The Biosynthetic Gene Cluster of Mushroom‐Derived Antrocin Encodes Two Dual‐Functional Haloacid Dehalogenase‐like Terpene Cyclases

Abstract

(−)‐Antrocin (1), produced by the medicinal mushroom Antrodia cinnamomea, is a potent antiproliferative compound. The biosynthetic gene cluster of 1 was identified, and the pathway was characterized by heterologous expression. We characterized a haloacid dehalogenase‐like terpene cyclase AncC that biosynthesizes the drimane‐type sesquiterpene (+)‐albicanol (2) from farnesyl pyrophosphate (FPP). Biochemical characterization of AncC, including kinetic studies and mutagenesis, demonstrated the functions of two domains: a terpene cyclase (TC) and a pyrophosphatase (PPase). The TC domain first cyclizes FPP to albicanyl pyrophosphate, and the PPase domain then removes the pyrophosphate to form 2. Intriguingly, AncA (94 % sequence identity to AncC), in the same gene cluster, converts FPP into (R)‐trans‐γ‐monocyclofarnesol instead of 2. Notably, Y283/F375 in the TC domain of AncA serve as a gatekeeper in controlling the formation of a cyclofarnesoid rather than a drimane‐type scaffold.

Class II terpene cyclases: structures, mechanisms, and engineering

“…Class II TCs exist exclusively in the early-stage of natural product biosynthetic routes. 5,37–42 The diphosphate group present in the cyclized ring system generated by class II diterpene cyclases (DTCs) or sesquiterpene cyclases (STCs) is known to facilitate the formation of carbocations, which can be further leveraged in synthetic chemistry approaches. This property makes a cyclized terpene skeleton bearing a diphosphate group an attractive starting point for carbocation chemistry and subsequent functional group decorations using both chemical strategies and enzymatic transformations.…”

Catalytic Mechanism and Heterologous Biosynthesis Application of Sesquiterpene Synthases

“…Both the H-α1 loop and the G helix play protective roles in shielding the active carbon moiety from potential premature solvent quenching which is often associated with water capture. Consequently, altering residues in these regions often leads to significant changes in the ratio of hydroxylated and nonhydroxylated products. ,, Previously, distal sites were believed to have no impact on protein functions, while recent findings have shown their association with catalytic activity. , Generally, such sites contribute to allosteric regulation of the enzyme through robust coupling to the active site. Nevertheless, the exact manner in which these sites affect the catalytic process in STSs remains undetermined.…”

“…17,71,89 Previously, distal sites were believed to have no impact on protein functions, while recent findings have shown their association with catalytic activity. 73,81 Generally, such sites contribute to allosteric regulation of the enzyme through robust coupling to the active site. Nevertheless, the exact manner in which these sites affect the catalytic process in STSs remains undetermined.…”

“…The residues Y283 and F375 of AncA were altered to F and L, which are at the corresponding positions on AncC. 73 AncA Y283F/F375L resulted in albicanol production, indicating that these two residues serve as gatekeepers for product scaffold formation. The narrower pocket of AncA enabled the cyclization of FPP to the monocyclic ring of the cyclofarnesoid, while the remaining carbon chain was constrained to extend in a linear conformation.…”

“…The ( R )- trans -γ-monocyclofarnesol synthase AncA and the albicanol synthase AncC show 94% sequence similarity. The residues Y283 and F375 of AncA were altered to F and L, which are at the corresponding positions on AncC . AncA Y283F/F375L resulted in albicanol production, indicating that these two residues serve as gatekeepers for product scaffold formation.…”