Novel triterpene oxidizing activity of Arabidopsis thaliana CYP716A subfamily enzymes

Abstract

Edited by Ulf-Ingo Fl€ uggeTriterpenoids have diverse chemical structures and bioactivities. Cytochrome P450 monooxygenases play a key role in their structural diversification. In higher plants, CYP716A subfamily enzymes are triterpene oxidases. In this study, Arabidopsis thaliana CYP716A1 and CYP716A2 were characterized by heterologously expressing them in simple triterpene-producing yeast strains. In contrast to the C-28 oxidative activity of CYP716A1 shown in several CYP716A subfamily enzymes, remarkably, CYP716A2 displayed 22a-hydroxylation activity against a-amyrin that has not been previously reported, which produces the cytotoxic triterpenoid, 22a-hydroxy-a-amyrin. Our results contribute to the enrichment of the molecular toolbox that allows for the combinatorial biosynthesis of diverse triterpenoids.Keywords: 22a-hydroxylation; Arabidopsis thaliana; combinatorial biosynthesis; CYP716A; cytochrome P450 monooxygenase; triterpenoids HighlightsThe full-length CDS of CYP716A2, miss-annotated on TAIR, is successfully cloned. Novel 22a-hydroxylation activity of CYP716A subfamily enzymes is reported. The chemical structure of 22a-hydroxy-a-amyrin is confirmed by NMR analysis.Triterpenoids represent one of the largest groups of plant specialized metabolites and are composed of six C5 (isopentenyl diphosphate) units [7]. Their carbon skeletons are synthesized from the common precursor 2,3-oxidosqualene by oxidosqualene cyclases (OSCs), and these skeletons are further oxidized by cytochrome P450 monooxygenases (P450s) and glycosylated by UDP-dependent glycosyltransferases (UGTs), which generate diverse triterpenoid/triterpenoid saponin structures [17,19]. Around 80 OSCs, 30 P450s, and 10UGTs are reportedly involved in triterpenoid biosynthesis in plants [22,25]. Among them, P450s have been described as the key players in diversifying triterpenoid scaffolds, and were recently identified in many triterpenoid-producing plant species [22]. For example, CYP88D6 and CYP72A154 from Glycyrrhiza uralensis were identified as components in the biosynthesis of glycyrrhetinic acid, the aglycone of glycyrrhizin [20,21]. In addition, CYP716A12 and other CYP716A subfamily enzymes have been identified as multifunc-

Integrating pathway elucidation with yeast engineering to produce polpunonic acid the precursor of the anti-obesity agent celastrol

“…CYP712K1 appears to produce higher absolute amounts of polpunonic acid and exhibits a higher polpunonic acid to 29-hydroxy-friedelin ratio than CYP712K2 or CYP712K3. This type of differences in the efficacy of conversion of the intermediate alcohols by P450s has previously also been observed in the case of α-amyrin, β-amyrin and lupeol C-28-oxidizing CYP716As [45,46,66].…”

Two CYP716A subfamily cytochrome P450 monooxygenases of sweet basil play similar but nonredundant roles in ursane‐ and oleanane‐type pentacyclic triterpene biosynthesis

“…1; Misra et al, 2014). Subsequently, amyrin C-28 oxidase (cytochrome P450 monooxygenase (CYP450) family member)mediated oxidation of a-amyrin and b-amyrin skeletons at the C-28 position, then leads to the formation of ursolic acid and oleanolic acid, respectively (Moses et al, 2013;Seki et al, 2015;Yasumoto et al, 2016). Previously, we have reported a b-amyrin synthase (ObAS1) and a mixed amyrin synthase (ObAS2), having product preference for a-amyrin (Misra et al, 2014).…”

Transcriptomic Comparison Reveals Candidate Genes for Triterpenoid Biosynthesis in Two Closely Related Ilex Species

“…CYP family is one of the largest and most diverse gene families in plants. Up to now, more than 20 CYPs were identified in pentacyclic triterpenoids biosynthesis (Shibuya et al, 2006; Seki et al, 2008; Huang et al, 2012; Fukushima et al, 2013; Geisler et al, 2013; Guo et al, 2013; Han et al, 2013; Moses et al, 2014a,b; Moses et al, 2015a,b; Yasumoto et al, 2016). Phylogenetic analysis of these CYPs was showed in Figure S9, which suggests that the members in CYP71 clan may act as C24 oxidase, while in CYP72 clan and CYP85 clan may exist multi-functional enzymes in pentacyclic triterpenoids biosynthesis.…”