# Active-site loop variations adjust activity and selectivity of the cumene dioxygenase

## TL;DR

Researchers engineered flexible enzyme loops to improve activity and selectivity of a biocatalyst, enabling new product formation.

## Contribution

A novel loop engineering approach called Linker In Loop Insertion is introduced, enhancing enzyme activity and selectivity.

## Key findings

- Loop modifications led to up to 16-fold activity increases and new enantiomer production.
- CAVER analysis suggests expanded active site volume and new tunnels for substrate flexibility.
- Combining hotspot engineering with the new insertion method improves biocatalyst design.

## Abstract

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.

Active-site loops are important for catalytic properties of enzymes, but challenging to engineer due to their high flexibility and diversity. Here, the authors identify and engineer hot-spots in the loops of cumene dioxygenase, obtain variants with changed activity, regio- and enantioselectivity, and present a Linker In Loop Insertion approach for loop modification.