The roles of a flavone-6-hydroxylase and 7-O-demethylation in the flavone biosynthetic network of sweet basil

Abstract

Lipophilic flavonoids found in the Lamiaceae exhibit unusual 6- and 8-hydroxylations whose enzymatic basis is unknown. We show that crude protein extracts from peltate trichomes of sweet basil (Ocimum basilicum L.) cultivars readily hydroxylate position 6 of 7-O-methylated apigenin but not apigenin itself. The responsible protein was identified as a P450 monooxygenase from the CYP82 family, a family not previously reported to be involved in flavonoid metabolism. This enzyme prefers flavones but also accepts flavanones in vitro and requires a 5-hydroxyl in addition to a 7-methoxyl residue on the substrate. A peppermint (Mentha × piperita L.) homolog displayed identical substrate requirements, suggesting that early 7-O-methylation of flavones might be common in the Lamiaceae. This hypothesis is further substantiated by the pioneering discovery of 2-oxoglutarate-dependent flavone demethylase activity in basil, which explains the accumulation of 7-O-demethylated flavone nevadensin.

FIGURE 1.

Proposed pathways in relevant segments of the flavone metabolic network in basil, and structures of compounds used in this work. The framed flavonoid backbone in the top left corner illustrates flavonoid backbone numbering and ring nomenclature as well as compounds tested as substrates and mentioned in Tables 2 and 3. F4′OMT, F6/4′OMT, and F7OMT, flavonoid 4′-, 6/4′-, and 7-O-methyltransferases. Compound abbreviations are underlined. Solid arrows, demonstrated major steps; broken arrows, biochemically unfavorable reactions; crossed arrows, steps that are probably not physiologically relevant; question marks, steps not yet elucidated.

FIGURE 2.

Oxygenase activities in crude trichome protein extracts. A, crude desalted trichome protein extracts from basil (line EMX-1) or peppermint were incubated with API (traces 1–4) or GENK (traces 5–8) as substrates. Dotted lines connect identical compounds for better overview. UV (B) and mass (D) spectra of SCU7Me are identical with those of the authentic standard. The peak labeled m/z 317 (trace 7) was tentatively identified as 3′-OH-SCU7Me based on its UV (C) and mass (E) spectra. Note the shift of peak I maximum from 336 to 345 nm, characteristic of the 3′,4′-di-substituted pendant ring. Ob, basil; Mp, peppermint; FeAKG, Fe²⁺, ascorbate, ketoglutarate. The scale is equal for all traces. The displayed chromatograms are representative of three independent trichome preparations.

FIGURE 3.

