Identification, sequence analysis, cloning and functional overexpression of the maltokinase gene (mak) from Mycobacterium bovis BCG, strains and growth conditions
To identify the mak gene in mycobacterial genomes we used the amino acid sequences of the Actinoplanes missouriensis and Streptomyces coelicolor maltokinases (GenPept accession number AAQ01690 and CAA04602, respectively) in BLAST searches at the National Center for Biotechnology Information (NCBI, http://blast.ncbi.nlm.nih.gov/Blast.cgi) database. Promoter identification was carried out using the prokaryotic promoter prediction software from Berkeley University, available at http://www.fruitfly.org. The mak gene was amplified from the chromosomal DNA from M. bovis BCG (DSM 43990) obtained from the Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH (DSMZ, Braunschweig, Germany), with a pair of primers based on the gene sequence retrieved from the Institut Pasteur database http://genolist.pasteur.fr. An NdeI restriction site (highlighted in bold) was added to the forward primer MtuNde (5'-CTTACATATG ACTCGGTCGGACACGC-3'). A HindIII restriction site (highlighted in bold) was added to the reverse primer MtuHind (5'-ATTAAGCTT GCTAGCGGTCAGGCGGG-3'). The stop codon was removed from the reverse primer to allow the translation of a C-terminal 6 × His-tag encoded by the expression vector pET30a (Novagen). PCR was carried out with the AccuPrime GC-Rich DNA Polymerase (Invitrogen). DNA (200 ng) was denatured by 95°C for 5 min followed by 30 cycles of 1 min denaturation step at 95°C, 1 min annealing step at 62°C, and 1.5 min extension step at 72°C. The PCR product was purified from agarose gel (NZYTech, Portugal), digested and cloned into pET30a. The construct was sequenced to confirm the identity of the insert (AGOWA, Berlin, Germany), and transformed into E. coli BL-21, which was used as host for expression of the mak gene. Recombinant E. coli was grown in a 5L fermentor, with continuous aeration and stirred at 180 rpm at 37°C, pH 7.0 in LB medium with kanamycin (30 μg/ml), to mid-exponential phase of growth (OD610 = 0.8). IPTG was added at a final concentration of 0.5 mM to induce gene expression, and temperature was reduced to 20°C. The cells were harvested 18 h later by centrifugation (9000 × g, 10 min, 4°C).
Preparation of cell-free extracts
Escherichia coli cells carrying the recombinant maltokinase (Mak) from M. bovis BCG were suspended in 25 mM Bis-tris propane buffer (BTP) at pH 7.5 with 50 mM NaCl for enzyme assays, or in 20 mM sodium phosphate buffer at pH 7.4 with 0.5 M NaCl and 20 mM imidazole, for protein purification. A protease inhibitor cocktail (Roche), 10 μg/ml DNAse I and 5 mM MgCl2 were added to the suspension. Cells were disrupted twice in a French-press cell followed by centrifugation (15000 × g, 4°C, 30 min).
The activity of the recombinant Mak from M. bovis BCG in E. coli extracts and during purification was detected after 15 min at 37°C in reaction mixtures (50 μl) containing 25 μl of cell-free extract, 3.0 mM (each) of ATP and maltose, and 10 mM MgCl2 in 50 mM BTP, pH 8.0. The synthesis of maltose-1-phosphate was monitored by thin-layer chromatography (TLC) with solvent systems composed by acetic acid/ethyl acetate/water/ammonia 25% (6:6:2:1, v/v) and butanol/ethanol/water (5:3:2, v/v). Trehalose-6-phosphate, ATP, GTP, UTP, ADP, GDP, AMP and maltose standards were used for comparative purposes. The maltose-1-phosphate formed was identified by NMR as described below. E. coli cell-free extracts carrying an empty vector were used as negative controls. Protein concentration was determined by the Bradford method .
Purification of recombinant Mak from M. bovis BCG
The His-tagged recombinant Mak from M. bovis BCG was purified in a prepacked Ni-Sepharose high-performance column (His-Prep FF 16/10) equilibrated with 20 mM sodium phosphate, pH 7.4, 0.5 M NaCl, and 20 mM imidazole. Elution was carried out with 500 mM imidazole and the purity of the fractions was determined by SDS-PAGE. The purest active fractions were pooled, diluted ten times with 25 mM BTP at pH 7.4 and loaded into a Q-Sepharose fast-flow column (Hi-Load FF 16/10), equilibrated with 25 mM BTP at pH 7.4 with 50 mM NaCl, and eluted by a linear gradient of NaCl (50 to 500 mM). The purity of the fractions was determined by SDS-PAGE and the purest active fractions were pooled, concentrated by ultracentrifugation in 30 kDa cutoff centricons (Amicon), equilibrated with 25 mM BTP at pH 7.4 with 200 mM NaCl, and loaded into a Superdex 200 fast-flow column equilibrated with the same buffer. After SDS-PAGE analysis the active pure fractions were concentrated and equilibrated with 50 mM BTP at pH 7.4 with 50 mM NaCl. Protein content of the samples was determined by the Bradford assay . The identity of the purified maltokinase from M. bovis BCG was confirmed by Peptide Mass Fingerprinting (IPATIMUP Proteomics Unit, Porto, Portugal).
