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

  • Anna Berim
    Affiliations
    From the Institute of Biological Chemistry Washington State University, Pullman, Washington 99164-6340
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  • David R. Gang
    Correspondence
    To whom correspondence should be addressed. Tel.: 509-335-0550; Fax: 509-335-7643;
    Affiliations
    From the Institute of Biological Chemistry Washington State University, Pullman, Washington 99164-6340
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  • Author Footnotes
    * This work was supported by Department of Energy Biological and Environmental Research Program Grant DE-SC0001728 (to D. R. G.).
    This article contains supplemental Figs. 1–5.
Open AccessPublished:November 26, 2012DOI:https://doi.org/10.1074/jbc.M112.420448
      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.
      Background: Late steps of lipophilic flavone biosynthesis in mints are unknown.
      Results: CYP82D monooxygenases catalyze 6-hydroxylation of 7-O-methylated precursor, whose 7-methyl group is subsequently removed by demethylation in basil but not in peppermint.
      Conclusion: Flavone biosynthesis in basil involves an unusual loop.
      Significance: Novel mechanisms elucidated in basil suggest a new hypothesis for similar metabolic networks in other plants.

      Introduction

      Flavonoids are a class of physiologically and ecologically essential compounds found ubiquitously in higher plants (
      • Pollastri S.
      • Tattini M.
      Flavonols. Old compounds for old roles.
      ). Their structural variation, reflected by more than 9,000 compounds reported to date, is achieved through “decorative” steps, such as glycosylations, acylations, prenylations, methylations, hydroxylations, and combinations of the above (
      • Veitch N.C.
      • Grayer R.J.
      Flavonoids and their glycosides, including anthocyanins.
      ). Because chemodiversity correlates with a diversity of function, specific strategies and mechanisms of chemical structural modification are of great interest (
      • Vogt T.
      Phenylpropanoid biosynthesis.
      ,
      • Matsuba Y.
      • Sasaki N.
      • Tera M.
      • Okamura M.
      • Abe Y.
      • Okamoto E.
      • Nakamura H.
      • Funabashi H.
      • Takatsu M.
      • Saito M.
      • Matsuoka H.
      • Nagasawa K.
      • Ozeki Y.
      A novel glucosylation reaction on anthocyanins catalyzed by acyl-glucose-dependent glucosyltransferase in the petals of carnation and delphinium.
      ). Numerous bioactivities have been reported for various flavonoids, turning them into attractive targets for bioengineering experiments (
      • Wang Y.
      • Chen S.
      • Yu O.
      Metabolic engineering of flavonoids in plants and microorganisms.
      ,
      • Chemler J.A.
      • Koffas M.A.
      Metabolic engineering for plant natural product biosynthesis in microbes.
      ).
      Lipophilic methylated flavonoids occur frequently in several eudicot families, such as the Lamiaceae, Asteraceae, and Rutaceae, but are also found in representatives of other plant families, including ferns and monocots (
      • Wollenweber E.
      • Dietz V.H.
      Occurrence and distribution of free flavonoid aglycons in plants.
      ,
      • Valant-Vetschera K.M.
      • Roitman J.N.
      • Wollenweber E.
      Chemodiversity of exudate flavonoids in some members of the Lamiaceae.
      ). Hydroxylation at positions 6 and 8 of ring A and multiple regiospecific O-methylations appear to be common modifications underlying the high lipophilicity. Notably, identical highly decorated compounds are known to be accumulated by quite distantly related species, prompting the question of whether the respective biosynthetic mechanisms in different species share any similarity.
      Sweet basil (Ocimum basilicum L.) is a popular culinary herb from the mint family. The production of many characteristic flavor- and fragrance-defining compounds is restricted to peltate glandular trichomes on its aerial surfaces (
      • Werker E.
      • Putievsky E.
      • Ravid U.
      • Dudai N.
      • Katzir I.
      Glandular hairs and essential oil in developing leaves of Ocimum basilicum L. (Lamiaceae).
