE

The maximum velocity (Vmax) and Michaelis constant (Km) of M. tuberculosis CysE were measured by a colorimetric assay coupled with DTNB. Based on the concentration curves and time-course curves of CysE, the range of CysE initial velocities was measured. The concentration curves of CysE were plotted by measuring the reaction velocities at varying CysE concentrations and reaction times. The reactions were performed in 50 mM Tris-HCl buffer (pH 7.5) containing AcCoA, L-Ser and different concentrations of purified CysE (0.74, 1.48, 2.22, 2.96 and 3.70 μg/ml) at 37°C for 5, 15 and 25 min. The time-course curves were plotted by measuring the amount of CoA at different reaction times (5, 10, 15, 20 and 25 min) and different concentrations of CysE (0.74, 2.22 and 3.70 μg/ml) at 37°C. To further characterize the CysE, the effect of pH, temperature, and Mg2+ concentration on CysE were evaluated by measuring CysE activity in different pH buffers (3–11), at various temperatures (16–80°C) and concentrations of Mg2+ (0–20 mM), respectively.

In dual-substrate reactions, the steady-state kinetic parameters Km and Vmax were calculated by double reciprocal plots prepared by varying the concentration of one substrate while the second substrate was in excess under optimal conditions.

Results

Cloning of the M. tuberculosis cysE gene

The PCR product for the cysE gene was obtained from the genomic DNA of M. tuberculosis H37Rv (Fig. 1A). The size of the PCR product (cysE gene plus NdeI and XhoI recognition sites) was 700 bp.

Figure 1 Analysis of cysE PCR products and purified M. tuberculosis CysE protein. (A) Electrophoretic analysis of cysE PCR product by 1% agarose. Lane 1, GeneRuler 1 kb DNA ladder (Fermentas); lane 2, PCR product of cysE gene amplified from M. tuberculosis H37Rv genomic DNA. (B) SDS-PAGE analysis of the purified CysE protein. Lane 1, PageRuler pre-stained protein ladder (Fermentas); lane 2, the purified CysE protein. (C) Western blotting analysis of purified CysE protein. Lane 1, PageRuler pre-stained protein ladder; lane 2, the purified CysE protein.

Expression, purification and identification of the CysE protein

The soluble M. tuberculosis CysE protein was expressed in E. coli BL21 (DE3) by induction with 1 mM IPTG. The purified CysE protein was detected by SDS-PAGE (Fig. 1B) and western blotting (Fig. 1C). The band of the CysE protein appeared at 30 kDa, which was higher than the theoretical molecular mass (24.6 kDa) of the CysE protein. The purified CysE protein was further confirmed by MALDI-TOF-MS analysis (data not shown).

Serine acetyltransferase activity of M. tuberculosis CysE protein

The serine acetyltransferase activity of M. tuberculosis CysE protein was detected. The specific activity of the serine acetyltransferase was 10.66±0.44 μmol/min/mg (Table I).

Table I Specific activity and kinetic parameters of M. tuberculosis CysE.

Table I

Specific activity and kinetic parameters of M. tuberculosis CysE.

	Specific activity (μmol·min−1·mg−1)	Vmax (mM·min−1)	KAcCoA (mM)	Kser (mM)	Kcat (sec−1)
M. tuberculosis CysE	10.66±0.44	0.0073±0.0005	0.0513±0.0050	0.0264±0.0006	81.36±5.22

[i] The activity was determined under specific conditions. The K_m and V_max of CysE were determined under the optimal conditions by double reciprocal plot. CysE, serine acetyltransferase. V_max, maximum velocity; K_ser, K_m for L-serine; K_AcCoA, K_m for acetyl-CoA.

Characterization of M. tuberculosis CysE

The reaction velocity was proportional to the concentration of M. tuberculosis CysE when the reaction time was 5 min (Fig. 2A). At 15 or 25 min reaction times, the reaction velocity gradually slowed and became non-linear with the CysE concentration. Therefore, the initial velocity of CysE was within 5 min.

Figure 2 (A) CysE concentration curves and (B) time-course curves. (A) Reaction velocity vs. concentration of CysE at different reaction times: ♦, 5 min; ■, 15 min; ▲, 25 min. (B) Concentration of CoA vs. reaction time at different concentrations of CysE: ◆, 0.74 μg/ml; ■, 2.22 μg/ml; ▲, 3.70 μg/ml.

Within a maximum concentration limit of 0.74 μg/ml, the concentration of CoA was proportional to the reaction time (Fig. 2B). As the CysE concentration reached 2.22 or 3.70 μg/ml, the rate of CoA formation gradually decreased with reaction time. The optimal concentration for characterizing CysE was 0.74 μg/ml.

