Natural products biosynthesis often requires the action of multiple CYPs (cytochromes P450), whose ability to introduce oxygen, increasing solubility, is critical for imparting biological activity. In previous investigations of rice diterpenoid biosynthesis, we characterized CYPs that catalyse alternative hydroxylation of ent-sandaracopimaradiene, the precursor to the rice oryzalexin antibiotic phytoalexins. In particular, CYP76M5, CYP76M6 and CYP76M8 were all shown to carry out C-7β hydroxylation, whereas CYP701A8 catalyses C-3α hydroxylation, with oxy groups found at both positions in oryzalexins A–D, suggesting that these may act consecutively in oryzalexin biosynthesis. In the present paper, we report that, although CYP701A8 only poorly reacts with 7β-hydroxy-ent-sandaracopimaradiene, CYP76M6 and CYP76M8 readily react with 3α-hydroxy-ent-sandaracopimaradiene. Notably, their activity yields distinct products, resulting from hydroxylation at C-9β by CYP76M6 or C-7β by CYP76M8, on different sides of the core tricyclic ring structure. Thus CYP76M6 and CYP76M8 have distinct non-redundant roles in orzyalexin biosynthesis. Moreover, the resulting 3α,7β- and 3α,9β-diols correspond to oryzalexins D and E respectively. Accordingly, the results of the present study complete the functional identification of the biosynthetic pathway underlying the production of these bioactive phytoalexins. In addition, the altered regiochemistry catalysed by CYP76M6 following C-3α hydroxylation has some implications for its active-site configuration, offering further molecular insight.
- cytochrome P450
- enzyme specificity
CYPs (cytochromes P450) (EC 1.14.13.x) serve important roles not only in xenobiotic metabolism/detoxification, but also in natural product biosynthesis. In particular, these haem-thiolate mono-oxygenases are often responsible for the insertion of oxygen into unreactive carbon–hydrogen bonds of hydrophobic intermediates. The resulting hydroxy groups then serve to both increase solubility and provide hydrogen-bonding potential that increases the specificity (i.e. binding) of the resulting natural product to its molecular target. Often multiple CYPs are required for the production of bioactive natural compounds and, given their sometimes broad substrate specificity, it can be difficult to determine whether there is a defined or preferred order of reactions, and what effect initial hydroxylation reactions may have on subsequently catalysed reactions. In addition, the homology among CYP paralogues, which can be inferred easily from their grouping into related numbered families and even more closely related lettered subfamilies, and that often exhibit similar biochemical activity, further complicates such analysis, and raises the potential of redundancy, e.g. in plants .
Plants produce a vast array of natural products that serve various ecological roles, such as in defence against microbial pathogens. In many cases, the biosynthesis of these compounds is induced by infection with the microbial pathogens against which they exhibit antibiotic activity, leading to their designation as phytoalexins . In the important cereal crop plant rice (Os; Oryza sativa), these phytoalexins are largely members of the LRD (labdane-related diterpenoid) superfamily .
LRD biosynthesis is characterized by its initiation via sequential cyclization reactions . First, (bi)cyclization of the general diterpenoid precursor GGPP [(E,E,E)-geranylgeranyl diphosphate], generally to the eponymous labdadienyl/CPP (copalyl diphosphate), catalysed by class II diterpene cyclases (EC 5.5.1.x) then termed CPSs (CPP synthases). This is followed by further cyclization, generally to an olefin, catalysed by class I (di)terpene synthases (EC 4.2.3.x) often termed KSL (ent-kaurene synthase-like) for their derivation from the ancestral enzymatic family member found in all plants for gibberellin phytohormone metabolism. The multicyclic olefins resulting from these initial cyclization reactions are quite hydrophobic, e.g. their logP (partition coefficient) is generally ≥8.5. Accordingly, these are invariably transformed further by the addition of oxygen, generally catalysed by CYPs, to increase their solubility, enabling exertion of biological activity. For example, it has been shown that gibberellin biosynthesis depends on ent-kaurene oxidases from the CYP701A subfamily and subsequently acting ent-kaurenoic acid oxidases from the CYP88A subfamily [5,6]. Similarly, conifer resin acid biosynthesis is dependent on CYP720B subfamily members that act on LRD precursors [7,8].
