# Structural Revision of the C16 Sesquiterpene Hegelenether and the Mechanism of C6‐Methylation in Terpene Biosynthesis

## TL;DR

The structure of a natural compound called hegelenether was corrected to marxdiol, and the mechanism of a key methylation step in its biosynthesis was revealed using enzyme crystal structures and labeling experiments.

## Contribution

The study provides a revised structure for hegelenether and a structural and mechanistic explanation for a non-canonical C6-methylation in terpene biosynthesis.

## Key findings

- The structure of hegelenether was revised to marxdiol with determined absolute configuration.
- The crystal structure of the methyltransferase C6-FPP-MT revealed a mechanism for stereoselective C6 methylation.
- Site-directed mutagenesis confirmed the role of Glu165 in the methylation reaction.

## Abstract

Non‐canonical methylation events generate terpene structures that evade classical biosynthetic predictions, as exemplified by the proposed C16 terpene hegelenether. Here, we show that this natural product is misassigned and revise its structure to the dihydroxylated sesquiterpenoid marxdiol. Its absolute configuration and that of its precursor prekantenol pyrophosphate were determined through terpene synthase‐mediated incorporation of stereoselectively labeled probes. To explain the initiating C6 methylation, we solved the crystal structure of the methyltransferase C6‐FPP‐MT with SAH and FPP, revealing a compact aromatic pocket that enforces Si‐face methylation and Glu165‐mediated deprotonation. These insights define how the active site controls regio‐ and stereochemistry and provide a structural basis for identifying related methyl‐modified terpenes in uncharacterized biosynthetic pathways.

The structure of the non‐canonical C16 sesquiterpene named hegelenether was revised to the structure of marxdiol, and its absolute configuration was assigned. Isotopic labeling experiments revealed the stereochemical course of the methyltransferase involved in its biosynthesis. The enzyme mechanism was further addressed through protein X‐ray crystallography and structure‐based site‐directed mutagenesis.