Probing the Role of Active Site Water in the Sesquiterpene Cyclization Reaction Catalyzed by Aristolochene Synthase

Abstract

Aristolochene synthase (ATAS) is a high-fidelity terpenoid cyclase that converts farnesyl diphosphate exclusively into the bicyclic hydrocarbon aristolochene. Previously-determined crystal structures of ATAS complexes revealed trapped active site water molecules that could potentially interact with catalytic intermediates: water "w" hydrogen bonds with S303 and N299, water molecules "w1" and "w2" hydrogen bond with Q151, and a fourth water molecule is coordinated to the Mg2+c ion. There is no obvious role for water in the ATAS mechanism, since the enzyme exclusively generates a hydrocarbon product. Thus, these water molecules are tightly controlled so that they cannot react with carbocation intermediates. Steady-state kinetics and product distribution analyses of eight ATAS mutants designed to perturb interactions with active site water molecules (S303A, S303H, S303D, N299A, N299L, N299A/S303A, Q151H, and Q151E) indicate relatively modest effects on catalysis but significant effects on sesquiterpene product distributions. X-ray crystal structures of S303A, N299A, N299A/S303A, and Q151H mutants reveal minimal perturbation of active site solvent structure. Seven of the eight mutants generate farnesol and nerolidol, possibly resulting from addition of the Mg2+c-bound water molecule to the initially formed farnesyl cation, but no products are generated that would suggest enhanced reactivity of other active site water molecules. However, intermediate germacrene A tends to accumulate in these mutants. Thus, apart from the possible reactivity ofMg2+c-bound water, active site water molecules in ATAS are not directly involved in the chemistry of catalysis, but instead contribute to the template that governs the conformation of the flexible substrate and carbocation intermediates.

Structural and Chemical Biology of Terpenoid Cyclases

“…Moreover, mutagenesis of residues that hydrogen bond to this water molecule does not lead to the generation of hydroxylated products, so this water molecule appears to be located in a position where it simply cannot react with carbocation intermediates. 25 Thus, as long as a water molecule is held in a location where it cannot react with the empty p orbital on a carbocation intermediate, then there is no risk of prematurely quenching the cyclization cascade. How terpenoid cyclases control water for organic synthesis remains a largely unanswered question.…”

Molecular dynamics study of taxadiene synthase catalysis

“…It should be noted that the involvement of water as a base in the catalysis of terpene synthase reactions is not a commonly accepted notion, except for enzymes generating hydroxylated products . The crystal structures of terpene synthases show only one or a few water molecules to be trapped in the active site close to the carbocations, and they are usually well stabilized by their surroundings in positions where they are not well oriented to react . Unfortunately, crystal structures of TXS complexed with analogues of the carbocation intermediates have not yet been reported, and there is no experimental information on the potential reactivity of water during TXS catalysis.…”

Substrate Pre‐Folding and Water Molecule Organization Matters for Terpene Cyclase Catalyzed Conversion of Unnatural Substrates

“…Moreover, we analyzed the MD simulations with respect to water molecules and their networks. This is particularly interesting since the dynamic behavior of water molecules and their impact on terpene cyclase function is a mainly unexplored field . Our studies suggest that in addition to efficient pre‐folding, the organization of water molecules is a crucial element for catalysis.…”