In the course of functionally characterizing the rice CPS and KSL, we found that consecutively acting OsCPS and OsKSL are close together in the rice genome, along with CYPs [9,10]. Functional characterization of these CYPs revealed their ability to oxygenate diterpene olefins produced by the co-clustered OsCPS and OsKSL [11–13], as well as those resulting from the action of OsKSL found elsewhere in the rice genome , with oxy groups found at the targeted positions in the derived natural products. In addition, similar activity has been demonstrated for CYP701A8, a paralogue of the ent-kaurene oxidase required for gibberellin metabolism, that is found elsewhere in the rice genome .
Given the alternative positions targeted by the functionally characterized CYPs, it is unclear how even diols, which seem to be the minimal degree of modification required to form bioactive LRDs, are generated. For example, although previous isolation of 3α-hydroxy-ent-sandaracopimaradiene and re-feeding in rice plant cell-free assays indicated its intermediacy in oryzalexin D and E biosynthesis , and we have characterized CYP that will catalyse such C-3α (CYP701A8) , as well as C-7β (CYP76M5, CYP76M6 and CYP76M8) , hydroxylation of the olefin precursor ent-sandaracopimaradiene, their ability to act consecutively remains uncertain (Figure 1). Moreover, the known CYP-catalysed reactions are insufficient to generate the observed range of LRDs, e.g. formation of orzyalexin E requires hydroxylation at C-9, but no CYP catalysing such a reaction with ent-sandaracopimaradiene has been found.
In the present study, we provide evidence indicating that oryzalexins D and E are generated by initial C-3α hydroxylation catalysed by CYP701A8, which alters the subsequent reaction catalysed by CYP76M6, such that it hydroxylates C-9β instead of C-7β, generating oryzalexin E (i.e. the 3α,9β-diol), whereas CYP76M8 continues to hydroxylate C-7β, generating oryzalexin D instead (i.e. the 3α,7β-diol). In addition, we examine the implications of the observed change in regiochemistry for configuration of the CYP76M6 active site.
Unless otherwise noted, chemicals were purchased from Fisher Scientific, and molecular biology reagents were from Invitrogen. The logP values presented in the present study were obtained from the SciFinder database (American Chemical Society). GC was performed with a Varian 3900 GC with Saturn 2100 ion trap MS in electron ionization (70 eV) mode for GC–MS analyses, or with an Agilent 6890N GC with FID (flame-ionization detection) for GC–FID analyses. Samples (1 μl) were injected in splitless mode at 50°C and, after holding for 3 min at 50°C, the oven temperature was increased at a rate of 14°C/min to 300°C, where it was held for an additional 3 min. MS data from 90 to 600 m/z were collected starting at 12 min after injection until the end of the run.
The CYP701A8 and CYP76M5–CYP76M8 genes used in the present study are the synthetic fully-codon-optimized and N-terminally modified constructs described recently [14,15]. However, to optimize metabolic flux, a new CYP expression vector was created by insertion of a DEST cassette into the first MCS (multiple cloning site) of pETDuet (Novagen), using the NcoI and NotI restriction sites, as described previously . To enable dual CYP expression, CYP701A8 was inserted into the second MCS of pETDuet/DEST using the NdeI and XhoI restriction sites, creating a pETDuet/DEST/CYP701A8 vector. Alternatively, CYP76M6 or CYP76M8 were inserted into the second MCS of pETDuet/DEST using the NdeI and XhoI restriction sites. In all three of these cases, a rice CPR (CYP reductase) (OsCPR1) was inserted via directional recombination into the DEST cassette, creating pETDuet/DEST::OsCPR1/(CYP701A8, CYP76M6 or CYP76M8) constructs, to enable co-expression for feeding studies. CYP76M5–CYP76M8 were transferred into the pETDuet/DEST/CYP701A8 vector for recombinant expression via directional recombination into the DEST cassette, creating pETDuet/DEST::CYP76M5–CYP76M8)/CYP701A8 constructs for balanced CYP expression. To enable co-production of the CYP olefin substrates investigated in the present study, OsKSL7 and OsKSL10 (producing ent-cassadiene or ent-sandaracopimaradiene respectively) were inserted via directional recombination into the DEST cassette of the pCDFDuet/DEST/OsCPR1 vector described previously , creating pCDFDuet/DEST::(OsKSL7 or OsKSL10)/OsCPR1 constructs.
