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

Abstract

Members of the cytochromes P450 superfamily (P450s) catalyze a huge variety of oxidation reactions in microbes and higher organisms. Most P450 families are highly divergent, but in contrast the cytochrome P450 14α-sterol demethylase (CYP51) family is one of the most ancient and conserved, catalyzing sterol 14α-demethylase reactions required for essential sterol synthesis across the fungal, animal, and plant kingdoms. Oats (Avena spp.) produce antimicrobial compounds, avenacins, that provide protection against disease. Avenacins are synthesized from the simple triterpene, β-amyrin. Previously we identified a gene encoding a member of the CYP51 family of cytochromes P450, AsCyp51H10 (also known as Saponin-deficient 2, Sad2), that is required for avenacin synthesis in a forward screen for avenacin-deficient oat mutants. sad2 mutants accumulate β-amyrin, suggesting that they are blocked early in the pathway. Here, using a transient plant expression system, we show that AsCYP51H10 is a multifunctional P450 capable of modifying both the C and D rings of the pentacyclic triterpene scaffold to give 12,13β-epoxy-3β,16β-dihydroxy-oleanane (12,13β-epoxy-16β-hydroxy-β-amyrin). Molecular modeling and docking experiments indicate that C16 hydroxylation is likely to precede C12,13 epoxidation. Our computational modeling, in combination with analysis of a suite of sad2 mutants, provides insights into the unusual catalytic behavior of AsCYP51H10 and its active site mutants. Fungal bioassays show that the C12,13 epoxy group is an important determinant of antifungal activity. Accordingly, the oat AsCYP51H10 enzyme has been recruited from primary metabolism and has acquired a different function compared to other characterized members of the plant CYP51 family-as a multifunctional stereo-and regio-specific hydroxylase in plant specialized metabolism.

Unearthing a sesterterpene biosynthetic repertoire in the Brassicaceae through genome mining reveals convergent evolution

“…Compounds 2-8 were then isolated for structural elucidation. To isolate compounds 2-9 for structural elucidation, we scaled up the transient expression of these different GFPPS-TPS pairs in N. benthamiana (34). Following extraction, repeated chromatography, and recrystallization, we obtained quantities of pure compounds 2 (102.0 mg), 3 (13.0 mg), 4 (3.7 mg), 5 (3.5 mg), 6 (4.2 mg), 7 (21.3 mg), 8 (17.3 mg), and 9 (2.1 mg) for collecting full 1D and 2D NMR datasets (Dataset S1).…”

Combinatorial biosynthesis of sapogenins and saponins in Saccharomyces cerevisiae using a C-16α hydroxylase from Bupleurum falcatum

“…Through transcript profiling of MeJA-treated B. falcatum roots, we identified CYP716Y1, encoding a P450 that is transcriptionally coregulated with known triterpene saponin biosynthetic genes and that catalyzes the C-16α hydroxylation of amyrin, a catalytic activity previously not linked to any gene product in the plant kingdom. Hence, the P450 compendium that can oxidize the amyrin backbone is expanded, now covering seven positions on the triterpene backbone (11)(12)(13)(14)(15)(16)(17)(18) and including two enzymes that catalyze hydroxylation at C-16, in β-(CYP51H10) (12) and α-configuration (CYP716Y1). Like many reported P450s (15,17), CYP716Y1 can hydroxylate α-and β-amyrin, both pentacyclic triterpenes.…”

“…P450s that modify the β-amyrin backbone on C-11; C-12,13; C-16; C-22; C-23; C-28 or C-30 have been characterized from Glycyrrhiza uralensis, Avena strigosa, Medicago truncatula, Glycine max, Vitis vinifera, and Catharanthus roseus (11)(12)(13)(14)(15)(16)(17)(18). Hydroxylases from Panax ginseng that oxidize the dammarenediol-II backbone on C-6, C-12, or C-28 (19)(20)(21), and a C-20 hydroxylase from Lotus japonicus (22) that modifies lupeol, have also been identified.…”

A conserved amino acid residue critical for product and substrate specificity in plant triterpene synthases

“…Root tips were ground in protein extraction buffer [50 mM Tris·HCl pH 7.5, 150 mM NaCl, 5 mM EDTA, 10% (vol/vol) glycerol, 1% (wt/vol) PVPP, 1% (vol/vol) Triton X-100 (Boehringer Mannheim), 1× Complete protease inhibitor (Roche)] for 1 min with a plastic pestle followed by incubation at 4°C for 2 h. Proteins were denatured, separated on NuPAGE gels (4-12% acrylamide gradient) (Invitrogen), and blotted onto nitrocellulose membranes (Bio-Rad) by using the manufacturer's protocol. Membranes were probed with anti-SAD1 antisera (1:10,000 dilution) (16) followed by detection with a goat anti-rat IgG horseradish peroxidaselabeled secondary antibody (Sigma-Aldrich) according to the manufacturer's protocol.…”

“…The preparations were then incubated on ice for 2 h and then centrifuged at 21,130 × g for 20 min. The supernatants were used for protein and Western blot analysis as described previously (16).…”