Medicago truncatula CYP716A12 Is a Multifunctional Oxidase Involved in the Biosynthesis of Hemolytic Saponins

Abstract

Saponins, a group of glycosidic compounds present in several plant species, have aglycone moieties that are formed using triterpenoid or steroidal skeletons. In spite of their importance as antimicrobial compounds and their possible benefits for human health, knowledge of the genetic control of saponin biosynthesis is still poorly understood. In the Medicago genus, the hemolytic activity of saponins is related to the nature of their aglycone moieties. We have identified a cytochrome P450 gene (CYP716A12) involved in saponin synthesis in Medicago truncatula using a combined genetic and biochemical approach. Genetic loss-of-function analysis and complementation studies showed that CYP716A12 is responsible for an early step in the saponin biosynthetic pathway. Mutants in CYP716A12 were unable to produce hemolytic saponins and only synthetized soyasaponins, and were thus named lacking hemolytic activity (lha). In vitro enzymatic activity assays indicate that CYP716A12 catalyzes the oxidation of b-amyrin and erythrodiol at the C-28 position, yielding oleanolic acid. Transcriptome changes in the lha mutant showed a modulation in the main steps of triterpenic saponin biosynthetic pathway: squalene cyclization, b-amyrin oxidation, and glycosylation. The analysis of CYP716A12 expression in planta is reported together with the sapogenin content in different tissues and stages. This article provides evidence for CYP716A12 being a key gene in hemolytic saponin biosynthesis.

Biochemical analysis of a multifunctional cytochrome P450 (CYP51) enzyme required for synthesis of antimicrobial triterpenes in plants

“…Our experiments demonstrate that the CPMV-HT expression system provides a rapid and effective means of expressing enzymes for the synthesis of low molecular weight specialized compounds alone and in combination. Previous work on triterpene biosynthetic enzymes by other groups has focused on yeast as a heterologous expression system (24)(25)(26)(27)(28). Here, we have used the CPMV-HT system for the synthesis and modification of the simple triterpene β-amyrin, thereby reconstructing the first two committed steps in the avenacin biosynthetic pathway.…”

“…Several triterpene-modifying P450s from eudicots have been characterized recently by heterologous expression in yeast. These include CYP93E1 from soybean, which hydroxylates β-amyrin and sophoradiol at position C24 (24); two P450s from liquorice, one (CYP88D6) that converts β-amyrin to 11-oxo-β-amyrin and a second (CYP72A154) that converts 11-oxo-β-amyrin to glycyrrhizin acid (25,26); and CYP716A12 from Medicago truncatula, a β-amyrin 28-oxidase that converts β-amyrin to oleanolic acid (27,28). These enzymes belong to different P450 families, indicating that the ability to oxygenate β-amyrin has arisen multiple times during evolution.…”

Plant P450s as versatile drivers for evolution of species-specific chemical diversity

“…It is, therefore, not surprising that this clan has been used for recruitment of P450s for biosynthesis of saponins. Recently, a number of papers have been published on the roles of CYP716As in particular saponin biosynthesis [69][70][71]. Medicago truncatula CYP716A12 catalyses three sequential oxidation of b-amyrin at the C-28 position, yielding oleanolic acid in biosynthesis of haemolytic, but not soyasaponins, in M. truncatula [69].…”

“…Recently, a number of papers have been published on the roles of CYP716As in particular saponin biosynthesis [69][70][71]. Medicago truncatula CYP716A12 catalyses three sequential oxidation of b-amyrin at the C-28 position, yielding oleanolic acid in biosynthesis of haemolytic, but not soyasaponins, in M. truncatula [69]. Erythrodiol (C-28 hydroxylated b-amyrin) is similarly converted to oleanolic acid by CYP716A12 catalysing sequential hydroxylations from the alcohol to the aldehyde and further on to the carboxylic acid.…”

OSC2 and CYP716A14v2 Catalyze the Biosynthesis of Triterpenoids for the Cuticle of Aerial Organs of Artemisia annua

“…To date, nine CYP716 family proteins with oxidase activity on a-amyrin, b-amyrin, lupeol, or dammarenediol have been identified. CYP716A12 from Medicago truncatula (Carelli et al, 2011), CYP716A15 and CYP716A17 from Vitis vinifera (Fukushima et al, 2011), CYP716AL1 from C. roseus (Huang et al, 2012), CYP716A52v2 from P. ginseng , and CYP716A75 from Maesa lanceolata (Moses et al, 2015) are C-28 oxidases of a-amyrin, b-amyrin, and lupeol. CYP716Y1 from Bupleurum falcatum is a C-16a oxidase of a-amyrin and b-amyrin (Moses et al, 2014), and CYP716A47 and CYP716A53v2 from P. ginseng are C-12 and C-6 oxidases of dammaranes, respectively (Han et al, 2011(Han et al, , 2012.…”