All recombinant expression was carried out in the C41 Overexpress strain of Escherichia coli (Lucigen), using the modular diterpene metabolic engineering concept we have described previously , and the vectors described above. Specifically, we co-expressed CYP701A8 with each of CYP76M5–CYP76M8, using the pETDuet/DEST::CYP76M(x)/CYP701A8 vectors, which were co-transformed with the compatible pGGeC, to enable production of the upstream GGPP and, subsequently, ent-CPP, as well as the relevant pCDFDuet/DEST::(OsKSL7 or OsKSL10)/OsCPR1 vector, to enable production of the immediate ent-cassadiene or ent-sandaraco pimaradiene respectively, along with the reductase needed for CYP activity. These recombinant strains were grown, typically in 50 ml cultures in TB (12 g yeast extract, 24 g casein hydrolysate and 4 ml glycerol per litre) liquid medium at 37°C to D600 ~0.6, shifted to 16°C for 1 h before induction with 1 mM IPTG, and supplementation with 5 mg/l riboflavin, and 75 mg/l δ-aminolaevulinic acid, then grown for an additional 72 h. The resulting diterpenoids were extracted (from the medium and cells) with an equal volume of hexane, and analysed by GC–MS. In every case, the hydroxylated diterpenoid products were observed as reported previously [14,15], with diols detected in certain cases, as described below.
To obtain the observed dual CYP product in sufficient amounts for NMR analysis, flux was increased into isoprenoid metabolism and the recombinant culture volumes also simply scaled up. To increase flux, the recombinant strain was transformed further with the pMBI vector encoding the bottom half of the mevalonate dependent isoprenoid precursor pathway . This is compatible with those mentioned above, and feeding 10 mM mevalonolactone to the resulting recombinant cultures greatly increases (di)terpenoid yield, as previously described . The dihydroxylated diterpenoid was extracted and purified much as previously described , using GC–MS to detect the presence of the desired compound (i.e. in various fractions). Briefly, 20×1 litre cultures were extracted twice with equal volumes of hexanes, and the combined organic extract then dried by rotary evaporation. The resulting residue was dissolved in 10 ml of hexanes, which was then extracted thrice with equal volumes of acetonitrile, and the combined acetonitrile extract dried under a gentle stream of N2. This was resuspended in 5 ml of hexanes, which was then fractionated via flash chromatography over a 4-g silica column using a Reveleris system (Grace) with UV detection and a hexanes to acetone gradient as the mobile phase. The dihydroxylated diterpenoid eluted in the 20% acetone fraction, which was dried under N2 and resuspended in 1 ml of acetonitrile. Further purification was carried out using an Agilent 1200 series HPLC instrument equipped with autosampler, fraction collector and diode array UV detection, over a ZORBAX Eclipse XDB-C8 column (4.6 mm×150 mm; 5 μm) at a 0.5 ml/min flow rate. The column was pre-equilibrated with 50% acetonitrile/ddH2O (double-distilled water), sample loaded, then the column washed with 50% acetonitrile/ddH2O (0–2 min), and eluted with 50–100% acetonitrile (2–7 min), followed by a 100% acetonitrile wash (7–10 min), leading to purification of a final estimated ~1 mg of the dihyroxylated diterpenoid.
