Molecular Insight\ninto the Catalytic Mechanism of\nthe Sesquiterpene Cyclase BcABA3

BcABA3 is an unusual sesquiterpene synthase that lacks\nthe conserved\nDDxxD and DTE/NSE motifs. Despite this, it can catalyze the conversion\nof farnesyl diphosphate to 2Z,4E-α-ionylideneethane. We used\nstructure prediction, multiscale simulations, and site-directed mutagenesis\nexperiments to investigate BcABA3 and its catalytic mechanism. BcABA3\nhas structural similarity to typical class I terpenoid cyclases in\nits active site. Based on simulation results, we identified two discontinuous\nglutamate residues, E124 and E88, which compensate for the absence\nof the aspartate-rich DDxxD motif. Quantum chemical calculations show\nthat BcABA3 adopts a direct rotation mechanism for allyl cation isomerization\nrather than via the nerolidyl diphosphate. Then, it can achieve a\nsuccessive proton transfer reaction, which is difficult to achieve\nby intramolecular rearrangement via the protruding outward carbonyl\noxygen of A206. This reaction is then directed forward by two relatively\nstable intermediates containing a cation-conjugated double-bond structure.\nE124 is also proposed as the proton receptor in the final deprotonation\nto couple this step with 2Z,4E-α-ionylideneethane release. These\nfindings provide valuable insight into the catalytic mechanisms of\nBcABA3 and can aid in its engineering, which will facilitate studies\nof abscisic acid biosynthesis.