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CYP51) Enzyme Required for Synthesis of Antimicrobial Triterpenes in Plants

CYP51) Enzyme Required for Synthesis of Antimicrobial Triterpenes in Plants

Biochemical analysis of a multifunctional PNAS PLUS P450 (CYP51) required for synthesis of antimicrobial triterpenes in

Katrin Geislera,b, Richard K. Hughesc, Frank Sainsburyc,1, George P. Lomonossoff c, Martin Rejzekc, Shirley Fairhurstc, Carl-Erik Olsenb, Mohammed Saddik Motawiab, Rachel E. Meltona, Andrew M. Hemmingsd,e, Søren Bakb, and Anne Osbourna,2

Departments of aMetabolic Biology and cBiological Chemistry, John Innes Centre, Norwich NR4 7UH, United ; bDepartment of and Environmental Sciences, VKR Research Centre Pro-Active Plants, Faculty of Sciences, University of Copenhagen, Thorvaldsensvej 40, DK-1871 Frederiksberg C, Copenhagen, Denmark; and Schools of dChemistry and eBiological Sciences, University of East Anglia, Norwich NR4 7TJ, United Kingdom

Edited by Joseph R. Ecker, The Salk Institute, La Jolla, CA, and approved July 18, 2013 (received for review May 15, 2013) Members of the P450 superfamily (P450s) catalyze with sterol 14α- activity are known as obtusifoliol a huge variety of oxidation reactions in microbes and higher 14α- and constitute the CYP51G subfamily (7). organisms. Most P450 families are highly divergent, but in contrast The triterpenes are one of the largest classes of plant-derived the 14α-sterol demethylase (CYP51) family is one natural products. Previously we reported the discovery of a of the most ancient and conserved, catalyzing sterol 14α-demethy- encoding a divergent plant CYP51 (AsCyp51H10) that is dis- lase reactions required for essential sterol synthesis across the pensable for the synthesis of essential sterols but is required for fungal, , and plant kingdoms. Oats (Avena spp.) produce production of specialized antimicrobial triterpene glycosides antimicrobial compounds, avenacins, that provide protection known as avenacins that confer disease resistance in oats (15). against disease. Avenacins are synthesized from the simple triter- AsCyp51H10 (also known as Saponin-deficient 2 or Sad2) was β fi pene, -amyrin. Previously we identi ed a gene encoding a mem- first identified in a screen for mutants of diploid oat that were ber of the CYP51 family of cytochromes P450, AsCyp51H10 (also

