Changing substrate specificity and stereoselectivity applying Trp128 mutants of wtMeHNL

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

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