Mechanistic Investigation Of Biosythesis Selectivity Of Terpene Cyclase

Terpenoid cyclases catalyze a variety of terpene biosynthesis,and produce the most chemically and structurally diverse family of natural products.Mutation investigations show aberrant product arrays that are different or even more diverse than,the wild-type enzyme.The occurrence of novel terpenes and the alteration of product preference,coincides with the protein engineering target to design and remodel enzymes,to enhance catalytic activity or obtain new catalytic functions.Therefore,terpene enzymes have attracted extensive research interests.Herein,we selected a representative enzyme to explore the main product and one byproduct reaction pathways which both catalyzed by the sesquiterpene synthase,trichodiene synthase,and deciphered the role of key residues in catalysis by using theoretical simulation and calculation method.We explored the structural and energetic features of biosynthesis pathway for trichodiene and ?-barbatene by trichodiene synthase(TDS)through the dual-level QM/MM MD simulation,and the carbocation reaction mechanism in the gas-phase using density functional theory.The two-ring main product trichodiene and tricyclic byproduct ?-barbatene were selected as comparison to explore the determinants controlling of product preference of TDS.The results have shown that the key point of divergence for these two products lies in the intermediate cuprenyl cation,where different methyl transfers lead to different reactions.In the synthesis of trichodiene,the transfer of vertical C12 methyl group requires an energy barrier of 8.19 kcal/mol in the enzyme system,and the gas phase reaction experiences an energy barrier of 4.6 kcal/mol.The free energy barrier for the transfer of vertical C13 methyl group by TDS is 10.11 kcal/mol,and the energy barrier in the gas phase is 5.36 kcal/mol.The C13 methyl group transfer produces the isotropic methyl structure and facilitates the formation of the third ring product ?-barbatene.The conformation change of fivemembered ring needs to cross the energy barrier of 2.90 kcal/mol,making the transition from the vertical C12 methyl group to the vertical C13 methyl group.Compared with the much higher energy barrier of methyl transfer,the five-membered ring conformation change can be very easy in the enzyme system.The simulation results suggested that performing C12 methyl transfer in TDS is more advantageous,that is,TDS has more selectivity for the main product trichodiene.In order to find the key possible residues that control the selectivity of different methyl group transfer reaction in the TDS active center,three mutants——T96I,T96 S and T96V——were designed.Compared with the wild-type TDS,the free energy barriers of the three mutants all differ.In particular,the energy barriers of T96 V and T96 I for C13 methyl transfer increased more than that for C12 methyl transfer,which may reduce the selectivity to the product ?-barbatene.It is mainly caused by the mutation of the hydrophilic residue threonine to the hydrophobic residue valine/isoleucine and the disappearance of the electron pushing group hydroxyl.T96 S only changed the position of the hydroxyl group,resulting in an increase of free energy barrier of both two methyl transfer processes.Hence,the non-acid/basic amino acids near the active site in terpenoid cyclases,such as hydroxyl residues,are helpful for the catalytic process,and require more attention in site-directed mutation design.Specifically,it is helpful to narrow the range of key residues in protein design engineering and reduce experiment workload by by integrating insights from purposeful computational studies on key residues..