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  • Plasmid construction and site directed

    2021-09-27

    Plasmid construction and site-directed mutagenesis of M. luteus B-P 26 UPS. For the expression of N-terminus His6-tagged M. luteus B-P 26 UPS, the expression plasmid pMluUEX [7] was cleaved with the restriction enzymes NdeI/BamHI. The gene fragment was inserted into an NdeI/BamHI-treated pET-15b to construct pET-HisMlUPS. For the construction of UPS mutant, a QuikChange Mutagenesis Kit was used according to the protocol of the manufacturer. The pET-HisMlUPS was used as a template for the construction of mutants. Mutagenic oligonucleotides designed to produce the desired mutant enzymes were: 5′-CTTAACGCTGTACTTATTTTCCACGGAAAATTGGTC-3′ (for A72L); 5′-CTTAACGCTGTACTTTTTTTCCACGGAAAATTGGTC-3′ (for A72F); 5′-CTTAACGCTGTACTGGTTTTCCACGGAAAATTGGTC-3′ (for A72W). Expression and purification of wild type and mutant enzymes. For the expression of Rv1086 and its mutants, E. coli BL21(DE3) was transformed with pET-Rv1086 or with each expression plasmid. The transformants were cultivated in 50mL M9YG broth supplemented with ampicillin (50mg/L). When the OD600 of the culture reached 0.5, the transformed bacteria were induced with 0.5mM isopropyl 1-thio-β-d-galactoside. The further info were incubated overnight and then harvested. The cells were disrupted in lysis buffer containing 20mM sodium phosphate buffer (pH 7.4), 100mM imidazol, and 0.5M NaCl. The homogenate was centrifuged at 6000g for 15min at 4°C. The supernatant was then applied on a HisTrap column (GE Healthcare, USA) previously equilibrated with the lysis buffer. The resin was washed with washing buffer containing 20mM sodium phosphate buffer (pH 7.4), 150mM imidazol, and 0.5M NaCl. The protein bound to the resin was then eluted with elution buffer containing 20mM sodium phosphate buffer (pH 7.4), 300mM imidazol, and 0.5M NaCl. Fractions with more than 90% protein purity as well as with high enzymatic activity were used for the experiments described below. For the expression and purification of M. luteus UPS, similar procedures as those described above were used. Measurement of prenyltransferase activity. The assay mixture for wild type and mutant Rv1086s contained 4μM [1-14C]IPP (2GBq/mmol), 10μM of an allylic primer (GPP or E,E-FPP), 0.1mM MgCl2, 2.5mM dithiothreitol, 0.3% (w/v) Triton X-100, 50mM Tris–HCl buffer (pH 8.0), and a suitable amount of each enzyme in a final volume of 100μL. This mixture was incubated at 30°C for 30min, and the reaction was stopped by chilling the mixture in an ice bath. The mixture was treated with 500μL of 1-butanol saturated with H2O. The butanol layer was washed with water saturated with NaCl, and the radioactivity in 50μL of the butanol layer was measured with a LS 6500 Liquid Scintillation Counting System (BECKMAN COULTER, USA). The residual butanol layer was used for product analysis. The assay mixture for wild type and mutant M. luteus B-P 26 UPSs contained, 10μM [1-14C]IPP (2GBq/nmol), 10μM of an allylic primer (GPP or E,E-FPP), 0.5mM MgCl2, 0.05% (w/v) Triton X-100, 100mM Tris–HCl buffer (pH 7.5), and a suitable amount of each enzyme in a final volume of 100μL. The mixture was incubated at 30°C for 30min and then processed as described above. Product analysis. Prenyl diphosphates in the residual 1-butanol layer were treated with acid phosphatase, according to the method of Fujii et al. [17]. The hydrolysates were extracted with pentane and analyzed by reversed-phase TLC using a precoated plates, LKC-18F (Whatman, UK), which were developed with the solvent mixture methanol/acetone (8/2) (for Rv1086) or acetone/H2O (19/1) (for UPS from M. luteus B-P 26). Authentic standard alcohols were visualized with iodine vapor, and the distribution of radioactivity was detected with a Fuji BAS 1000 Mac Bioimage Analyzer (FUJIFILM, Japan).
    Results and discussion
    Acknowledgments
    Introduction Short-chain isoprenyl diphosphate synthases (scIPPSs) are a class of prenyltransferases that catalyze the condensation of isopentenyl diphosphate (IPP; C5) with an allylic co-substrate. These enzymes are distinguished by the specific chain length of the prenyl diphosphate product generated. Thus, geranyl diphosphate synthase (GPPS) catalyzes a single condensation of IPP with dimethylallyl diphosphate (DMAPP; C5) to form geranyl diphosphate (GPP; C10), whereas farnesyl diphosphate synthase (FPPS) and geranylgeranyl diphosphate synthase (GGPPS) catalyze one and two additional condensations with IPP, respectively, generating farnesyl diphosphate (FPP; C15) and geranylgeranyl diphosphate (GGPP; C20). These products are precursors to many biologically essential compounds classified into three groups: monoterpenes, sesquiterpenes and diterpenes, respectively [1] (Fig. 1).