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