A Set of Regioselective O-Methyltransferases Gives Rise to the Complex Pattern of Methoxylated Flavones in Sweet Basil      

Abstract

Polymethoxylated flavonoids occur in a number of plant families, including the Lamiaceae. To date, the metabolic pathways giving rise to the diversity of these compounds have not been studied. Analysis of our expressed sequence tag database for four sweet basil (Ocimum basilicum) lines afforded identification of candidate flavonoid O-methyltransferase genes. Recombinant proteins displayed distinct substrate preferences and product specificities that can account for all detected 7-/6-/49-methylated, 8-unsubstituted flavones. Their biochemical specialization revealed only certain metabolic routes to be highly favorable and therefore likely in vivo. Flavonoid O-methyltransferases catalyzing 49-and 6-O-methylations shared high identity (approximately 90%), indicating that subtle sequence changes led to functional differentiation. Structure homology modeling suggested the involvement of several amino acid residues in defining the proteins' stringent regioselectivities. The roles of these individual residues were confirmed by site-directed mutagenesis, revealing two discrete mechanisms as a basis for the switch between 6-and 49-O-methylation of two different substrates. These findings delineate major pathways in a large segment of the flavone metabolic network and provide a foundation for its further elucidation.

The Roles of a Flavone-6-Hydroxylase and 7-O-Demethylation in the Flavone Biosynthetic Network of Sweet Basil

“…Apparent maximal turnover and affinity constants imply that GENK is the genuine substrate for CYP82D33 and not an in vitro artifact. The apparent K m in the low micromolar range matches well with affinity constants of recently characterized basil flavonoid OMTs (12). Turnover of GENK is additionally favored by the high apparent K i value (Table 3).…”

“…One of the candidates encoded a protein belonging to the family CYP71 and was considered promising because the flavonoid 6-hydroxylase from soybean also belongs to this family (14). However, all of the encoding ESTs originated from just one of the four basil lines (MC), which contradicts flavone accumulation data in different lines (12). The recombinant protein was nevertheless tested but displayed no detectable activity with any of the offered substrates, including GENK, despite a visible peak in the CO difference spectrum (data not shown).…”

“…Therefore, it remains unclear whether this in vitro phenomenon will translate into a physiologically relevant regulatory mechanism. Remarkably, two flavonoid 4Ј-OMTs were also inhibited by higher concentrations of GENK (12).…”

“…Assay, reagents, instrumentation, primers for the reference gene (elongation factor-1) and 7-OMT, and general considerations were as described previously (12). CYP82D33 was detected using 5Ј-TGATCTCGGTGGTTAAAGGT-3Ј (forward) and 5Ј-AGTGTCAGTACCACCAGCAA-3Ј (reverse) as primers.…”

“…1) as characteristic compounds (11). We recently found that flavone biosynthesis and accumulation are also largely peltate trichome-specific (12). Within that investigation, which focused on methylation processes and pathways leading to accumulation of SALV, we identified scutellarein-7-methyl ether (SCU7Me) (Fig.…”