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キーワード:
Directed evolution Reduced amino acid alphabet Epoxide hydrolases Saturation mutagenesis Stereoselectivity
要旨:
Background: Recent methodology development in directed evolution of stereoselective enzymes has shown that
various mutagenesis strategies based on saturation mutagenesis at sites lining the binding pocket enable the generation
of small and smart mutant libraries requiring minimal screening.
Methods: In this endeavor, limonene epoxide hydrolase (LEH) has served as an experimental platform, the hydrolytic
desymmetrization of cyclohexene oxide being the model reaction with formation of (R,R)- and (S,S)-cyclohexane-
1,2-diol. This system has now been employed for testing reduced amino acid alphabets based on the Hecht concept
of binary patterning, with and without additional hydrophobic amino acids.
Results and Conclusions: It turns out that in binary pattern based saturation mutagenesis as applied to LEH, polar
amino acids are seldom introduced. When applying binary patterning in combination with additional hydrophobic
amino acids as building blocks in iterative saturation mutagenesis, excellent LEH variants were evolved for the
production of both (R,R)- and (S,S)-diols (80–97 % ee), but again the introduction of polar amino acids occurs rarely.
Docking computations explain the source of enhanced and inverted stereoselectivity. Some of the best variants are
also excellent catalysts in the hydrolytic desymmetrization of other meso-epoxides, although both enantiomeric diols
are not always accessible.