Chemical structure identification
NMR spectra were recorded at 25°C on a Bruker Avance 700 spectrometer equipped with a 5-mm HCN cryogenic probe for detection of 1H and 13C. The purified compound was dried under a gentle stream of N2, and then dissolved in 0.5 ml deuterated chloroform (CDCl3; Sigma–Aldrich), with this evaporation-resuspension process repeated once more to completely remove the protonated acetonitrile solvent. This sample was placed in a NMR microtube (Shigemi) for analysis, and chemical shifts were referenced using known chloroform-d (13C 77.23, 1H 7.24 p.p.m.) signals offset from TMS (tetramethylsilane). Structural analysis was performed using 1D 1H, and 2D DQF-COSY (double-quantum-filtered correlation spectroscopy), HSQC (heteronuclear single-quantum coherence), HMQC (heteronuclear multiple-quantum coherence), HMBC (heteronuclear multiple bond correlation) and NOESY experiment spectra acquired at 700 MHz, and 1D 13C and DEPT-135 (distortionless enhancement by polarization transfer 135) spectra (174 MHz) using standard experiments from the Bruker TopSpin v1.4 software. Correlations from the HMBC spectra were used to propose a partial structure, whereas connections between protonated carbons were obtained from DQF-COSY data to complete the partial structure and assign proton chemical shifts. The structure was further verified using HSQC and DEPT-135 to confirm the chemical shift assignments.
Substrates (ent-sandaracopimaradiene, 3α-hydroxy-ent-sandaracopimaradiene and 7β-hydroxy-ent-sandaracopimaradiene) were obtained via extraction and purification from metabolically engineered strains of E. coli as described recently [14,15]. Feeding studies with CYP701A8, CYP76M6 and CYP76M8 were then carried out as follows. The CYPs were co-expressed with OsCPR1, using the pETDuet/DEST::OsCPR1/(CYP701A8, CYP76M6 or CYP76M8) constructs described above, in the C41 Overexpress strain of E. coli. These recombinant cultures (50 ml) were supplemented with the addition of 1 mM thiamine, 5 mg/l riboflavin and 75 mg/l δ-aminolaevulinic acid at the time of induction, then grown for 24 at 16°C. The relevant substrates were then fed to a final concentration of 50 μM (added from 100× stocks in methanol). The cultures were allowed to ferment for an additional 48 h, and then extracted with an equal volume of hexanes, following which the resulting products were analysed by GC–MS. All studies were run in duplicate.
Substrate competition assays
For in vitro assays, cells co-expressing CYP76M6 or CYP76M8 with OsCPR1 were harvested 72 h after induction via centrifugation (15 min at 5000 g), with the cells then resuspended in one-tenth of the culture volume of buffer A (0.1 M Tris/HCl, pH 7.2, 20% glycerol and 0.5 mM EDTA) and passed through a French press homogenizer (Emulsiflex-C5; Avestin) three times at 15000 psi (1 psi=6.9 kPa). The resulting lysates were clarified via centrifugation (15 min at 15000 g), and the supernatant used for substrate competition assays. These contained 50 μM 3α-hydroxy-ent-sandaracopimaradiene, along with various concentration of ent-sandaracopimaradiene, ranging 5–80 μM, and were carried out in 0.5 ml volumes, containing 100 μl CYP preparation, with reactions initiated by the addition of NADPH (to 0.4 mM). After 10 min of incubations at 30°C, the reactions were terminated by the addition of 50 μl of 1 M HCl, then extracted thrice with an equal volume of hexanes. In all cases, the enzymatic products were analysed by GC–MS. The relative specificity constant (kcat/Km) for ent-sandaracopimaradiene compared with 3α-hydroxy-ent-sandaracopimaradiene was calculated from the resulting data, as described previously , with the observed R2>0.9 in each case.
