Abstract
In the world of natural products, terpenes reign as the most diverse category. The natural world has employed terpenes as signaling molecules, antifungals, and pigments while humans have found use for terpenes as biofuels, fragrances, and medicines. Due to the wide-spread prevalence of terpenes, it is useful to understand the enzymes that catalyze their formation, terpene synthases and cyclases. Most interesting about these enzymes are their strategy for carbocation formation and their ability to exhibit promiscuity. A subclass of these enzymes is sesquiterpene synthases, which catalyze the formation of 15-carbon terpene and terpenoid products. Pentalenene Synthase (PS) is a sesquiterpene synthase that makes pentalenene through an anti-Markovnikov mechanism, where it employs residue F76 to stabilize the resulting secondary carbocation on C9 through a π-cation interaction. African-1-ene Synthase (Af-1S) is a novel sesquiterpene synthase that catalyzes the formation of African-1-ene, also likely through an anti-Markovnikov mechanism that forms a secondary carbocation on C9. Unlike PS, however, Af-1S has an alanine rather than a phenylalanine residue at position 76. This calls into question how this enzyme stabilizes its high energy intermediate. Here we develop an effective expression and purification method, assay the protein with FPP, and use AlphaFold2 to infer the structure of the active site of Af-1S. We found that Af-1S makes several sesquiterpene products, suggesting enzymatic promiscuity. We also performed a large-scale assay of Af-1S and FPP to characterize each product with NMR. Finally, when the inferred structure of Af-1S was aligned with PS bound to an inactive substrate analog, we found that Af-1S has no aromatic residues spatially close to C9 of the inactive substrate analog, suggesting that Af-1S must employ some other unidentified stabilizing mechanism.