Fig 1.
Biosynthetic pathway for menaquinone in mycobacteria.
The reaction catalyzed by MenA is shown. Rv numbers have been provided for enzymes for which the activity has been empirically demonstrated.
Fig 2.
LC/MS analysis of neutral lipids.
E. coli WT (CGSC # 7636) (Panel A), E. coli ΔmenA (CGSC # 10816) (Panel B) and E. coli ΔmenA (CGSC # 10816) complemented with pVV16:MenA (Panel C). Data shown are extracted ion chromatograms for m/z values of 703.54 (dotted line) and 717.56 (solid line), DMK-8 and MK-8, respectively.
Fig 3.
MenA activity in E. coli membranes.
Membranes were prepared from E. coli BL21 (DE3) transformed with pET:MenA (open circles) or pET28a(+) (filled circles) grown to mid log phase. Reaction mixtures contained 0.1 M Tris-HCl buffer (pH 8.0), 5 mM MgCl2, 2.5 mM dithiothreitol, 0.1% CHAPS, membrane protein preparation (100 μg), DHNA (500 μM) and [1-3H]farnesyl diphosphate (10 μM). Mixtures were incubated at 37°C for 30 min and processed as described in Materials and Methods section. Data was fit to a single rectangular, 2 parameter hyperbola using SigmaPlot 13, the regression lines (solid) and the 95% confidence intervals (dotted) are shown. Statistical analysis can be found in Supporting Information.
Fig 4.
Effect of MgCl2 concentration on MenA activity.
Reaction mixtures contained 0.1 M Tris-HCl buffer (pH 8.0), 2.5 mM dithiothreitol, 0.1% CHAPS, membrane protein preparation (100 μg) from M. tuberculosis, DHNA (500 μM), [1-3H]farnesyl diphosphate (10 μM) and MgCl2 at the indicated concentration. Mixtures were incubated at 37°C for 30 min and processed as described in Materials and Methods section. The inset shows the effect of various divalent cations on MenA activity when added to the reaction mixture at a final concentration of 5 mM. In all cases endogenous divalent cations were removed with Bio-Rex 70. Data was fit to a single rectangular, 2 parameter hyperbola using SigmaPlot 13, the regression line (solid) and the 95% confidence intervals (dotted) are shown. Statistical analysis can be found in Supporting Information.
Fig 5.
Effect of pH on MenA activity.
Reaction mixtures contained 50 mM buffer (at the indicated pH), 5 mM MgCl2, 2.5 mM dithiothreitol, 0.1% CHAPS, membrane protein preparation (100 μg), DHNA (500 μM) and [1-3H]farnesyl diphosphate (10 μM). Mixtures were incubated at 37°C for 30 min and processed as described in Materials and Methods section. Three buffers, adjusted to the indicated pH with appropriate counter ions, were used: 50 mM MES (filled circles), 50 mM TAPS (open circles) or 50 mM CAPS (filled triangles).
Fig 6.
Effect of Ro 48–8071 on MenA activity.
Double reciprocal plots showing the effect of Ro 48–8071 (structure Panel A) on the reaction rate at the indicated concentrations of [1-3H]farnesyl diphosphate and 250 μM DHNA (Panel B) or the indicated concentrations of DHNA and 20 μM [1-3H]farnesyl diphosphate (Panel C). In each case Ro 48–8071 was added at 0 (closed circles), 3 (open circles) or 7.5 (closed triangles) μg/ml. Reaction mixtures also contained 0.1 M Tris-HCl buffer (pH 8.0), 5 mM MgCl2, 2.5 mM dithiothreitol, 0.1% CHAPS and membrane protein preparation (90 μg) from M. tuberculosis, in a final volume of 0.1 ml. Mixtures were incubated at 37°C for 30 min and processed as described in Materials and Methods section. Linear regression lines were generated using SigmaPlot 13. Regression analysis resulted in r2 values greater than 0.98 for all lines.