Bioinformatic analysis of subcellular localization
The potential differential subcellular localization of CYP701A8 compared with CYP76M6 and CYP76M8 was investigated by bioinformatics analysis using four distinct algorithms: CholoroP (http://www.cbs.dtu.dk/services/ChloroP/), Predotar (http://urgi.versailles.inra.fr/predotar/predotar.html), PCLA (http://www.andrewschein.com/cgi-bin/pclr/pclr.cgi) and iPSORT (http://ipsort.hgc.jp/). As controls, the immediately upstream OsKSL7 and OsKSL10 were included, as was the CYP701A3 family member shown previously to be in the plastid . The corresponding sequences are CYP76M6 (NP_001047192), CYP76M8 (NP_001047184), CYP701A8 (NP_001057905), CYP701A3 (NP_197962), OsKSL7 (NP_001047186) and OsKSL10 (NP_001066799).
Probing consecutive hydroxylation reactions via metabolic engineering
The potential sequential activity of CYP701A8 with CYP76M5- CYP76M8 in oryzalexins A–F and/or phytocassanes A–E biosynthesis (e.g. Figure 1) was investigated via a synthetic biology approach. In particular, we used codon-optimized genes for these CYP to enable functional expression in E. coli [14,15]. Using a modular metabolic engineering system developed previously , CYP701A8 was then co-expressed with each of CYP76M5–CYP76M8, along with the requisite CPR, and biosynthetic enzymes for production of the corresponding LRD olefin precursor (i.e. either ent-cassadiene or ent-sandaracopimaradiene). Although only trace amounts of any dihydroxylated product were observed, in the course of these initial studies we noted that the amount of hydroxylated product was dependent on which plasmid the relevant CYP was cloned into, with higher yields observed when this was the pET-derived pDEST14 vector as opposed to pCDFDuet. Accordingly, we incorporated use of the pETDuet vector to enable expression of dual CYPs, subcloning the invariable CYP701A8 into the second MCS and a DEST cassette into the first MCS to enable modular assembly of constructs for balanced co-expression of CYP701A8 with each of CYP76M5–CYP76M8.
Using these improved constructs, it was possible to observe reasonable production of both of the expected hydroxylated products from CYP701A8 and any of CYP76M5–CYP76M8 with either ent-cassadiene or ent-sandaracopimaradiene in the appropriately engineered E. coli. Moreover, although no dihydroxylated products were observed with ent-cassadiene, such products were observed with ent-sandaracopimaradiene. In particular, products were observed resulting from the activity of CYP701A8 with either CYP76M6 or CYP76M8 (Figure 2). Notably, despite the fact that CYP76M6 and CYP76M8 both catalyse C-7β hydroxylation with ent-sandaracopimaradiene , the dihydroxylated products were not the same, indicating a change in regiochemistry for one of the hydroxylation reactions. Comparison of these products with an authentic standard of oryzalexin E (a gift from Professor Robert M. Coates emeritus, Department of Chemistry, University of Illinois, Urbana-Champaign, IL, U.S.A.), demonstrated that CYP701A8 and CYP76M6 together produce this 3α,9β-diol, indicating a change in regiochemistry, from C-7 to C-9, of the CYP76M6 catalysed hydroxylation reaction following C-3α hydroxylation of ent-sandaracopimaradiene by CYP701A8.
Identification of CYP701A8 and CYP76M8 product as oryzalexin D
To enable production of sufficient amounts of the unknown dihydroxylated diterpenoid product of CYP701A8 and CYP76M8 acting on ent-sandaracopimaradiene for structural analysis by NMR, we increased flux into terpenoid metabolism in the metabolically engineered E. coli. Specifically, the endogenous methylerythritol phosphate isoprenoid precursor supply pathway was supplemented by incorporation of the bottom ‘half’ of the mevalonate-dependent pathway, along with feeding of mevalonate, which significantly increases yield, as described previously . This enabled production of approximately 1 mg of protein from not unreasonable quantities of recombinant culture (20 litres), with the resulting diterpenoid extracted and purified by flash chromatography and HPLC. From the subsequent NMR analysis (Figure 3 and Table 1), it was possible to assign the position of the two hydroxy groups to C-3α and C-7β, with the resulting diol then corresponding to the oryzalexin D expected from the originally observed olefin hydroxylation reactions (see Figure 1).