unable to make avenacins (16). Subsequent analysis revealed PLANT BIOLOGY known as Saponin-deficient 2, Sad2), that is required for avenacin fi that this gene forms part of a metabolic gene cluster for avenacin synthesis in a forward screen for avenacin-de cient oat mutants. synthesis (15). AsCYP51H10 (SAD2) belongs to a newly defined sad2 mutants accumulate β-amyrin, suggesting that they are and as yet functionally uncharacterized subfamily of CYP51 blocked early in the pathway. Here, using a transient plant expres- , the CYP51H subfamily, which also includes nine sion system, we show that AsCYP51H10 is a multifunctional P450 fi capable of modifying both the C and D rings of the pentacyclic members of unknown function from rice (7, 15, 17). The rst β β β committed step in the synthesis of avenacins is the cyclization of triterpene scaffold to give 12,13 -epoxy-3 ,16 -dihydroxy-olea- β β nane (12,13β-epoxy-16β-hydroxy-β-amyrin). Molecular modeling 2,3-oxidosqualene to -amyrin, catalyzed by the oat -amyrin and docking experiments indicate that C16 is likely synthase AsbAS1 (also known as SAD1) (18, 19). Biochemical β to precede C12,13 epoxidation. Our computational modeling, in analysis has shown that sad2 mutants accumulate -amyrin, β combination with analysis of a suite of sad2 mutants, provides suggesting that -amyrin may be the for AsCYP51H10 insights into the unusual catalytic behavior of AsCYP51H10 and (15, 20). Partial characterization of AsCYP51H10 in is its mutants. Fungal bioassays show that the C12,13 consistent with this observation (21). However, full character- epoxy group is an important determinant of activity. Accordingly, the oat AsCYP51H10 enzyme has been recruited from Significance primary and has acquired a different function compared — to other characterized members of the plant CYP51 family as We carried out functional analysis of the oat enzyme AsCYP51H10, fi a multifunctional stereo- and regio-speci c hydroxylase in plant which is a divergent member of the CYP51 cytochrome P450 specialized metabolism. family and showed that this enzyme is able to catalyze both hydroxylation and epoxidation of the simple triterpene β- CPMV-HT transient expression | cytochrome P450 CYP51 amyrin to give 12,13β-epoxy-3β,16β-dihydroxy-oleanane (12,13β- family | disease resistance | neofunctionalization | epoxy-16β-hydroxy-β-amyrin). In contrast, the canonical CYP51 enzymes are highly conserved and catalyze only sterol deme- igher plants produce a huge array of low molecular weight thylation. We further show that the C12,13 epoxy group is Hspecialized compounds (natural products) that have im- critical for antifungal activity, a discovery that has important portant functions in biotic and abiotic stress tolerance (1, 2) and implications for triterpene metabolic engineering for food, that also provide a matchless starting point for and agro- health, and industrial biotechnology applications. chemical discovery (3). The cytochrome P450 (P450) superfamily is the largest family of plant metabolic enzymes. The majority of Author contributions: K.G., A.M.H., S.B., and A.E.O. designed research; K.G., R.K.H., S.F., fl fi C.-E.O., M.S.M., R.E.M., and A.M.H. performed research; K.G., F.S., G.P.L., and R.E.M. plant P450 families are highly divergent, re ecting diversi cation contributed new reagents/analytic tools; K.G., M.R., S.F., C.-E.O., M.S.M., R.E.M., A.M.H., and neofunctionalization as new metabolic pathways evolve (4– S.B., and A.E.O. analyzed data; and K.G., G.P.L., M.R., A.M.H., S.B., and A.E.O. wrote 6). In contrast, the cytochrome P450 14α-sterol demethylase the paper. (CYP51) family is one of the most ancient of the P450 families, Conflict of interest statement: A.E.O. is a co-inventor on a patent filing on AsCYP51H10. and the function of CYP51 enzymes is highly conserved across This article is a PNAS Direct Submission. fungi, plants, and (7, 8). These enzymes are sterol Freely available online through the PNAS open access option. – demethylases required for the synthesis of essential sterols (9 1Present address: Australian Institute for Bioengineering and Nanotechnology, University 14). Although different sterol substrates are used (e.g., of Queensland, St Lucia QLD 4072, Australia. in mammals and yeast and obtusifoliol in plants), the reaction 2To whom correspondence should be addressed. E-mail: [email protected]. — α mechanism 14 - and subsequent formation of This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. a Δ14–15 double bond—is preserved. In plants, CYP51 enzymes 1073/pnas.1309157110/-/DCSupplemental.

