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.

Keywords: CPMV-HT transient expression; cytochrome P450 monooxygenase CYP51 family; disease resistance; neofunctionalization; terpenes.

Fig. 1.

Expression of AsbAS1 and AsCYP51H10 in N. benthamiana leaves. (A) Biosynthesis of avenacin A-1 in oat. Potential oxidation sites for AsCYP51H10 are highlighted in red. (B) CPMV-HT expression constructs and the silencing suppressor construct pBIN61-P19. Black boxes indicate transfer DNA borders; white arrows indicate the 35S promoter sequence; solid black lines indicate CPMV RNA-2 UTRs; light gray arrows indicate the coding sequence; dark gray arrows indicate terminator sequences. (C) Detection of AsbAS1 protein by immunoblot analysis. Leaf material was infiltrated with A. tumefaciens cultures containing the empty vector control (EV) or expression constructs for AsbAS1, AsCYP51H10, or AsbAS1 + AsCYP51H10. Total soluble protein was extracted from infiltrated leaf material. (Left) Coomassie blue-stained replica gel. (Right) Immunoblot analysis with polyclonal antisera raised against AsbAS1. The expected molecular mass of AsbAS1 is 87 kDa (arrow). (D) GC-MS analysis of extracts from infiltrated N. benthamiana leaves. Total ion chromatograms (TICs) and extracted ion chromatograms (EICs) at an m/z of 218 (EIC 218) and 189 (EIC 189) are shown. Accumulation of β-amyrin was detected in AsbAS1-expressing leaves. Coexpression of AsbAS1 and AsCYP51H10 resulted in lower levels of a β-amyrin and accumulation of a new peak [retention time (R_t) =19.1 min]. B, betulin (internal standard; R_t =18.7 min). The unlabeled peaks are sterols. Data are representative of at least three separate expression experiments. Corresponding chromatograms are drawn at the same scale, as indicated in the top left corners.

Fig. 2.

Identification of the product generated by coexpression of AsbAS1 and AsCYP51H10. (A) Automated mass spectral deconvolution and identification system-extracted ion component spectra of the trimethylsilyated compound at the indicated retention time (RT). The compound has a predicted molecular ion at m/z = 602 that is consistent with the molecular equation C36H66O3Si2. The signal at m/z = 512 is consistent with the loss of C3H9OSi, resulting in the molecular equation C30H56O2Si. (B) Calculated m/z values for β-amyrin derivates, considering trimethylsilyl (TMS) derivatization (+72) and introduction of oxygen atoms (+16). (C) Structure, chemical formula, and molecular mass of 12,13β-epoxy-3β,16β-dihydroxy-oleanane (12,13β-epoxy-16β-hydroxy-β-amyrin). TMS-derivatized 12,13β-epoxy-16β-hydroxy-β-amyrin has a predicted molecular ion at m/z = 602. Potential intermediates in the synthesis of 12,13β-epoxy-16β-hydroxy-β-amyrin from β-amyrin are shown also.

Fig. 3.

Potential precatalytic binding modes of β-amyrin and 16-hydroxy β-amyrin. (A) Alignment of sequences in substrate recognition sites (38) of representative members of CYP51 subfamilies. Sequences shown are those of human lanosterol 14α-demethylase (HsCYP51A1, sequence database entry U23942); Mycobacterium tuberculosis (MtCYP51B1, P0A512); Trypanosoma brucei brucei lanosterol 14α-demethylase (TbCYP51E1, AF363026); Saccharomyces cerevisiae lanosterol 14α-demethylase (ScCYP51F1, M18109); Avena strigosa obtusifoliol 14α-demethylase (AsCYP51G1, DQ680850), and Avena strigosa AsCYP51H10 (DQ680852) (see Fig. S4 for full sequence alignment). Residues that are predicted to line the active site in AsCYP51H10 and that interact with the substrate in either or both precatalytic binding modes are indicated by black triangles. Residues identified in sad2 mutants of A. strigosa that are defective in avenacin synthesis (15) are indicated by red triangles. Residue Thr113 falls into both categories and is indicated by a blue triangle. SRS3 is omitted because no residues from this site in AsCYP51H10 are predicted to interact with the substrate. Absolutely conserved residues are shown in white with a red background. Residues that are conserved in other families but differ in AsCYP51H10 have an orange background. (B and C) Putative precatalytic binding modes of (B) β-amyrin and (C) 16-hydroxy β-amyrin in the active site of AsCYP51H10. Carbon atoms of the substrates are colored yellow, and oxygen is shown in red. The molecular surfaces of the substrates are shown. The heme iron distal ligand [FeO]³⁺ is shown as a red sphere (partly obscured by residue F287). Heme carbon atoms are colored cyan. The coloring of residue labels follows that of A. (D and E) The distance from the heme iron distal ligand [FeO]³⁺ to the potential site of substrate modification as monitored during 1-ns molecular dynamics simulations of (D) β-amyrin (distance to C16 monitored) and (E) 16β-hydroxy β-amyrin (distance to C12 monitored). Distances of 5 Å or less (horizontal dashed line) between the metabolized carbon atoms and the heme iron oxygen ligand indicate a possible oxidation event, and the corresponding binding modes (shown in B and C) are classified as precatalytic.

Fig. 4.

The C12/C13 epoxide is important for antifungal activity. (A) Structures of avenacin A-1 and the 12-oxo-avenacin A-1 derivative (OA). (B) Disk assays for antifungal activity. The avenacin-sensitive fungus G. graminis var. tritici was grown in presence of avenacin A-1 (A-1) and 12-oxo-avenacin A-1 (OA). The amounts of compound applied to the discs are indicated. The control disk (c) was treated with 75% methanol only. A concentration-dependent zone of inhibition can be observed for avenacin A-1 but not for the modified compound.

Fig. 5.

Examples of microbial multifunctional P450s catalyzing epoxidations. (A) MycG, a P450 involved in mycinamicin biosynthesis in the actinomycete, Micromonspora griseorubida. MycG catalyzes hydroxylation of mycinamycin IV (M-IV) followed by epoxidation of mycinamycin V (M-V) to mycinamycin II (M-II). (B) GfsF is required for biosynthesis of the antibiotic FD-891 in Streptomyces. GfsF first catalyzes the epoxidation of 25-O-methyl FD-892 and then the hydroxylation, yielding FD-891. (C) TamI is required for tirandamycin (Tir) biosynthesis in Streptomyces and catalyzes the hydroxylation of TirC to TirE. TirE is further modified to TirD by the flavoprotein TamL. Subsequently TamI catalyzed epoxidation and hydroxylation of TirD to form TirA and TirB. (D) The P450 Tri4 is involved in four successive oxygenation reactions during trichothecene synthesis in Fusarium. After the trichodiene formation, Tri4 catalyzes hydroxylation at C2, epoxidation at C12/13, hydroxylation at C11, and hydroxylation at C3.