Lamiaceae

Bathe, Ulschan & Tissier, Alain, 2019, Cytochrome P 450 enzymes: A driving force of plant diterpene diversity, Phytochemistry 161, pp. 149-162 : 153-155

publication ID	https://doi.org/10.1016/j.phytochem.2018.12.003
persistent identifier	https://treatment.plazi.org/id/039987DD-FFCA-5047-FCB4-E1248155A4CE
treatment provided by	Felipe
scientific name	Lamiaceae
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Treatment

4.1. The CYP 76 family is a dominant catalyzer of labdane diterpene diversification in Lamiaceae View in CoL View at ENA

The Lamiaceae include more than 7800 species (www.theplantlist. org) many of which are used as spices or as medicinal plants ( Kokkini et al., 2003). Prominent examples are Rosmarinus officinalis , Salvia sp. or Coleus forskohlii which contain compounds of industrial relevance such as carnosic acid, carnosol, sclareol and forskolin, respectively ( Follin-Arbelet et al., 2015; Ibarra et al., 2011; Johnson, 2011). As fragrances, drug components or food additives – metabolites from Lamiaceae species have been of great interest for decades. Labdane-related diterpenoids, which can be found in more than 20 genera ( Vestri Alvarenga et al., 2001), contribute significantly to the interest in this plant family. A survey of diterpenes present in Lamiaceae predicted 91 different backbones ( Vestri Alvarenga et al., 2001). Knowing that each of these skeletons can be further oxidized at several positions, in different combinations, and that additional modifications such as acylation and glycosylation are possible, it is likely that the number of diterpenoids in Lamiaceae alone is close to or even in excess of 1000. In this structural diversification, CYPs of the CYP 76 family, and to a lesser extent from the CYP 71 family, play a dominant role in the oxidation of diterpenes in the Lamiaceae ( Fig. 3A View Fig , Table 1 View Table 1 ). The last five years have seen the characterization of CYPs from rosemary, sage species and C. forskohlii that participate in the biosynthesis of phenolic diterpenes, related tanshinones and the manoyl oxide derived forskolin, respectively. Most of them belong to the two distinct but closely related subgroups, CYP 76AH and CYP 76AK ( Fig. 2 View Fig ). The former are rather involved in the oxidation of olefinic or alcohol diterpenes. For example, CYP 76AH1 is a ferruginol synthase from Salvia miltiorrhiza which has been co-expressed with the required diTPS and cytochrome P450 reductase (SmCPR) in the model organism Saccharomyces cerevisiae to reconstruct ferruginol biosynthesis ( Guo et al., 2013). This step was proposed to be the first committed step in tanshinone biosynthesis in danshen ( S. miltiorrhiza ). CYP 76AH1 RNAi lines of hairy root cultures of S. miltiorrhiza exhibited accumulation of the precursor miltiradiene in parallel to a reduction of downstream tanshinones proving the role of ferruginol as starting point for further decorations in tanshinone biosynthesis ( Ma et al., 2015, 2016). Recent studies provided more insights into tanshinone biosynthesis by the characterization of SmCYP76AH3, which oxidizes ferruginol to 11-hydroxy ferruginol. The authors also demonstrated the promiscuity of these CYPs, which can oxidize their substrates at C 7, C 11 and C 12, leading to a spectrum of products ( Guo et al., 2016). The final steps in tanshinone biosynthesis have not been clarified so far but presumably require other types of reactions, C-C cleavage and decarboxylation, which may or may not be catalyzed by CYPs. Alternatively, it is conceivable that different enzyme families such as 2-oxoglutarate-dependent dioxygenases (2-ODD) participate in the downstream pathway as it can be inferred from 2-ODD RNAi lines of hairy root cultures which displayed reduced tanshinone content ( Xu and Song, 2017).

Species closely related to S. miltiorrhiza such as Salvia fruticosa , Salvia pomifera and R. officinalis also produce phenolic diterpenes. There, RoCYP76AH4, RoCYP76AH22-23 and Sf/SpCYP76AH24 are highly similar to SmCYP76AH1, but carry out two successive oxidations on the substrate miltiradiene/abietatriene at two different positions on the C-ring, leading to the synthesis of 11-hydroxy ferruginol (Bo ž i ć et al., 2015; Ignea et al., 2016; Scheler et al., 2016; Zi and Peters, 2013). Interestingly, these 11-hydroxy ferruginol synthases accept not only miltiradiene/abietatriene as substrate but also manoyl oxide, pointing to a certain degree of promiscuity of these enzymes not only with regards to the position but also to the diterpene substrate ( Pateraki et al., 2017).