www.pnas.org/cgi/doi/10.1073/pnas.1309157110 PNAS Early Edition | 1of8 Downloaded by guest on September 30, 2021 ization of the biochemical function of AsCYP51H10 has not yet the AsbAS1 and AsCYP51H10 constructs. Leaf tissue was har- been carried out. vested after 6 d, and extracts were analyzed by immu- Triterpenes have a wide range of commercial applications as noblot analysis using polyclonal antisera raised against AsbAS1 agrochemicals, food additives, and pharmaceuticals and as (attempts to raise antisera specific for AsCYP51H10 were un- foaming agents in the beverage, food, and cosmetics industries successful). AsbAS1 protein was readily detectable in protein (22). Commercial exploitation of triterpenes has been limited extracts from leaves infiltrated with the AsbAS1 construct alone thus far by their recalcitrance to synthetic chemistry and their or the AsbAS1 and AsCYP51H10 constructs together, as expec- occurrence in low abundance in complex mixtures in plants (23). ted (Fig. 1C). The triterpene content of infiltrated leaf material The availability of enzymes that can stereo- and regio-specifically then was analyzed using GC-MS. The total ion chromatogram functionalize triterpene scaffolds will open opportunities for the (TIC) from AsbAS1-infiltrated leaf extracts revealed a peak with production of novel triterpenes using synthetic biology ap- a retention time of 17.8 min that was not observed in the empty proaches. Several triterpene-modifying P450s from eudicots have vector control (Fig. 1D). The fragmentation pattern of this peak been characterized recently by heterologous expression in yeast. was identical to that of β-amyrin (Fig. S1A). β-Amyrin was not These include CYP93E1 from soybean, which hydroxylates detected in the empty vector-treated control leaves or leaves that β-amyrin and sophoradiol at position C24 (24); two P450s from had been infiltrated with the AsCYP51H10 construct alone (Fig. liquorice, one (CYP88D6) that converts β-amyrinto11-oxo- 1D). Coexpression of AsbAS1 and AsCYP51H10 resulted in β-amyrin and a second (CYP72A154) that converts 11-oxo- a substantial reduction in β-amyrin and the appearance of a new β-amyrin to glycyrrhizin acid (25, 26); and CYP716A12 from peak with a retention time of 19.1 min (Fig. 1D), indicating that Medicago truncatula,aβ-amyrin 28- that converts β-amyrin AsCYP51H10 is able to modify β-amyrin. to oleanolic acid (27, 28). These enzymes belong to different P450 families, indicating that the ability to oxygenate β-amyrin Identification of the AsCYP51H10 Product. The extracted ionization has arisen multiple times during evolution. spectrum for the new peak revealed a molecular ion at m/z 602 Here we show that AsCYP51H10 is a multifunctional CYP51 (retention time = 19.08 min; Fig. 2A). Given that we analyzed that is able to convert β-amyrin to a product that we determined trimethylsilyl (TMS)-derivatized extracts and the AsCYP51H10 by NMR spectrometry (NMR) to be 12,13β-epoxy-3β,16β-dihy- product is derived from β-amyrin, a mass of 602 is consistent with droxy-oleanane (12,13β-epoxy-16β-hydroxy-β-amyrin). Molecular the molecular formula C36H66O3Si2. This result suggests that two modeling and docking experiments indicate that C16 hydroxyl- additional atoms have been added to the β-amyrin ation is likely to occur first, followed by C12,13 epoxidation. Our backbone, of which only one can be TMS-derivatized (Fig. 2B). computational modeling in combination with mutant analysis has Under GC-MS conditions, the β-amyrin mass spectrum shows yielded insights into the structural features that are important for a characteristic base peak at m/z 218 because of retro-Diels– AsCYP51H10 function. We further show that the C12,13 epoxy Alder fragmentation typical for triterpenes that contain a C12/ group is critical for antifungal activity, a discovery that has im- C13 double bond (Fig. S1A) (33). Because this typical β-amyrin portant implications for triterpene metabolic engineering for base peak at m/z 218 was not prominent in the mass spectrum food, health, and