Figure 3.
MmCobD has L-Thr-P decarboxylase activity in vivo and in vitro. (A) Cobalamin-dependent growth assessment of S. enterica cobD+ (open squares) and ΔcobD (diamonds) strains with plasmids synthesizing MmCobD (triangles), MmCobD1-385 (inverted triangles), MmCobD386-497 (solid squares), or SeCobD (circles) proteins. Plasmid pBAD24 was introduced into the strains to monitor their growth behavior when the empty cloning vector was present in the cell. Cells were grown aerobically at 37°C in NCE minimal medium containing glycerol (22 mM) as the sole carbon and energy source, supplemented with (CN)2Cby (1 nM), arabinose (0.25 mM), ampicillin (0.1 mg mL−1), and MgSO4 (1 mM). Growth analysis was performed in triplicate and repeated in three independent experiments. Error bars represent the standard error of the mean (SEM). (B) Phosphor image of products and reactants resolved by TLC with retention factors (Rf) indicated. A sample (5 μL) of reactions containing HEPES buffer (50 mM, pH 8.5 at 25 °C), normoxically purified protein (72 nM), and a 1:10 ratio of [14C-U]-L-Thr-P and L-Thr-P (5 μM), was spotted onto a cellulose TLC plate. Plates were developed for 1 h with ammonium acetate (2.5 M):ethanol (95%; v/v) (30:70 ratio) mobile phase. Reactions and TLC separations were repeated in three independent experiments. AP-P, (R)-1-aminopropan-2-ol-O-phosphate; L-Thr-P, L-threonine-O-3-phosphate; L-Thr, L-threonine.