      ,
      • Gang D.R.
      • Wang J.
      • Dudareva N.
      • Nam K.H.
      • Simon J.E.
      • Lewinsohn E.
      • Pichersky E.
      An investigation of the storage and biosynthesis of phenylpropenes in sweet basil.
      ). Construction of EST
      The abbreviations used are: EST
      expressed sequence tag
      NEV
      nevadensin
      GARD B
      gardenin B
      SALV
      salvigenin
      SCU
      scutellarein
      SCU7Me
      scutellarein-7-methyl ether
      GENK
      genkwanin
      API
      apigenin
      NAR
      naringenin
      SAK
      sakuranetin
      C7Me
      carthamidin-7-methyl ether
      CdM
      carthamidin-7,4′-dimethyl ether
      AdM
      apigenin-7,4′-dimethyl ether
      NdM
      naringenin-7,4′-dimethyl ether
      LUT
      luteolin
      LAD
      ladanein
      APItriMe
      apigenin-5,7,4′-trimethyl ether
      ODD
      2-oxoglutarate dependent dioxygenase
      OMT
      O-methyltransferase
      CPR
      cytochrome P450 reductase
      FNS
      flavone synthase
      F6H
      flavonoid 6-hydroxylase
      RACE
      rapid amplification of cDNA ends
      7-OMT
      contig, group of overlapping clones.
      libraries selectively using this specialized cell type (
      • Gang D.R.
      • Wang J.
      • Dudareva N.
      • Nam K.H.
      • Simon J.E.
      • Lewinsohn E.
      • Pichersky E.
      An investigation of the storage and biosynthesis of phenylpropenes in sweet basil.
      ) facilitated fruitful studies of its metabolic and physiological processes. In addition to volatiles, basil produces specific lipophilic flavones, such as nevadensin (NEV), salvigenin (SALV), and gardenin B (GARD B) (Fig. 1) as characteristic compounds (
      • Grayer R.J.
      • Bryan S.E.
      • Veitch N.C.
      • Goldstone F.J.
      • Paton A.
      • Wollenweber E.
      External flavones in sweet basil, Ocimum basilicum, and related taxa.
      ). We recently found that flavone biosynthesis and accumulation are also largely peltate trichome-specific (
      • Berim A.
      • Hyatt D.C.
      • Gang D.R.
      A set of regioselective O-methyltransferases gives rise to the complex pattern of methoxylated flavones in sweet basil.
      ). Within that investigation, which focused on methylation processes and pathways leading to accumulation of SALV, we identified scutellarein-7-methyl ether (SCU7Me) (Fig. 1) as a central intermediate in the metabolic network, making flavone 6-hydroxylation, a step necessary for its formation, our next object of interest. In addition, characterization of specific O-methyltransferases (OMTs) provided clues regarding biochemically favorable routes, revealing that 7-O-methylation preferentially occurred on the 6-unsubstituted substrate apigenin (API) rather than scutellarein (SCU). The combined data from the initial OMT work pointed to two candidate substrates for 6-hydroxylation in basil, API, and its 7-O-methylated derivative genkwanin (GENK) (see Fig. 1 for all structures).
      Figure thumbnail gr1
      FIGURE 1Proposed 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 TABLE 2, TABLE 3. F4OMT, F6/4OMT, 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.
      Flavonoid-6-hydroxylases (F6Hs) have been insufficiently studied, in part due to their apparently restricted occurrence (
      • Halbwirth H.
      The creation and physiological relevance of divergent hydroxylation patterns in the flavonoid pathway.
      ). Interestingly, the only two publications reporting molecular data for F6Hs ascribed this activity to two different enzyme classes. A cytochrome P450-dependent monooxygenase from soybean was most active with liquiritigenin, a 7,4′-dihydroxyflavanone, and is likely to contribute to accumulation of 6-substituted isoflavonoids (
      • Latunde-Dada A.O.
      • Cabello-Hurtado F.
      • Czittrich N.