The CysE activity was determined at varying pHs with appropriate buffer systems (3–11) after the initial velocity and optimal CysE protein concentration were set (Fig. 3A). The optimal pH for CysE was 7.5. The optimal temperature for CysE was investigated from 16 to 80°C (Fig. 3B), with the highest activity observed as the temperature reached 37°C. The catalytic activity of CysE was not significantly changed by varying the Mg2+ concentration (Fig. 3C), indicating that Mg2+ had no effect on the CysE activity.

Figure 3 Determination of the optimal conditions for M. tuberculosis CysE activity. (A) pH curve; (B) temperature curve; (C) Mg2+ concentration curve.

The steady-state kinetic constants were determined under the optimal conditions and the initial velocity by a double reciprocal plot (Fig. 4). The Vmax value of CysE was 0.0073±0.0005 mM/min. The Km of CysE against AcCoA was 0.0513±0.0050 mM, while the Km value of L-Ser was 0.0264±0.0006 mM (Table I).

Figure 4 Determination of Vmax and Km values for M. tuberculosis CysE. (A) Vmax and Km values for AcCoA from a double reciprocal plot; (B) Vmax and Km values for L-Ser from a double reciprocal plot.

Discussion

Serine acetyltransferase is an enzyme involved in cysteine biosynthesis, and it plays an important role in the growth of M. tuberculosis (10). In addition, this enzyme only exists in microorganisms and plants (9), making serine acetyltransferase a potential anti-TB drug target.

M. tuberculosis Rv2335 is predicted to be a cysE gene encoding serine acetyltransferase. Bioinformatic analyses have shown that the M. tuberculosis Rv2335 protein is 45% identical to E. coli CysE, S. typhimurium CysE and H. influenzae CysE using the Basic Local Alignment Search Tool (BLAST). Serine acetyltransferase is a member of the hexapeptide acetyltransferase family (19). This protein family has a conserved active left-handed-β-helix (LβH) domain, which is composed of a six-peptide ([LIV]-[GAED]-X2[STAV]-X) tandem repeat (15,20,21). The M. tuberculosis Rv2335 protein contained the tandem repeat and showed LβH structure when modeled using the NCBI Conserved Domain Search (data not shown).

To identify the function of M. tuberculosis CysE, the M. tuberculosis cysE (Rv2335) gene was amplified with high fidelity DNA polymerase, and the soluble CysE protein was expressed in E. coli. SDS-PAGE and western blotting showed that the molecular weight of the expressed CysE protein (~30 kDa) was higher than predicted. This finding could be due to the auxiliary fusion of six histidines to the recombinant M. tuberculosis CysE protein generated from the pET29b vector. The six consecutive histidines impart a strong positive charge that may retard the mobility of the CysE protein in SDS-PAGE.

As indicated in Table I, M. tuberculosis CysE demonstrated serine acetyltransferase activity of 10.66 μmol/min/mg. The specific activity of E. coli serine acetyltransferase has been reported as 71.6 μmol/min/mg (22). The specific activity of M. tuberculosis CysE is lower than that of E. coli CysE, possibly because of the different methods of purification. M. tuberculosis CysE exhibited its highest acetyltransferase activity at pH 7.5 and 37°C. The optimal pH is consistent with those reported for other bacteria, but the optimal temperature is different from those reported for other bacteria such as S. typhimurium (25°C) (14), E. coli (25°C) (12) and H. influenzae (25°C) (15). The Km for L-serine (Kser) of M. tuberculosis CysE (0.026 mM) is lower than the Kser of S. typhimurium CysE (0.7 mM) and E. coli CysE (1.17 mM) (12,23). The Km for AcCoA (KAcCoA) of M. tuberculosis CysE (0.051 mM) is also lower than that of S. typhimurium CysE (0.1 mM) and E. coli CysE (0.2 mM) (12,23). In the present study, the KAcCoA of M. tuberculosis CysE was 0.051 mM, while the Kser was 0.026 mM. This finding suggests that CysE had higher affinity for L-Ser than AcCoA, and CysE was bound more easily to L-Ser than to AcCoA in M. tuberculosis. Cysteine is reported to inhibit the activity of serine acetyltransferase in its biosynthetic pathway by a feedback mechanism (7,12,15). Furthermore, cysteine was found to bind E. coli CysE at the serine substrate site rather than at the acetyl-CoA substrate site from the structural study on acetyltransferase (20). This finding indicates that it is preferable to screen and design compounds against the L-serine site to inhibit the activity of CysE.

In summary, serine acetyltransferase CysE was encoded by the cysE (Rv2335) gene in M. tuberculosis. We investigated the kinetic parameters and optimal catalytic conditions of CysE using simple and rapid enzyme assays. The CysE assay and kinetic properties of CysE will facilitate the high-throughput screening of inhibitors against CysE. However, there are currently no reports of the crystal structure and active sites of M. tuberculosis CysE. The expressed soluble CysE protein will be available to further elucidate its crystal structure and active sites.

Acknowledgements

This study was supported by a grant from the National Natural Science Foundation of China (31070066) and the National Basic Research Program of China (2012CB518803).

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References