industrial biotechnology applications. of the AsCYP51H10 product (Figs. 1D and 2A), loss of the β-amyrin double bond at C12/C13 is likely. Results Approximately 20 mg of the AsCYP51H10 product was puri- Coexpression of β-Amyrin Synthase and AsCYP51H10 in Nicotiana fied from AsbAS1/AsCyp51H10 coinfiltrated plant material (∼17 benthamiana Leaves. We investigated the biochemical function g dry weight) (Materials and Methods) and was analyzed by MS of AsCYP51H10 using the cowpea mosaic hyper-trans and NMR to determine the structure. A molecular mass of 458 cassette (CPMV-HT), a plant expression system based on cow- Da was determined using high-resolution MS. This result sug- pea mosaic virus. For CPMV-HT, constructs are generated so gests a chemical formula of C30H50O3 and is consistent with the that the sequence to be expressed is flanked by modified 5′ and 3′ previously obtained GC-MS data. The 1H and 13C NMR spectra UTR sequences from CPMV RNA-2 (the CPMV-HT cassette) of the purified compound were compared with available NMR under the control of the 35S promoter. The presence of the data for β-amyrin (34, 35). Characteristic signals for the olefinic CPMV-derived UTR sequences enhances mRNA translatability, carbons in β-amyrin (C12 δ 123; C13 δ 145) (34) were missing in thereby substantially increasing the amount of protein expressed. the 13C NMR spectrum of the analyzed compound, indicating Constructs then are transformed into Agrobacterium tumefaciens the loss of the double bond. Further distortionless enhancement and infiltrated into leaves of N. benthamiana for transient ex- by polarization transfer, heteronuclear multiple bond correla- pression in the presence of the gene-silencing suppressor, P19. tion, and selective NOE experiments revealed an be- CPMV-based systems previously have been used very success- tween C12 and C13 and a hydroxyl group attached to C16, leading fully for production of structural such as vaccines and to the identification of the compound as 12,13β-epoxy-3β,16β- antibodies (29, 30). We also have shown this approach to be ef- dihydroxy-oleanane (12,13β-epoxy-16β-hydroxy-β-amyrin) (Fig. fective for expression of three triterpene biosynthetic enzymes 2C and Table S1). (an acyltransferase, a methyl , and a sugar transferase) Potential reaction intermediates in the conversion of β-amyrin required for avenacin acylation (31, 32). to 12,13β-epoxy-16β-hydroxy-β-amyrin are 12,13β-epoxy-β-amyrin Our previous analysis of oat mutants defective in avenacin and 16β-hydroxy-β-amyrin (Fig. 2C). Neither of these was de- synthesis indicated that mutations in the AsCyp51H10 (Sad2) tectable in leaf extracts expressing AsbAS1 alone (Fig. S2), in- gene lead to accumulation of the first committed intermediate in dicating that N. benthamiana is not able to modify β-amyrin. We the avenacin pathway, β-amyrin, suggesting that β-amyrin may be cannot exclude the possibility that AsCYP51H10 may carry out the substrate for AsCYP51H10 (Fig. 1A) (15, 20). We therefore one modification to generate either 12,13β-epoxy-β-amyrin and coexpressed AsCYP51H10 with AsbAS1, the oat β-amyrin syn- 16β-hydroxy-β-amyrin and that an endogenous N. benthamiana thase that catalyses the first step in avenacin synthesis (18), to in- enzyme then may mediate the second modification. However, vestigate the biochemical function of AsCYP51H10. Constructs this possibility is unlikely, because previous preliminary analysis for transient expression were made containing the AsbAS1 and in yeast suggested that AsCYP51H10 is able to oxygenate β-amyrin AsCyp51H10 coding sequences, each under the control of the at two positions (although the full structure was not determined) 35S promoter (Fig. 1B). N. benthamiana leaves were infiltrated (21). Comparisons of the MS spectrum of our product with that with A. tumefaciens containing the empty vector control, AsbAS1, of the partially characterized product from yeast were in agree- and AsCYP51H10 constructs separately or were coinfiltrated with ment (Fig. S3). Thus, AsCYP51H10 is able to stereo- and regio-

