Molecular Insight into the Catalytic Mechanism of the Sesquiterpene Cyclase BcABA3.

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