      • Didierjean L.
      • Schopfer C.
      • Hertkorn N.
      • Werck-Reichhart D.
      • Ebel J.
      Flavonoid 6-hydroxylase from soybean (Glycine max L.), a novel plant P-450 monooxygenase.
      ). In contrast, a 2-oxoglutarate-dependent dioxygenase (ODD) from Chrysosplenium americanum preferred methylated flavonols as substrates and is involved in biosynthesis of polymethoxylated flavonols (
      • Anzellotti D.
      • Ibrahim R.K.
      Novel flavonol 2-oxoglutarate-dependent dioxygenase. Affinity purification, characterization, and kinetic properties.
      ,
      • Anzellotti D.
      • Ibrahim R.K.
      Molecular characterization and functional expression of flavonol 6-hydroxylase.
      ) that are similar to methoxylated flavones in sweet basil (
      • Grayer R.J.
      • Bryan S.E.
      • Veitch N.C.
      • Goldstone F.J.
      • Paton A.
      • Wollenweber E.
      External flavones in sweet basil, Ocimum basilicum, and related taxa.
      ). In addition, P450-dependent 6-hydroxylase activity was detected in protein extracts from Tagetes species, yet the molecular basis was not elucidated (
      • Halbwirth H.
      • Forkmann G.
      • Stich K.
      The A-ring specific hydroxylation of flavonols in position 6 in Tagetes sp. is catalyzed by a cytochrome P450 dependent monooxygenase.
      ). These few examples did not allow predictions regarding the nature of this reaction in basil.
      In this investigation, we found that flavonoid 6-hydroxylation in two Lamiaceae species, sweet basil and peppermint (Mentha × piperita), was catalyzed by monooxygenases belonging to the CYP82D family that used 7-O-methylated apigenin, but not apigenin itself, as substrate. Moreover, our results indicate that 7-O-unmethylated flavone nevadensin found in basil is produced via late 7-O-demethylation of gardenin B by an oxoglutarate-dependent dioxygenase, thereby supporting the conclusion that the pathway to 6-substituted flavones via a 7-O-methylated precursor is the only one existing in basil. These findings reveal an unprecedented loop in lipophilic flavone metabolism, further the delineation of the flavone metabolic network in basil, and add two important activities to the growing catalytic toolbox of flavonoid bioengineering.

      DISCUSSION

      Elucidation of biosynthetic pathways and mechanistic details of plant specialized metabolite accumulation is essential for further ecological, evolutionary, and metabolic engineering studies (
      • Gang D.R.
      Evolution of flavors and scents.
      ,
      • Facchini P.J.
      • Bohlmann J.
      • Covello P.S.
      • De Luca V.
      • Mahadevan R.
      • Page J.E.
      • Ro D.-K.
      • Sensen C.W.
      • Storms R.
      • Martin V.J.
      Synthetic biosystems for the production of high-value plant metabolites.
      ). In the biosynthesis of polymethylated flavonoids, the late hydroxylations at positions 6 and 8 provide a basis for alternative substitution patterns and thus for the expansion of chemodiversity. This report describes cytochrome P450 monooxygenases that catalyze the 6-hydroxylation of flavones in basil and peppermint. The two proteins share high identity levels (73%), which are higher than those found for peppermint and basil flavonoid O-methyltransferases (
      • Berim A.
      • Hyatt D.C.
      • Gang D.R.
      A set of regioselective O-methyltransferases gives rise to the complex pattern of methoxylated flavones in sweet basil.
      ), and show very similar substrate requirements. The few members of the CYP82 family (sharing 40–55% identity with CYP82D proteins; Fig. 3) that have been studied to date are not known to be involved in flavonoid metabolism but are involved in a variety of metabolic pathways. CYP82A2 from soybean yielded a type I binding spectrum with NAR and eriodictyol but did not convert either into any product (
      • Latunde-Dada A.O.
      • Cabello-Hurtado F.
      • Czittrich N.
      • Didierjean L.
