As mentioned earlier, it is known from the crystal structure of MeHNL that the active site of the enzyme is accessible by a narrow channel. The channel entrance is capped by the large amino acid Trp128. Substitution of Trp128 by amino acids with decreasing size gives the corresponding MeHNL mutants. The MeHNL-W128A mutant, for example, could be prepared and overexpressed in E. coli.49 Since the entrance to the active site of this mutant is significantly less hindered, the
Scheme 10: Mechanism of cyanogenesis catalyzed by MeHNL.45
access of sterically demanding substrates is facilitated considerably. A comparison of the reactions of 3-phenoxy benzaldehyde, which is a very bulky substrate, with the wt-enzyme and the W128A mutant, respectively, reveals the superiority of the mutant. The aldehyde was converted quantitatively in a much shorter time and with high enantiomeric excess using the mutant W128A, nearly independent of the amount of enzyme present.49
Was this article helpful?