Active-site loops play essential roles in various catalytically important enzyme properties like activity, selectivity, and substrate scope. However, their high flexibility and diversity makes them challenging to incorporate into rational enzyme engineering strategies. Here, we report the engineering of hot-spots in loops of the cumene dioxygenase from Pseudomonas fluorescens IP01 with high impact on activity, regio- and enantioselectivity. Libraries based on alanine scan, sequence alignments, and deletions along with a novel insertion approach result in up to 16-fold increases in activity and the formation of novel products and enantiomers. CAVER analysis suggests possible increases in the active pocket volume and formation of new active-site tunnels, suggesting additional degrees of freedom of the substrate in the pocket. The combination of identified hot-spots with the Linker In Loop Insertion approach proves to be a valuable addition to future loop engineering approaches for enhanced biocatalysts.

No abstract

In enzymes, the active site is the location where substrates are chemically converted. If this site is deeply buried within the protein, substrates must pass not only through the body of the protein via a tunnel, but also flexible, site-decorating loops to access the active site. These elements can act as filters that influence on both substrate specificity and activity. Identifying and understanding how they exert such control has been of growing interest over the past several years.