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Free keywords:
directed evolution · epoxide hydrolases · reduced
amino acid alphabet · saturation mutagenesis ·
stereoselectivity
Abstract:
Saturation mutagenesis at sites lining the binding pockets of
enzymes constitutes a viable protein engineering technique
for enhancing or inverting stereoselectivity. Statistical analysis
shows that oversamplin g in the screening step (the bottleneck)
increases astronomically as the number of residues in the ran-
domization site increases, which is the reason why reduced
amino acid alphabets have been employed, in addition to
splitting large sites into smaller ones. Limonene epoxide
hydrolase (LEH) has previously served as the experimental plat-
form in these methodological efforts, enabling comparisons
between single-code saturation mutagenesis (SCSM) and
triple-code saturation mutagenes is (TCSM); these employ
either only one or three amino acids, respectively, as building
blocks. In this study the comparative platform is extended by
exploring the efficacy of double-code saturation mutagenesis
(DCSM), in which the reduced amino acid alphabet consists of
two members, chosen according to the principles of rational
design on the basis of structural information. The hydrolytic
desymmetrization of cyclohexene oxide is used as the model
reaction, with formation of either (R,R)- or (S,S)-cyclohexane-
1,2-diol. DCSM proves to be clearly superior to the likewise
tested SCSM, affording both R,R-andS,S-selective mutants.
These variants are also good catalysts in reactions of further
substrates. Docking computations reveal the basis of enantio-
selectivity.