      • Schopfer C.
      • Hertkorn N.
      • Werck-Reichhart D.
      • Ebel J.
      Flavonoid 6-hydroxylase from soybean (Glycine max L.), a novel plant P-450 monooxygenase.
      ). CYP82E4 and CYP82E5 from Nicotiana tabacum were identified as nicotine N-demethylases (
      • Siminszky B.
      • Gavilano L.
      • Bowen S.W.
      • Dewey R.E.
      Conversion of nicotine to nornicotine in Nicotiana tabacum is mediated by CYP82E4, a cytochrome P450 monooxygenase.
      ). CYP82N2v2 acts as protopine 6-hydroxylase in sanguinarine biosynthesis in Eschscholzia californica (
      • Takemura T.
      • Ikezawa N.
      • Iwasa K.
      • Sato F.
      Molecular cloning and characterization of a cytochrome P450 in sanguinarine biosynthesis from Eschscholzia californica cells.
      ). CYP82G1 is the homoterpene synthase in Arabidopsis (
      • Lee S.
      • Badieyan S.
      • Bevan D.R.
      • Herde M.
      • Gatz C.
      • Tholl D.
      Herbivore-induced and floral homoterpene volatiles are biosynthesized by a single P450 enzyme (CYP82G1) in Arabidopsis.
      ). An in planta biocatalysis screen in Arabidopsis revealed that CYP82C2 and CYP82C4 are capable of 5-hydroxylating 8-hydroxypsoralen, although this might not be their natural function (
      • Kruse T.
      • Ho K.
      • Yoo H.-D.
      • Johnson T.
      • Hippely M.
      • Park J.-H.
      • Flavell R.
      • Bobzin S.
      In planta biocatalysis screen of P450s identifies 8-methoxypsoralen as a substrate for the CYP82C subfamily, yielding original chemical structures.
      ). Such functional divergence in CYP families is not unusual. For example, the family CYP71 encompasses proteins active with monoterpenes (
      • Mau C.J.D.
      • Croteau R.
      Cytochrome P450 oxygenases of monoterpene metabolism.
      ) and sesquiterpenes (
      • Ralston L.
      • Kwon S.T.
      • Schoenbeck M.
      • Ralston J.
      • Schenk D.J.
      • Coates R.M.
      • Chappell J.
      Cloning, heterologous expression, and functional characterization of 5-epi-aristolochene-1,3-dihydroxylase from tobacco (Nicotiana tabacum).
      ,
      • Teoh K.H.
      • Polichuk D.R.
      • Reed D.W.
      • Nowak G.
      • Covello P.S.
      Artemisia annua L. (Asteraceae) trichome-specific cDNAs reveal CYP71AV1, a cytochrome P450 with a key role in the biosynthesis of the antimalarial sesquiterpene lactone artemisinin.
      ) as well as indole alkaloids (
      • Schröder G.
      • Unterbusch E.
      • Kaltenbach M.
      • Schmidt J.
      • Strack D.
      • De Luca V.
      • Schröder J.
      Light-induced cytochrome P450-dependent enzyme in indole alkaloid biosynthesis. Tabersonine 16-hydroxylase.
      ), coumarins (
      • Larbat R.
      • Kellner S.
      • Specker S.
      • Hehn A.
      • Gontier E.
      • Hans J.
      • Bourgaud F.
      • Matern U.
      Molecular cloning and functional characterization of psoralen synthase, the first committed monooxygenase of furanocoumarin biosynthesis.
      ), and flavonoids (
      • Latunde-Dada A.O.
      • Cabello-Hurtado F.
      • Czittrich N.
      • Didierjean L.
      • Schopfer C.
      • Hertkorn N.
      • Werck-Reichhart D.
      • Ebel J.
      Flavonoid 6-hydroxylase from soybean (Glycine max L.), a novel plant P-450 monooxygenase.
      ) (Fig. 3). Further functional analysis of members of the CYP82 family will reveal the degree of its divergence and the occurrence of other flavonoid-modifying enzymes within it.