2of8 | www.pnas.org/cgi/doi/10.1073/pnas.1309157110 Geisler et al. Downloaded by guest on September 30, 2021 A PNAS PLUS E E AsbAS1 E AsCYP51H10 CD CD CD Sad1 Sad2 AB AB AB

β-Amyrin Des-acyl-avenacin A Avenacin A-1

B C CPMV-HT-AsbAS1

CPMV-HT-AsCYP51H10

pBIN61-P19

D PLANT BIOLOGY

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 ter- minator 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 (Rt) =19.1 min]. B, betulin (internal standard; Rt =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.

specifically catalyze both hydroxylation at C16 and epoxide for- zymes from , protozoa, fungi, animals, and plants, six are mation at C12/C13 in a β configuration, modifications that are not conserved in AsCYP51H10 (Fig. S4) (15). Our previous mo- both present in the pathway end-product, avenacin A-1 (Fig. 1A). deling of AsCYP51H10 and the oat sterol demethylase AsCYP51G1 based on the Mycobacterium tuberculosis MtCYP51B1 crystal Molecular Modeling and Analysis of Mutants. AsCYP51H10 is a structure revealed that the active site volume of AsCYP51H10 is highly divergent CYP51 enzyme and has only 46% relatively large compared with the active site of CYP51G en- sequence identity with the bona fide oat sterol demethylase, zymes, consistent with acquisition of a different function (15). AsCYP51G1. This identity drops to 32% for the subset of resi- We used molecular modeling and docking approaches to gain dues that form the active site cavity (15). Of a total of 34 amino further insights into the potential reaction mechanism of acid residues that are completely conserved across CYP51 en- AsCYP51H10, this time building a model based on the

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modification m/z β-amyrin 426 β-amyrin + TMS 498 β-amyrin + TMS + oxygen 514 β-amyrin + 2 x TMS + oxygen 586 β-amyrin + 2 x TMS + 2 x oxygen 602

C 12,13β-epoxy- β-amyrin

12 13 16 β-amyrin

12,13β-epoxy- 16β-hydroxy-β-amyrin

C30H50O3 458 Da 16β-hydroxy- β-amyrin

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.

crystal structure of human CYP51 in complex with orientations of β-amyrin and 16β-hydroxy-β-amyrin as observed (36). Human CYP51 has a higher sequence identity to AsCYP51H10 in these modeled complexes are necessary to allow the close (34%) than does MtCYP51B1 (28%) and so represents a more approach of the reactive oxygen O1 to the site of hydroxylation appropriate template. The active site of this model also is large or epoxidation on the substrate. Residues from SRS1, SRS2, relative to that of CYP51G enzymes and points to a central role SRS4, SRS5, and SRS6 are likely to contribute to the precat- for SRS5, one of the proposed substrate recognition sites (SRSs), alytic complex with β-amyrin (Fig. 3B), whereas those from in determining the size of the active site cavity. Amino acids with SRS1, SRS4, SRS5, and SRS6 are likely to contribute to binding small side chains are found at positions 4 and 