| By screening a low number of Pseudomonas fluorescens (PFE) mutants, directed evolution identified a mutant with modestly improved enantioselectivity (from Ewild-type = 12 to EThr230Ile = 19) toward the useful chiral synthon methyl 3-bromo-2-methylpropionate (MBMP). A homology model of HE revealed that the mutation was far away from the enzyme active site and may contribute to increased enzyme flexibility.; In a second approach to improving enantioselectivity of PFE toward MBMP, the homology model was used to select amino acid residues near the stereocenter of the docked tetrahedral intermediate of the substrate. Randomization of these residues yielded a Trp29Leu mutant with E increased to 58, as well as a Phe199Trp mutant with E decreased to 2. |