      The identity of the F6H in these species as a P450-dependent enzyme places it far apart from the F6H from C. americanum, an oxoglutarate-dependent dioxygenase that is, however, also involved in the metabolism of polymethoxylated flavonoids and prefers methylated flavonols as substrates, with 7-O-methylation recognized as a preeminently important structural element (
      • Anzellotti D.
      • Ibrahim R.K.
      Novel flavonol 2-oxoglutarate-dependent dioxygenase. Affinity purification, characterization, and kinetic properties.
      ,
      • Anzellotti D.
      • Ibrahim R.K.
      Molecular characterization and functional expression of flavonol 6-hydroxylase.
      ). At the same time, the F6Hs from basil and peppermint share only low protein identity of ∼31% with the F6H from soybean (
      • Latunde-Dada A.O.
      • Cabello-Hurtado F.
      • Czittrich N.
      • Didierjean L.
      • Schopfer C.
      • Hertkorn N.
      • Werck-Reichhart D.
      • Ebel J.
      Flavonoid 6-hydroxylase from soybean (Glycine max L.), a novel plant P-450 monooxygenase.
      ), which is placed in a different P450 family (CYP71; Fig. 3), indicating that their similar functions evolved independently. It is worth mentioning that the substrate preferences of CYP71D-F6H are very different from those of CYP82D-F6Hs; for example, 7-O-methylation abolishes conversion by the former enzyme, and a 5-OH group is not required for activity. Thus, the three distinct types of F6Hs studied to date at the molecular level present an interesting case of convergent evolution (
      • Pichersky E.
      • Lewinsohn E.
      Convergent evolution in plant specialized metabolism.
      ). Because the respective compounds produced by both saxifrage and soybean (
      • Yuk H.J.
      • Curtis-Long M.J.
      • Ryu H.W.
      • Jang K.C.
      • Seo W.D.
      • Kim J.Y.
      • Kang K.Y.
      • Park K.H.
      Pterocarpan profiles for soybean leaves at different growth stages and investigation of their glycosidase inhibitions.
      ,
      • Ibrahim R.K.
      A forty-year journey in plant research. Original contributions to flavonoid biochemistry.
      ) belong to different flavonoid subclasses than the basil and mint flavones, the independent origins of analogous catalytic capacities are not quite unexpected. It remains intriguing what type of catalyst mediates the 6-hydroxylation of the same flavonoid subclass, the flavones, in distantly related species and genera accumulating identical compounds, such as, for example, Tamarix, Citrus, Ononis, and others (
      • Wollenweber E.
      • Dietz V.H.
      Occurrence and distribution of free flavonoid aglycons in plants.
      ,
      • Parmar V.S.
      • Bisht K.S.
      • Sharma S.K.
      • Jain R.
      • Taneja P.
      • Singh S.
      • Simonsen O.
      • Boll P.M.
      Highly oxygenated bioactive flavones from Tamarix.
      ,
      • Wollenweber E.
      • Dörr M.
      • Rivera D.
      • Roitman J.N.
      Externally accumulated flavonoids in three Mediterranean Ononis species.
      ). So far, our BLAST searches of Citrus sinensis and Citrus clementina genomes and EST collections, as well as Helianthus annuus ESTs (using Phytozome and NCBI GenBankTM online tools) only yielded protein sequences with less than 55% identity to CYP82D33, which precludes function prediction.
      The finding that the P450-dependent flavone 6-hydroxylation in basil occurs via 7-O-methylated substrates matches well with our previous results (
      • Berim A.
      • Hyatt D.C.
      • Gang D.R.
      A set of regioselective O-methyltransferases gives rise to the complex pattern of methoxylated flavones in sweet basil.
      ). Based on these two extensive studies, we can complement our proposed pathway leading to SALV and expand it to reach out to NEV (Fig. 1). Pivotal roles in directing metabolite flux within this network are attributed to 7-OMT and flavone synthase. The properties of basil flavonoid 7-OMTs strongly suggest that API and not NAR is the early intermediate undergoing 7-O-methylation (
      • Berim A.