5 (residues 353 and of 16β-hydroxy-β-amyrin (Fig. 3C). Taken together, these ob- 354) following the characteristic ExxR motif of SRS5 (Fig. 3A), servations are consistent with a scenario in which AsCYP51H10 leading to a significant increase in cavity volume over that ob- acts as a multifunctional enzyme capable of catalyzing two re- served in AsCYP51G1 (with proline and leucine at these posi- actions sequentially, i.e., C16 hydroxylation of β-amyrin followed tions). Alanines are observed infrequently at these positions in by C12,C13 epoxidation. The predicted precatalytic binding modes P450 enzymes (37). Substitutions in SRS5 have been shown to are markedly different and can be accommodated only because alter the regiospecificity and selectivity of CYP enzymes from of the large active site relative to that of CYP51G enzymes. The a range of families (39–42), including CYP51 (43). The model absence of a detectable intermediate in the in planta expression also provided the basis for three molecular docking experiments experiments further suggests that these reactions occur without using geometry-optimized models of β-amyrin and the potential releasing an intermediate. reaction intermediates 16β-hydroxy-β-amyrin and 12,13β-epoxy- Previously we reported on the identification of a collection of β-amyrin. Molecular dynamics trajectories initiated from low- oat mutants defective in avenacin synthesis (15, 16). We next energy docking poses were used to identify precatalytic binding mapped the locations of the mutations in a suite of seven sad2 modes as those complexes in which the average distance of ap- mutants onto the homology model for AsCYP51H10 (Fig. S4). proach of the distal oxygen (atom O1) to ep- Four of the mutants carry single substitutions involving residues oxidation sites (C12, C13) or the hydroxylation site (C16) on the that are critical for heme binding or that play structural roles, ligand is 5 Å or less (44). This result led to the identification of being located at the closely packed interfaces of major structural single, unique precatalytic binding modes for both β-amyrin and α-helices, and so were not considered further. The three remaining 16β-hydroxy-β-amyrin to AsCYP51H10 suggesting the formation mutants involve substitutions to residues either within or adja- of the 16β-hydroxy intermediate (Fig. 3 B and D) and the 16β- cent to substrate recognition sites critical to our catalytic model hydroxy-12,13β-epoxy product (Fig. 3 C and E), respectively. In (Fig. 3A). Mutant #1027 contains the mutation A124V within contrast, none of the simulations involving low-energy docked SRS1. This residue lies immediately adjacent to Y123 whose side complexes of 12,13β-epoxy-β-amyrin yielded conformations con- chain makes contact with ring E of the substrate in both pre- sistent with a precatalytic complex. Significant differences in the catalytic binding modes (Fig. 3 B and C). Mutant #791 has the

