Structural Revision of the C ₁₆ Sesquiterpene Hegelenether and the Mechanism of C6‐Methylation in Terpene Biosynthesis

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.