Downstream of the hydroxy ferruginol synthases ( CYP 76AHs), four enzymes from Salvia sp. and R. officinalis of the CYP 76AK subfamily have been identified. They were collectively named C 20 oxidases because they oxidize their substrates at position C 20 ( Guo et al., 2016; Ignea et al., 2016; Scheler et al., 2016). Like CYP 76AHs, CYP 76AKs are promiscuous and therefore form a variety of metabolites. For example, Sf/SpCYP76AK6 and RoCYP76AK7-8 are involved in the biosynthesis of carnosic acid in R. officinalis , S. fruticosa and S. pomifera and catalyze three successive oxidations at C 20 on 11-hydroxy ferruginol to carnosic acid via the alcohol and the aldehyde (carnosaldehyde) as intermediates. Although, C 20 oxidases also have activity on ferruginol, yielding pisiferol, pisiferal and pisiferic acid, we showed that CYP 76AK8 favors 11-hydroxy ferruginol instead of ferruginol ( Scheler et al., 2016).

Next to C 20 oxidases from the carnosic acid pathway, there is one characterized C 20 oxidase from S. miltiorrhiza, SmCYP 76AK1. It shares high sequence similarity with CYP 76AK6-8 (more than 68% on protein level) and is a promiscuous CYP that accepts several substrates and is proposed to be involved in tanshinone biosynthesis. SmCYP76AK1 introduces a single hydroxyl group at position C 20, unlike the C 20 oxidase involved in the biosynthesis of carnosic acid. This results in the production of 11,20-dihydroxy sugiol and 11,20-dihydroxy ferruginol, the latter spontaneously oxidizing to 10-hydroxymethyltetrahydromiltirone ( Guo et al., 2016). Spontaneous oxidation has also been shown for other phenolic diterpenes such as carnosic acid to carnosol and 11-hydroxy ferruginol to the corresponding quinone, which is a manifestation of the high antioxidant activities of these compounds ( Loussouarn et al., 2017; Scheler et al., 2016). It is still not clear whether the in planta conversion of carnosic acid to carnosol occurs via spontaneous oxidation or enzymatically.

Forskolin is a diterpene from C. forskohlii which has been long known for its positive effect on glaucoma, as inotrope and because of its vasodilatory activities ( Bristow et al., 1984; Caprioli et al., 1984; Wysham et al., 1986). Its biosynthesis has recently been clarified by Pateraki and co-workers including the characterization of involved CYPs and an acetyl transferase. Interestingly, as in the biosynthesis of carnosic acid, CYPs from the CYP 76AH subfamily, namely CfCYP76AH11 and CfCYP76AH15-16, are involved and catalyze five successive oxidations on manoyl oxide. CfCYP76AH15 was found to have activity on miltiradiene to form ferruginol , while CYP 76AH4 and CYP 76AH22-24 from rosemary and sage accepted manoyl oxide as substrate, providing nice examples of the promiscuity of this enzyme family ( Pateraki et al., 2017).

CYPs from the CYP 71 family were until now more known for their activities in mono- and sesquiterpene biosynthesis ( Mau and Croteau, 2006). In labdane-related diterpene biosynthesis, only two CYP 71s were found to be involved. CYP 71BE52 from S. pomifera and CYP 71D381 from C. forskohlii have hydroxylation activity on position C 2 of ferruginol and manoyl oxide, respectively ( Ignea et al., 2016). In addition, CfCYP71D381 hydroxylates at position C 19 ( Ignea et al., 2016; Pateraki et al., 2017). Interestingly, these CYPs share only 49% amino acid sequence similarity but can be collectively considered as C 2 oxidases.

Most recently, CYP 76BK1 from Vitex agnus-castus has been reported to oxidize peregrinol, a bicyclic diterpene, at position C 16 to labd-13Z- ene-9,15,16-triol. This compound is presumed to be an intermediate en route to furan- and lactone-containing diterpenoids ( Heskes et al., 2018). This constitutes yet another addition of a diterpene CYP to the CYP 76 family from Lamiaceae , further supporting the important role of this CYP family in diterpene diversification in Lamiaceae .

In Arabidopsis CYP 76 enzymes have been shown to be involved in the oxidation of monoterpenes ( Boachon et al., 2015; Hofer et al., 2014). Also, CYP 76 enzymes exist in Gymnosperms ( Nelson and Werck-Reichhart, 2011), and it remains to be seen whether in these species as well they are involved in the biosynthesis of similar compounds, such as pisiferic acid, a ferruginol derived carboxylic acid, from Chamaecyparis pisifera ( Kobayashi et al., 1987) .