Characterization of the recombinant Mak from M. bovis BCG
The substrate specificity of the recombinant Mak from M. bovis BCG was determined using glucose, galactose, mannose, maltose, isomaltose, trehalose, maltotriose, maltotetraose, maltopentaose and maltoheptaose as possible acceptors and with ADP, CDP, GDP, TDP, UDP, ATP, CTP, GTP, TTP and UTP as possible phosphate donors (all from Sigma-Aldrich). Since the Mak protein had high sequence identity (>40%) with putative aminoglycoside phosphotransferases, this activity was also tested using the aminoglycoside antibiotics gentamicin, kanamycin, streptomycin and hygromycin B as possible phosphate acceptors (all from Sigma-Aldrich). The reaction mixtures (50 μl) containing (0.5 μg) pure recombinant Mak, 3 mM of each substrate, and 10 mM MgCl2 in 50 mM BTP at pH 8, were incubated at 37°C for 10 min. The products were visualized by TLC as described above.
Temperature and pH profile, effect of cations and thermal stability of Mak were determined by the addition of 0.5 μg of Mak to 50 μl reaction mixtures containing the appropriate buffer, 3 mM maltose and 3 mM NTP and stopped at different times by cooling on ethanol-ice. The Mak was inactivated by the addition of 5 μl of 1N HCl and neutralized by 5 μl of 1N NaOH. Controls were performed to account for possible NTP degradation following acid treatment. The amount of NDP released was determined at 340 nm after incubation of the sample with 3 U of pyruvate kinase and lactate dehydrogenase, 0.3 mM NADH and 2.5 mM phosphoenolpyruvate (all from Sigma-Aldrich) in 1 ml mixture (total volume) for 10 min at 30°C . The temperature profile was determined between 20 and 65°C in 50 mM BTP at pH 7.0, with 10 mM MgCl2. The effect of pH was determined at 37°C in 50 mM BTP (pH 6.0 to 9.0) and in 50 mM CAPS (pH 9.0 to 11.0), with 10 mM MgCl2. The effect of cations was examined by incubating reaction mixture containing the appropriate substrates, with the chloride salts of Mg2+, Mn2+, Co2+, Zn2+ (0.5 to 50 mM) or without cations, at 37°C.
The kinetic parameters for the recombinant Mak were determined by measuring the amount of NDP released, as described above. The Km values for the substrates ATP, GTP, UTP and maltose were determined at 37°C, from Lineweaver-Burk plots. All experiments were performed in triplicate.
The molecular mass of the recombinant Mak from M. bovis BCG was estimated by gel filtration on a Superdex 200 column and the molecular mass standards were aprotinin (6.5 kDa), ribonuclease (13.7 kDa), carbonic anhydrase (29 kDa), ovalbumine (43 kDa), conalbumine (75 kDa), aldolase (158 kDa). Blue Dextran 2000 was used to determine the void volume (Amersham).
Enzyme stabilization assays
Pure recombinant Mak was stored in several aliquots containing 50 mM BTP at pH 8.0, and one of the following substances (final concentration) was added: 20% glycerol, 50% glycerol, 10 mM maltose, 10 mM trehalose or 50 mM NaCl. The initial Mak activity was measured at 37°C in 50 mM BTP at pH 8.0, with 10 mM MgCl2 and 3 mM (each) of ATP and maltose. Aliquots were stored at 4 and -20°C for seven days. Control aliquots were kept at 4° and -20°C only in 50 mM BTP at pH 8.0. After seven days storage the residual activity was measured for each of the conditions described above.
For the NMR experiments, 10% (v/v) of D2O was added to the reaction mixture containing 5 mM (each) of ATP and maltose, 5 mM MgCl2 in 10 mM BTP at pH 8.0. After spectral acquisition, 15 μg of enzyme were added to the NMR tube and incubated for 10 min at 37°C. All spectra were acquired on a Bruker AVANCE III 800 spectrometer (Bruker, Rheinstetten, Germany) working at a proton operating frequency of 800.33 MHz, equipped with a four channel 5 mm inverse detection probe head with pulse-field gradients along the Z axis.
Spectra were run at 25°C using standard Bruker pulse programs. 1H and 13C chemical shifts are referenced to 3-(trimethylsilyl)propane sulfonic acid, and 31P resonances to external 85% phosphoric acid. In the 1H-13C and the 1H-31P heteronuclear two-dimensional single quantum coherence (HSQC) spectra, delays of 3.44 and 71.4 ms were used for evolution of the JH, C and JH, P couplings, respectively; proton decoupling was, in both cases, achieved using the GARP4 sequence .