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#1412 #1027

#791

B C PLANT BIOLOGY

D E

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-deme- thylase (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.

substitution P463S, involving a residue lying at the beginning of a long C). The sad2 mutant #1412 (T113I) (marked with a blue triangle polypeptide loop which extends into the active site, presenting in Fig. 3A) is predicted to lie on helix B′ and contributes to SRS1 residues that contribute to SRS6-contacting ring A (Fig. 3 B and (Fig. 3 A and B) (15). The side chain of T113 faces into the active

Geisler et al. PNAS Early Edition | 5of8 Downloaded by guest on September 30, 2021 site cavity and is predicted to be in van der Waals contact with droxyl group at C16 of the D ring and introduction of an epoxide bound β-amyrin and 16β-hydroxy-β-amyrin at rings D and E. These at the C12/C13 positions of the C ring. Our molecular modeling mutants were tested individually by computational modeling, and and docking approaches demonstrate that both possible inter- none was predicted to be able to form precatalytic complexes mediates, 12,13β-epoxy-β-amyrin and 16β-hydroxy-β-amyrin, fit (Fig. S5). Consistent with the computational analysis, although the into the active site cavity. However, when a distance of heme seven sad2 mutants all accumulate β-amyrin (15, 20), none was iron distal ligand to reaction sites of 5 Å or less is used as a cri- found to accumulate either 16β-hydroxy-β-amyrin or 12,13β- terion (44), the proposed first reaction step is likely to be the epoxy-β-amyrin. Our computational structural model in combi- hydroxylation of β-amyrin at C16, followed by an epoxidation of nation with mutant analysis thus provides insights into the unusual 16β-hydroxy-β-amyrin at C12/C13. The active site of AsCYP51H10 catalytic behavior of AsCYP51H10 and its active site mutants. is large compared with that of conserved CYP51G proteins (15), consistent with our findings that AsCYP51H10 introduces two The Epoxide Group Is Required for Antifungal Activity of Avenacin A- successive modifications to the β-amyrin backbone. We further 1. β-Amyrin itself is not antifungal. We therefore were interested show that none of the seven sad2 mutants examined was predicted in establishing whether the action of AsCYP51H10 was likely to to be able to form precatalytic complexes. Consistent with this influence the antimicrobial properties of the triterpene scaffold. observation, these mutants all accumulated β-amyrin but not 16β- Structural variants of avenacin A-1 lacking one or more mod- hydroxy-β-amyrin or 12,13β-epoxy-β-amyrin. That a relatively large ifications are not commercially available, and so the significance active site allows sequential reactions at different positions has of the C12/13 epoxide and the C16 hydroxide for the antimi- been discussed previously for CYP71D20, a 5-epiaristolochene crobial activity of avenacin A-1 was not known. However, we were 1,3-dihydroxylase that catalyzes two successive on able to generate and purify 12-oxo-avenacin A-1, in which the a sesquiterpene without releasing an intermediate (46). We C12/13 epoxide is replaced by a C12 carbonyl group (Fig. 4A). further show that the epoxide group is critical for the antifungal Assays of antifungal activity were carried out using Gaeumanno- activity of avenacin A-1. This result has important implications myces graminis var. tritici, a that is sensitive to avenacin A-1 for metabolic engineering of crop plants for enhanced disease (45). Avenacin A-1 was clearly inhibitory to fungal growth, but resistance and also for the generation of antimicrobial triterpenes 12-oxo-avenacin A-1 had no effect (Fig. 4B), indicating that for other applications. the presence of the C12/C13 epoxide group is critical for an- Multifunctional P450 enzymes from other P450 families that tifungal activity. Thus, AsCYP51H10 is important in determining catalyze both hydroxylation and epoxidation reactions have the of avenacin A-1 through modification of the been described recently from bacteria (47–51) and fungi (52, 53). triterpene scaffold. Examples of both possible reaction orders (hydroxylation fol- lowed by epoxidation and epoxidation followed by hydroxyl- Discussion ation) have been reported (Fig. 5). Here, we have shown that CYP51 enzymes are regarded as one of the most ancient and highly a plant P450 AsCYP51H10 is also able to catalyze both of these conserved cytochrome P450 families. These enzymes are well types of modification. An exciting future challenge will be to es- known to have functions in the synthesis of essential sterols in tablish which amino acid residues are important in converting animals, fungi, and plants and are important targets for antifungal a canonical CYP51 into an enzyme with the properties of agents, herbicides, and -lowering . Here we show AsCYP51H that is able to catalyze both hydroxylation and ep- that AsCYP51H10 has acquired a different function compared formation of β-amyrin stereo- and regio-specifically. Given to other characterized members of the CYP51 family and carries the many differences between these two types of enzymes, this out two modifications to the β-amyrin scaffold: addition of a hy- conversion in activity is unlikely to be trivial and may require

A Avenacin A-1 (A1) 12-Oxo-avenacin A-1 (OA)

B 2μg5μg10μg A1 A1 A1

c c cc c c

OA OA OA

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.

6of8 | www.pnas.org/cgi/doi/10.1073/pnas.1309157110 Geisler et al. Downloaded by guest on September 30, 2021 PNAS PLUS

A MycG MycG

M-IV M-V M-II

B

GfsF GfsF

25-O-Methyl FD-892 FD-891

C TamI TamL TamI TamI

TirC TirE TirD TirA TirB

D Tri4

Trichodiene PLANT BIOLOGY 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 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 hy- droxylation at C2, epoxidation at C12/13, hydroxylation at C11, and hydroxylation at C3.

