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  • Methods and materials Escherichia coli JM DE cells containin


    Methods and materials Escherichia coli JM109(DE3) Pam3CSK4 containing a derivative plasmid of pUCmod that encodes E. coli FDPS (IspA) previously described by Schmidt-Dannert and co-workers [22] were grown in LB media containing 150μg/mL of ampilicin. E. coli was grown directly from stock cells stored at −80°C. Initially, they were grown overnight at 37°C with shaking at 240rpm. The next morning, 1L flasks were inoculated with 10mL of the overnight culture and grown to an OD600 of approximately 0.8. Cells were harvested by centrifugation at 5400g, and the cell pellets (one pellet equivalent to 1L of cell growth) were frozen and stored at −80°C. FDPS was purified using a previously reported procedure [22] with minor modifications. Briefly, E. coli cell pellets expressing FDPS were thawed and resuspended in 50mL of 50mM phosphate buffer (pH 8.0), 50mM NaCl, and 1mL of protease inhibitor cocktail, a cocktail developed for His-tagged proteins (Sigma Aldrich, No. P8849). This was loaded onto a 25mL Ni-NTA column bed that had been preequilibrated with the cell suspension buffer. This column was then washed with 100mL of a 50mM phosphate buffer (pH 8.0) containing 300mM NaCl, followed by a second wash with 200mL of a 50mM phosphate buffer containing 300mM NaCl and 20mM imidiazole. The enzyme was eluted from the column with a 50mM phosphate buffer (pH 8.0) containing 300mM NaCl and 300mM imidazole. Fractions containing the enzyme were pooled together and concentrated and then diluted three times with a 12-fold dilution with 50mM Tris–HCl (pH 8.0) using an Amicon Ultra-15 centrifugal filter device (Millipore). After concentration, the enzyme was diluted to 50% glycerol (final enzyme concentration of 2mg/mL) and stored at −80°C. This purification typically yielded 2mg/L of liquid culture of FDPS with a purity of 80%.
    Results To determine the feasibility of this assay for measuring FDPS activity, initial studies were performed to examine the increase in fluorescence in the presence and absence of both enzymes (Fig. 2). When the reaction mixture contains only DMAPP, IPP, and N-dansyl-GCVIA substrates, there was no significant increase in fluorescence; additionally, no fluorescence increase was observed when only one of the two necessary enzymes (FDPS or PFTase) was present in the reaction. Only when both of the enzymes were present was there an increase in fluorescence consistent with the generation of FPP by FDPS. Next, to establish appropriate reaction conditions, the concentrations of the two FDPS substrates, DMAPP (Fig. 3) and IPP (Fig. 4), were varied in the FDPS assay. That data shows that as the concentration of DMAPP is increased, the reaction rate increases up to 25μM DMAPP before leveling off. This is reasonable because the IPP concentration in the assay was held constant at 50μM; since FDPS uses IPP and DMAPP in a 2:1 ratio to generate FPP, IPP is the limiting reagent; this means that increasing the concentration of DMAPP beyond 25μM (half of the amount of IPP) would not result in additional FPP production or an increase in rate (provided that the KM for DMAPP is not significantly greater than 25μM [25]). The relationship between FDPS activity and the concentration of IPP is more complex. A significant increase in reaction rate was observed as IPP was increased to 50μM, followed by a decrease when the IPP concentration was increased further. This is consistent with previous observations of substrate inhibition [26] and is presumably due to binding of IPP in the DMAPP binding site at high IPP concentrations. Based on the above experiments and previous work with FDPS enzymes, concentrations of 50μM were chosen for both DMAPP and IPP concentrations. Finally, the concentrations of the two enzymes were varied to determine appropriate concentrations and ensure that the rate of the FPP synthesis reaction and not the subsequent coupling reaction was being monitored. Thus, as can be seen when the amount of FDPS was increased in the assay (Fig. 5), the reaction rate increased in a linear manner. When varying the concentration of PFTase, an increase in the reaction rate was observed at low concentrations (Fig. 6), indicating that in that regime, the coupling reaction was controlling the overall rate. However, at higher PFTase concentrations, no further increase in overall reaction rate was observed, indicating that the coupling reaction was no longer limiting the process and that the rate being observed reflected the rate of FPP production. Based on those experiments, enzyme concentrations of 5μM and 120nM were chosen for FDPS and PFTase, respectively.