The N267D substitution conferring an increased thermal stability<

The N267D substitution conferring an increased thermal stability

to the MetA enzyme has been previously described [11]. 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.[14] 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.

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