Circular permutation of enzymes offers an alternative protein engineering strategy for changing the substrate specificity, as well as improving enantioselectivity and catalytic performance of biocatalysts. These functional changes are linked to structural rearrangements altering active site accessibility and backbone flexibility. To more broadly explore the potential functional benefits of circular permutated enzymes, we have expanded our engineering studies to old yellow enzyme from Saccharomyces cerevisiae (OYE). OYE is a member of the flavin-dependent oxidoreductase family with an (beta/alpha)8 barrel topology that catalyzes the highly enantioselective trans-hydrogenation of activated alkenes. The study marks a departure from traditional protein engineering protocol; we have replaced the random circular permutation method with a fully synthetic protein library design which dramatically reduces library size and eliminates the need for library oversampling. In combination with cell-free protein production, it also greatly enhances the efficiency and versatility of library analysis. Furthermore, the impact of circular permutation on the structure and function of selected OYE variants will be presented. The effects of active site reorganization in OYE are not limited to substrate specificity and enantioselectivity but can potentially be extended to changes in chemistry, due to the remodeling of the flavin cofactor environment.

New tricks with old yellow enzyme

245th National Meeting (2013)

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