Alternative Active Site Confinement in Squalene–Hopene Cyclase Enforces Substrate Preorganization for Cyclization

Confinement of an enzyme's active site is critical to the efficiency of chemical reactions and has been recognized as an important tool for catalysis to accelerate reactions by proximity and via specific interactions with the substrate to control the course of the reaction. Despite these benefits, a strongly confined active site is inherently limited to compounds that resemble the native substrate, with only small deviations tolerated. To unlock the potential of modifying the terpene scaffold as building blocks of interest with branched isoprene/terpene motifs, the present study demonstrates a simple structure-guided strategy to create alternative confinement in the squalene–hopene cyclase from Alicyclobacillus acidocaldarius (AacSHC). This strategy aims for proximity and shape complementarity between the protein and substrate to enforce preorganization of geranyl acetone analogs with deviant isoprene patterns. Screening of AacSHC variants affecting the substrate tunnel allowed us to detect starting activity with the AacSHC G600M variant. Structural analysis led to the identification of reduced tunnel radii that resulted in steric interactions and proximity by a decrease in the average distances between the double bond of the substrate's terminal isoprene unit and the catalytic site. Iterative saturation mutagenesis with SHC variants and screening with the geranyl acetone analog dihydropseudoirone were used to generate complementarity and demonstrated further evolvability. The final variant showed a 1174-fold increase in total turnover number and 111-fold increased catalytic efficiency compared to the wild-type. This approach to create alternative active site confinement demonstrates great potential to overcome limitations in the engineering of biocatalysts and generation of interesting building blocks.