Defining CYP reaction order
Although the observed change in CYP76M6 catalytic regiochemistry presumably results from action on 3α-hydroxy-ent-sandaracopimaradiene rather than ent-sandaracopimaradiene, the order of reactions in oryzalexin D biosynthesis was uncertain. Feeding experiments demonstrated the expected conversion of 3α-hydroxy-ent-sandaracopimaradiene to oryzalexin E by CYP76M6. Similarly, CYP76M8 readily converts this to oryzalexin D. By contrast, CYP701A8 only poorly reacts with 7β-hydroxy-ent-sandaracopimaradiene, producing only trace amounts of oryzalexin D (Figure 4).
The increased solubility of 3α-hydroxy-ent-sandaracopimaradiene relative to ent-sandaracopimaradiene complicated comparison of the kinetic constants obtained in reactions with these as substrates. Thus we turned to direct comparison in substrate competition assays . In particular, running reactions with the CYP76M6 and CYP76M8 that are active with both, in the presence of a constant amount of 3α-hydroxy-entsandaracopimaradiene (50 μM) and varying levels of ent-sandaracopimaradiene. These studies revealed that CYP76M6 exhibits a clear preference for 3α-hydroxy-ent-sandaracopimaradiene compared with ent-sandaracopimaradiene, with a relative catalytic efficiency for ent-sandaracopimaradiene relative to 3α-hydroxy-ent-sandaracopimaradiene of 0.03 (i.e. ~33-fold increase in kcat/Km). By contrast, CYP76M8 seems to exhibit a slight preference for ent-sandaracopimaradiene, with a relative catalytic efficiency of 3.3 for this compared with 3α-hydroxy-ent-sandaracopimaradiene.
Although this kinetic analysis leaves some uncertainty in preferred reaction order, at least for biosynthesis of the 3α,7β-diol, 3α-hydroxy-ent-sandaracopimaradiene is readily detectable in planta , whereas 7β-hydroxy-ent-sandaracopimaradiene was not detected previously . In the present study, through pre-fractionation, we were able to detect 7β-hydroxy-ent-sandaracopimaradiene, but it is only present at ~1% of the amount of 3α-hydroxy-ent-sandaracopimaradiene. These results then indicate that oryzalexin biosynthesis proceeds via initial hydroxylation of ent-sandaracopimaradiene by CYP701A8 at the C-3α position, followed by alternative hydroxylation reactions catalysed by CYP76M6 or CYP76M8 at the C-9β or C-7β positions, producing oryzalexins E and D respectively (Figure 5).
Plant diterpenoid, as well as monoterpenoid, biosynthesis is initiated in plastids, whereas CYPs are almost invariably localized to the endoplasmic reticulum . For example, the limonene (monoterpene) hydroxylases (CYP71B subfamily members) from Mentha species  and the CYP720B subfamily members involved in conifer diterpenoid resin acid biosynthesis . However, it has been reported previously that CYP701A3, the ent-kaurene oxidase required for gibberellin phytohormone biosynthesis in Arabidopsis thaliana [25,26], is localized on the plastid membrane, providing it ready access to its ent-kaurene substrate . Hypothesizing that similar plastid localization and increased substrate access for the rice CYP701A8 might help explain the observed higher levels of 3α-hydroxy-ent-sandaracopimaradiene relative to 7β-hydroxy-ent-sandaracopimaradiene in planta, we carried out bioinformatics analysis to predict subcellular localization. Strikingly, CYP701A8 is predicted to be in the plastid by two of the four utilized algorithms, just as is CYP701A3, whereas CYP76M6 and CYP76M8 are not (Table 2). Given the clear plastid localization of the immediately upstream OsKSL10 (as well as OsKSL7), such co-localization of CYP701A8 may provide preferential access to the diterpene olefins, leading to the observed accumulation of its product, relative to that of CYP76M6 and CYP76M8, in planta.
