Characterization of CYP76AH4 clarifies phenolic diterpenoid biosynthesis in the Lamiaceae

Abstract

Miltiradiene (1) is the precursor of phenolic diterpenoids such as ferruginol (2), requiring aromatization and hydroxylation. While this has been attributed to a single cytochrome P450 (CYP76AH1), characterization of the rosemary ortholog CYP76AH4 led to the discovery that these CYPs simply hydroxylate the facilely oxidized aromatic intermediate abietatriene (3).

Figure 1

Representative phenolic diterpenoids.

Figure 2

GC-MS chromatogram of extract from E. coli co-expressing CYP76AH4 and enzymes for the production of 1.

Figure 3

GC-MS chromatograms of in vitro assays of CYP76AH4 with either A) purified 1 (selected ions m/z = 272 + 286) or B) 3 (selected ions m/z = 255 + 286).

Figure 4

GC-MS chromatogram of rosemary extract.

Scheme 1

Reactions putatively catalysed by CYP76AH1.

Scheme 2

Actual role of CYP76AH sub-family members in plant phenolic diterpenoid biosynthesis.