      • Hyatt D.C.
      • Gang D.R.
      A set of regioselective O-methyltransferases gives rise to the complex pattern of methoxylated flavones in sweet basil.
      ). Here, we show that although a flavone synthase (CYP93B23) is capable of converting SAK into GENK at low rates, the enzyme strongly prefers NAR as substrate (Fig. 5B), further validating the in vivo route via API. Because no flavone synthase activity was detected with the 6-substituted flavanones, C7Me and CdM, these compounds would not be converted into major accumulated flavones if formed by side activities of upstream enzymes. Their occurrence has not been reported for basil to date, suggesting that such parallel steps that seem possible in vitro are not physiologically significant. The detection of GARD B 7-O-demethylation lends critical support to the proposed biosynthetic pathway, because it allows for the accumulation of NEV in a pathway operating entirely on the level of 7-O-methylated intermediates. A detailed biochemical characterization of the underlying protein is necessary to advance our understanding of the intricate metabolic loop it facilitates and will help to determine the biosynthetic origin and position in the metabolic network of pilosin, a 7,8-dihydroxylated flavone occurring in basil (
      • Grayer R.J.
      • Veitch N.C.
      • Kite G.C.
      • Price A.M.
      • Kokubun T.
      Distribution of 8-oxygenated leaf-surface flavones in the genus Ocimum.
      ). So far, we could not investigate the substrate preferences of the 7-demethylase due to lack of appropriate substrate (8-hydroxylated SALV). Furthermore, the identification of the molecular data for the flavonoid 7-O-demethylase in basil will be very instructive, because it will reveal whether it is more closely related to flavonoid-pathway dioxygenases or to Papaver alkaloid demethylases. The ODDs involved in morphine biosynthesis share ∼55% identity with ODDs involved in the flavonoid pathway (flavonol synthase, flavonoid 3-hydroxylase, etc.). The basil EST database contains several highly expressed putative ODDs that also share ∼50% identity with Papaver demethylases and are ∼60% identical with some of the flavonoid-related ODDs. Remarkably, their identity with F6H from Chrysosplenium (
      • Anzellotti D.
      • Ibrahim R.K.
      Molecular characterization and functional expression of flavonol 6-hydroxylase.
      ), the only ODD known to act upon the A-ring of flavonoids, is only about 30–35%. However, heterologously expressed proteins encoded by the three most promising genes did not catalyze the 7-demethylation of GARD B (not shown). We will further pursue the isolation of the responsible gene and protein in order to fill in this missing puzzle piece in the flavone biosynthetic network.
      These unusual steps found in the flavone metabolic network in basil raise questions of whether similar processes occur in other species, which are both mechanistically and evolutionarily interesting. Because it appears that flavonoid 7-O-methylation is a prerequisite for 6-hydroxylation in these two mint species, the first question is whether this route is common in the Lamiaceae and whether it is shared by non-mints producing identical or very similar flavones, such as Alnus (
      • Wollenweber E.
      • Wassum M.
      Salvigenin und ein neues natürliches flavon aus den knospen von alnus Japonica.
      ), Rubus (
      • Wollenweber E.
      • Doerr M.
      Flavonoid aglycones from the lipophilic exudates of some species of Rosaceae.
      ), and Eupatorium (
      • Talapatra S.K.
      • Bhar D.S.
      • Talapatra B.
      Flavonoid and terpenoid constituents of Eupatorium odoratum.
      ), all of which are reported to accumulate SALV. In the Lamiaceae, phytochemical data for some groups, such as Thymus (
      • Ferreres F.
      • Barberan F.A.T.
      • Tomas F.
      5,6,4′-Trihydroxy-7,8-dimethoxyflavone from Thymus membranaceus.
      ,
      • Vandenbroucke C.O.
      • Dommisse R.A.
      • Esmans E.L.
      • Lemli J.A.
