(Focused) Directed Evolution to Unterstand Relationships in alpha/beta-Hydrolase-Fold Enzymes

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Esterases, epoxide hydrolases and haloalcohol dehalogenases have a highly conserved protein scaffold ((/(-hydrolase fold) with (apparently) only minor differences in key amino acid residues. However, changing conserved amino acids (i.e. the serine in the esterase to an aspartate to create epoxide hydrolase activity) does not lead to functional changes. In this proposal, a combination between rational protein design and directed evolution will be applied to create catalytically promiscuous activity into the esterase from Pseudomonas fluorescens (PFE). Preliminary experiments already confirmed that epoxide hydrolase activity could be gained by this approach. This will be extended by further rounds of directed evolution, back mutations and saturation mutagenesis. We expect to get a detailed picture of the mechanism and key residues required to confer the promiscous activity. This insight into the structure-function basis should not only extend our understanding how epoxide hydrolases work, but also provide information about a/ß-hydrolase function in general. The same concept will be applied to confer haloalcohol dehalogenase activity in PFE.