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Abstract:
A new directed evolution approach is presented to enhance the activity of an enzyme and to manipulate
stereoselectivity by focusing iterative saturation mutagenesis (ISM) simultaneously on residues lining the
entrance tunnel and the binding pocket. This combined mutagenesis strategy was applied successfully to
the monoamine oxidase from Aspergillus niger (MAO-N) in the reaction of sterically demanding
substrates which are of interest in the synthesis of chiral pharmaceuticals based on the benzo-piperidine
scaffold. Reversal of enantioselectivity of Turner-type deracemization was achieved in the synthesis of
(S)-1,2,3,4-tetrahydro-1-methyl-isoquinoline, (S)-1,2,3,4-tetrahydro-1-ethylisoquinoline and (S)-1,2,3,4-
tetrahydro-1-isopropylisoquinoline. Extensive molecular dynamics simulations indicate that the altered
catalytic profile is due to increased hydrophobicity of the entrance tunnel acting in concert with the
altered shape of the binding pocket.