      Three methylated flavones from Thymus vulgaris.
      ), Origanum (
      • Bosabalidis A.
      • Gabrieli C.
      • Niopas I.
      Flavone aglycones in glandular hairs of Origanum x intercedens.
      ), and Orthosiphon (
      • Tezuka Y.
      • Stampoulis P.
      • Banskota A.H.
      • Awale S.
      • Tran K.Q.
      • Saiki I.
      • Kadota S.
      Constituents of the Vietnamese medicinal plant Orthosiphon stamineus.
      ), indicate that they only accumulate 6-substituted flavonoids possessing 7-O-methylated hydroxyl moieties and therefore could well follow the same pattern. Likewise, the discovery of the flavonoid 7-O-demethylation prompts an investigation into the frequency of demethylation in flavonoid metabolism. On the one hand, the occurrence or even prevalence of regiospecifically unmethylated compounds within the mint family (4′-OH in Thymus, 6-OH in Mentha) could result either from lack of OMT activity or from the corresponding demethylation, although we did not detect the latter in peppermint under the conditions tested and with GARD B as substrate (Fig. 6). On the other hand, it is even more interesting to consider whether a 7-O-demethylation is involved in the production of NEV in other species, such as H. annuus (Asteraceae) (
      • Rieseberg L.H.
      • Soltis D.E.
      • Arnold D.
      Variation and localization of flavonoid aglycones in Helianthus annuus (Compositae).
      ), Limnophila aromatica (Plantaginaceae) (
      • Bui M.L.
      • Grayer R.J.
      • Veitch N.C.
      • Kite G.C.
      • Tran H.
      • Nguyen Q.C.K.
      Uncommon 8-oxygenated flavonoids from Limnophila aromatica (Scrophulariaceae).
      ), Lysionotus pauciflorus (Gesneriaceae) (
      • Liu Y.
      • Wagner H.
      • Bauer R.
      Phenylpropanoids and flavonoid glycosides from Lysionotus pauciflorus.
      ), and Biebersteinia orphanidis (family unassigned) (
      • Greenham J.
      • Vassiliades D.D.
      • Harborne J.B.
      • Williams C.A.
      • Eagles J.
      • Grayer R.J.
      • Veitch N.C.
      A distinctive flavonoid chemistry for the anomalous genus Biebersteinia.
      ). To date, there are no data regarding the formation of lipophilic flavones in any of the above species. Our findings, together with previous results, provide a solid starting point for the elucidation of analogous processes in flavone metabolic networks across the plant kingdom.
      Finally, the identification of a very efficient F6H adds an important catalytic activity for use in biotechnological applications. Because of their potential benefits to plants' fitness and reported pharmacological activities, there is considerable interest in engineering production of flavonoids in both plants and microorganisms (
      • Wang Y.
      • Chen S.
      • Yu O.
      Metabolic engineering of flavonoids in plants and microorganisms.
      ,
      • Chemler J.A.
      • Koffas M.A.
      Metabolic engineering for plant natural product biosynthesis in microbes.
      ). Although the spectrum of compounds accessible through use of F6H is limited by its substrate specificity, many of those, such as SALV, LAD, or SCU7Me, are scarce and valuable. Tests with further potential precursors and in combination with other flavonoid-modifying enzymes, including recently isolated regiospecific flavonoid OMTs from basil (
      • Berim A.
      • Hyatt D.C.
      • Gang D.R.
      A set of regioselective O-methyltransferases gives rise to the complex pattern of methoxylated flavones in sweet basil.
      ), will be necessary to fully investigate and exploit the potential of basil F6H for the production of both natural and non-natural, novel compounds.

      Acknowledgments

      We thank Dr. R. Grayer (Kew Gardens, London, UK) for samples of authentic basil flavones, Dr. D. Nelson (University of Tennessee, Memphis, TN) for assignment of CYP designations, and the greenhouse staff of the Institute of Biological Chemistry (Pullman, WA) for raising the plants.

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