The N267D substitution conferring an increased thermal stability
to the MetA enzyme has been previously described . The double LY and triple LYD mutant strains were cultured at 45°C in M9 glucose medium and compared with single mutants L124 and Y229 and the wild-type strain WE (Additional file 3: Figure S2). The temperature 45°C was chosen because no significant differences between the strains harboring single and multiple VS-4718 clinical trial mutated MetA enzymes were detected at 44°C (data not shown). The wild-type strain did not grow at 45°C (Additional file 3: Figure S2). The double LY and triple LYD mutants grew faster than the single mutant strains L124 and Y229, which had specific growth rates of 0.37 and 0.42 h-1 versus 0.18 and 0.3 h-1, respectively. The highest growth rate at 45°C was
observed in the LYD strain (0.42 h-1), in which the selleck inhibitor Selleckchem OICR-9429 effects of the MetA enzyme were combined the maximal number of the stabilizing mutations. However, the mutant LYD still grew slower than in the presence of L-methionine (specific growth rate 0.53 h-1; data not shown). This result might reflect the presence of another thermolabile protein in the methionine biosynthetic pathway. Previously, Mogk et al. showed that MetE, which catalyzes the last step in methionine biosynthesis, was also thermally sensitive and tended to form aggregates at a 45°C heat shock. Mutant MetAs enabling E. coli growth at higher temperatures did not display an increased thermal transition midpoint To determine whether the accelerated growth observed
at 44°C for the single mutant MetA strains is due to increased thermal stability of MetA, the protein melting temperature (T m) was measured using differential scanning calorimetry (DSC). The wild-type and mutant MetA enzymes containing a C-terminal six-histidine tag were purified as described in the Methods section. The T m of the wild-type MetA was 47.07 ± 0.01°C (Table 1), and the T ms of the stabilized MetA proteins were slightly higher than that of the wild-type enzyme (Table 1). Table 1 Differential scanning calorimetric data for the wild- type and mutant MetA enzymes Enzyme T m (°C) ∆H* ∆Hv * ∆H/∆Hv MetA, wt Atezolizumab 47.01 ± 0.26 5.93 x 104 1.18 x 105 0.5 I124L 48.65 ± 0.06 6.51 x 104 1.86 x 105 0.35 I229Y 50.68 ± 0.06 8.99 x 104 2.38 x 105 0.38 *The errors associated with the data were <2% for ∆H and ∆Hv. The calorimetric heat (∆H) is the heat change per mole of enzyme. The van’t Hoff heat (∆Hv) is the heat change per cooperative unit. The ratio ∆H/∆Hv is a measure of the number of thermally transited cooperative units per mole of enzyme. All measurements were performed in triplicate. Because the stabilized mutants displayed T m values similar to the native enzyme, we hypothesized that the catalytic activity was enhanced in the MetA mutants.