Similarity tree of selected cytochrome P450 families. Protein sequences were retrieved from NCBI GenBank^TM using the following accession numbers (source organism and proposed function, if any, are given in parentheses): CYP82N2v2, BAK20464 (E. californica, protopine 6-hydroxylase); CYP82G1, NP 189154 (Arabidopsis thaliana, geranyllinalool catabolism); CYP82C2, O49394 (A. thaliana); CYP82E4v1, ABA07805 (N. tabacum, nicotine demethylase); CYP82D33, JX162212 (O. basilicum, flavonoid 6-hydroxlase); CYP82D62, JX162214 (M. piperita, flavonoid 6-hydroxylase); CYP82A2, CAA71515 (Glycine max); CYP82B1, AAC39454 (E. californica); AAS90126, CYP82H1 (Ammi majus); ABB20912, CYP82Q1 (Stevia rebaudiana); CYP93B23, JX162213 (O. basilicum, flavone synthase); CYP93B1, BAA22423 (Glycyrrhiza echinata, flavone synthase); CYP93B6, BAB59004 (Perilla frutescens var. crispa, flavone synthase); AAD39549, CYP93B2 (Gerbera hybrida, flavone synthase); CYP93A1, NP_001241186 (G. max, 3,9-dihydroxypterocarpan 6a-hydroxylase); CAA80266, CYP75A1 (Petunia × hybrida, flavonoid 3′,5′-hydroxylase); AAD56282, CYP75B1 (P. hybrida, flavonoid 3′-hydroxylase); CYP73A1, CAA78982 (Helianthus tuberosus, trans-cinnamate 4-hydroxylase); CYP80A1, AAC48987 (Berberis stolonifera, berbamunine synthase); CYP80F1, ABD39696 (Hyoscyamus niger, littorine mutase); CYP51G1, BAB61873 (A. thaliana, obtusifoliol 14-demethylase); CYP71AJ1, AAT06911 (A. majus, psoralen synthase); CYP71D9, CAA71514 (G. max, flavonoid 6-hydroxylase); CYP71D18, AAD44150 (M. piperita, limonene 6-hydroxylase); CYP71E7, AAP57704 (Manihot esculenta, cyanogenic glucoside oxim metabolism); CYP71D12, CAB56503 (Catharanthus roseus, tabersonine 16-hydroxylase); CYP71AV1, ABB82944 (Artemisia annua, amorpha-4,11-diene C-12 oxidase); CYP98A13, AAL99200 (O. basilicum, p-coumaroyl shikimate 3′-hydroxylase); CYP719A1, BAB68769 (C. japonica, methylenedioxy bridge-forming enzyme); CYP72A1, AAA33106 (C. roseus, secologanin synthase); CYP79A1, AAA85440 (Sorghum bicolor, tyrosine N-hydroxylase); CYP701A1, AAG41776 (Cucurbita maxima, ent-kaurene oxidase); CYP86A1, P48422 (A. thaliana, fatty acid ω-hydroxylase); CYP90A1, Q42569 (A. thaliana, 6-oxo-cathasterone 23a-hydroxylase); CYP88A1, AAC49067 (Zea mays, ent-kaurenoic acid oxidase); CYP703A1, BAA92894 (P. hybrida lauric acid hydroxylase); CYP74A1, AAA03353 (Linum usitatissimum, allene oxide synthase); CYP74B1, AAA97465 (Capsicum annuum, fatty acid hydroperoxide lyase). Sequences were aligned using ClustalW (version 18.3) and default settings, and an unrooted neighbor-joining tree was constructed using Mega 5 (59). Only bootstrap values for nodes supported by >70% of 1,000 replicates are shown. The scale is in changes per amino acid. Boldface type, proteins described in this report; underlined, F6H from soybean (14).

FIGURE 4.

Expression of F6H in seven successive leaf pairs of basil lines SD and EMX-1. Expression of F6H was co-analyzed with basil flavonoid 7-OMT (F7OMT) (12), using quantitative real-time RT-PCR. Results are means of five biological replicates ± S.E. (error bars). The key indicates leaf pairs, where 1 is the oldest.

FIGURE 5.

Activities of basil flavone synthase (CYP93B23) with higher substituted flavanones. A, representative chromatograms of enzyme assays containing an excess of protein for qualitative detection of turnover. Respective substrate peak labels are underlined. Arrows indicate retention time of expected products (shown in parentheses) if none are detected (traces 4 and 5). B, relative activities of CYP93B23 with different flavanones under linear assay conditions. Turnover rates are relative to those with NAR, which was 5.97 pkat mg⁻¹ protein and set as 100%. Results are means ± S.E. (error bars) (n = 3). ND, no activity detected under all tested assay conditions.

FIGURE 6.

Flavone demethylase activity in basil and peppermint. A, GARD B was incubated with crude desalted trichome protein extracts from basil (traces 2–7) or peppermint (traces 8–10) in the presence of various cofactors. Trace 1 contained only the NEV standard. FeAKG, Fe²⁺, ascorbate, α-ketoglutarate. All traces for the same ion and species are scaled equally. The displayed chromatograms are representative of three independent trichome preparations. Product peak was identified as NEV based on its retention time, UV spectrum (B), and fragmentation pattern (C), which are identical with those of the authentic standard.