systematic combinatorial of multiple amino acid Materials and Methods residues. The development of methods for the synthesis of the Expression of AsbAS1 and AsCYP51H10 in N. benthamiana. CPMV expression two possible intermediates 12,13β-epoxy-β-amyrin and 16β-hy- constructs carrying the AsbAS1 or AsCyp51H10 coding sequence were droxy-β-amyrin, neither of which is commercially available, transformed into A. tumefaciens strain LBA4404 and leaves of N. benthamiana also will be important for further mechanistic insights into plants were infiltrated as previously described (29–32). Full details of cloning AsCYP51H10 function. procedures, including primer sequences (Table S2) and expression in N. Our experiments demonstrate that the CPMV-HT expression benthamiana are provided in SI Materials and Methods. system provides a rapid and effective means of expressing en- Protein Extraction and Immunoblot Analysis. Details of methods for protein zymes for the synthesis of low molecular weight specialized com- extraction and immunoblot analysis are provided in SI Materials and Methods. pounds alone and in combination. Previous work on triterpene biosynthetic enzymes by other groups has focused on yeast as Triterpene Analysis and Purification of 12,13β-Epoxy-16β-Hydroxy-β-Amyrin. a heterologous expression system (24–28). Here, we have used The triterpene content of N. benthamiana leaves expressing AsbAS1 and/ the CPMV-HT system for the synthesis and modification of the or AsCYP51H10 cDNA was analyzed by GC-MS as previously described (54). simple triterpene β-amyrin, thereby reconstructing the first two For further purification of 12,13β-epoxy-16-hydroxy-β-amyrin from N. ben- committed steps in the avenacin biosynthetic pathway. Unlike thamiana leaf material, large-scale triterpene extraction was performed. fi yeast, there was no requirement for introduction of a heterologous Further puri cation was achieved using medium pressure liquid chroma- tography. Full details of triterpene extraction methods, purification of cytochrome P450 because endogenous N. benthamiana β β β fi 12,13 -epoxy-16 -hydroxy- -amyrin, and analysis of oat mutants are pro- cytochrome P450 reductase activity was able to provide suf cient vided in SI Materials and Methods. transfer to enable catalysis. Furthermore, even without substantial scale-up and optimization, we were able to purify ∼20 Molecular Modeling. Development of an AsCYP51H10 model was carried mg of the triterpene 12,13β-epoxy-16β-hydroxy-β-amyrin and deter- out as previously described (15), except the crystal structure of human mine its structure by NMR. The CPMV-HT expression system CYP51 in complex with ketoconazole ( ID code 3LD6) recently has been refined to give the pEAQ series of transient (37) was used as a template. Docking of substrate molecules was per- expression vectors (30), some of which are Gateway-based. It also formed using Autodock Vina (55). Molecular dynamics simulations were can be made to be modular, permitting the expression of several carried out using GROMACS (56). Full details are provided in SI Materials from multiple CPMV-HT cassettes within the same transfer and Methods. DNA. Our proof-of-concept experiments now open the possi- Fungal Growth-Inhibition Assays. Assays were carried out as described previously bility of using the CPMV-HT system for synthetic biology-based (57). Plugs of mycelium from an actively growing colony of the ascomycete metabolic engineering approaches to enable the production of fungus G. graminis var. tritici isolate T5 (45) were placed onto potato dextrose known and novel triterpenes and other high-value compounds in agar. Avenacin A-1 and carbonyl-avenacin A-1 were dissolved in 70% methanol “green factories.” (1 mg/mL stock solution) and applied to filter paper discs. The discs were placed

Geisler et al. PNAS Early Edition | 7of8 Downloaded by guest on September 30, 2021 equidistant from the fungal plug of inoculum. Plates were incubated at 22 °C puting Cluster supported by the Research and Specialist Computing Support in the dark, and growth was monitored after 1 wk. Details of avenacin A-1 and Service at the University of East Anglia. This work was supported by the United carbonyl-avenacin A-1 preparation are provided in SI Materials and Methods. Kingdom Biotechnological and Biological Sciences Research Council Institute Strategic Programme Grant “Understanding and Exploiting Plant and Microbial ” ACKNOWLEDGMENTS. We thank Alan Jones and Lionel Hill of John Innes Secondary Metabolism BB/J004561/1 and by the John Innes Foundation (R.K.H., Metabolite Services for metabolite analysis. The computer modeling and sim- G.P.L., M.R., S.F., R.E.M., and A.E.O.). K.G. was supported by a Danish Research ulation presented in this paper were carried out on the High Performance Com- School for Biotechnology International PhD studentship.

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