Unexpected roles for ancient proteins: flavone 8‐hydroxylase in sweet basil trichomes is a Rieske‐type, PAO‐family oxygenase

Abstract

SUMMARYMost elucidated hydroxylations in plant secondary metabolism are catalyzed by oxoglutarate-or cytochrome P450-dependent oxygenases. Numerous hydroxylations still evade clarification, suggesting that they might be performed by alternative enzyme types. Here, we report the identification of the flavone 8-hydroxylase (F8H) in sweet basil (Ocimum basilicum L.) trichomes as a Rieske-type oxygenase. Several features of the F8H activity in trichome protein extracts helped to differentiate it from a cytochrome P450-catalyzed reaction and identify candidate genes in the basil trichome EST database. The encoded ObF8H proteins share approximately 50% identity with Rieske-type protochlorophyllide a oxygenases (PTC52) from higher plants. Homology cloning and DNA blotting revealed the presence of several PTC52-like genes in the basil genome. The transcripts of the candidate gene designated ObF8H-1 are strongly enriched in trichomes compared to whole young leaves, indicating trichome-specific expression. The full-length ObF8H-1 protein possesses a predicted N-terminal transit peptide, which directs green fluorescent protein at least in part to chloroplasts. The F8H activity in crude trichome protein extracts correlates well with the abundance of ObF8H peptides. The purified recombinant ObF8H-1 displays high affinity for salvigenin and is inactive with other tested flavones except cirsimaritin, which is 8-hydroxylated with less than 0.2% relative activity. The efficiency of in vivo 8-hydroxylation by engineered yeast was improved by manipulation of protein subcellular targeting. BLAST searches showed that occurrence of several PTC52-like genes is rather common in sequenced plant genomes. The discovery of ObF8H suggests that Rieske-type oxygenases may represent overlooked candidate catalysts for oxygenations in specialized plant metabolism.

Two CYP82D Enzymes Function as Flavone Hydroxylases in the Biosynthesis of Root-Specific 4′-Deoxyflavones in Scutellaria baicalensis

“…Additionally, in 2014 Berim and colleagues isolated an 8-hydroxylase from sweet basil that could convert salvigenin or crisimaritin to 8-hydroxysalvigenin or 8-hydroxycrisimaritin. This plastid-localized enzyme belongs to the protein family of Rieske-type oxygenases and is highly expressed in the trichomes of sweet basil leaves ( Berim et al., 2014 ).…”

Bioenergetics of Monoterpenoid Essential Oil Biosynthesis in Nonphotosynthetic Glandular Trichomes

“…This is a logical consequence of the fact that Fd is a cofactor for electron transfer reactions toward two reductive enzymes of the MEP pathway (Seemann et al, 2006;Rekittke et al, 2013) and flavone hydroxylases (Berim et al, 2014), which are involved in the formation of the major products of mint GTs (monoterpenes and methoxylated flavones, respectively). In earlier versions of menpiGT_2015, Fd served as a substrate for FNR, which is the direction of electron flow in photosynthetic cells.…”

Unleashing the Synthetic Power of Plant Oxygenases: From Mechanism to Application

“…A recent study identified a novel RSO from Ocimum basilicum (basil) trichomes that functions as a flavone-8-hydroxylase with strong selectivity toward salvigenin ( Fig. 5B; Berim et al, 2014). Additional uncharacterized RSO homologs found in plant genomes may be proven to operate in specialized metabolism as well.…”