The use of CYPs is widespread in natural product biosynthesis, and is especially prevalent with terpenoids, where the production of highly hydrophobic olefin intermediates essentially necessitates the action of these membrane-associated mono-oxygenases. However, we have relatively limited understanding of how these operate, and it can be particularly opaque how multiple CYPs act in common biosynthetic processes. For example, although gibberellin biosynthesis follows a defined CYP reaction order [5,6], as does biosynthesis of the sesquiterpenoid lactone costunolide , the reaction order for the multiple relevant CYPs is not entirely clear in biosynthesis of the diterpenoid paclitaxel/taxol , as well as the triterpenoid glycyrrhizin .
Although the work reported in the present study directly applies to biosynthesis of the rice LRDs, which act as phytoalexins and allelochemicals [30,31], the use of LRDs, at least as phytoalexins, seems to extend beyond rice and be widespread throughout the Poaceae plant family . For example, wheat (Triticum aestivum) contains expanded CPS and KSL gene families, and the transcription of certain members is induced by at least UV irradiation, with the encoded enzymes further exhibiting biochemically diverse function [32–34], much like that of the rice homologues involved in phytoalexin biosynthesis . Moreover, maize (Zea mays) not only has been found to contain at least a CPS whose transcription is induced by fungal infection , but also more directly demonstrated to produce LRD phytoalexins, i.e. kauralexins A1–3 and B1–3, which exhibit anti-fungal activity . Thus understanding LRD biosynthesis has broader importance.
Although oxygen can be incorporated during the reactions catalysed by class II diterpene cyclases [37–40] and/or class I diterpene synthases [41–47], LRD biosynthesis generally proceeds through a diterpene olefin intermediate . These are strongly hydrophobic (logP≥8.5), requiring the incorporation of oxygen to increase polarity and solubility. Moreover, although addition of a single oxygen does increase solubility, the resulting compounds resemble cholesterol (e.g. 3α-hydroxy-ent-sandaracopimaradiene), and are then expected to still partition into membranes. Hence it perhaps is not surprising that the bioactive rice LRDs contain a minimum of two oxygen molecules, which reduces their logP to ~5 (e.g. 5.0 and 4.9 for oryzalexins D and E respectively), indicating that these will exhibit significant solubility in natural settings.
Given the invariable use of CYPs to introduce these oxygen molecules, investigation of their consecutive action is then critical. In the present study, we not only provide evidence elucidating the biosynthesis of bioactive LRDs, but also demonstrate flux through dual plant CYP-mediated steps in bacteria engineered to complete such metabolism, further supporting the utility of this synthetic approach to investigating biosynthetic pathways.
We have hypothesized that phytocassane biosynthesis would proceed via early C-3α and C-11α hydroxylation , which can be catalysed with the ent-cassadiene olefin precursor by CYP701A8, and CYP76M7 and CYP76M8 respectively [11,14,15]. However, we do not observe production of any dihydroxylated ent-cassadiene in cultures co-expressing CYP701A8 and CYP76M7 (or CYP76M8), despite the presence of the expected C-3α- and C-11α-(mono)hydroxylated-ent-cassadiene, demonstrating that both CYPs are active. This suggests that phytocassane biosynthesis may not proceed via this pair of reactions and/or utilizes other enzymes for this purpose.
On the other hand, the production of dihydroxylated ent-sandaracopimaradiene products, with co-expression of CYP701A8 and CYP76M6 or CYP76M8, does provide some insight into orzyalexin biosynthesis. Interestingly, this extends beyond the potential roles indicated by their activity with the ent-sandaracopimaradiene olefin precursor (Figure 1). In particular, the presence of the C-3α-hydroxy group changes substrate orientation in CYP76M6 such that it targets C-9β instead of C-7β, which is targeted by both CYP76M6 and CYP76M8 with ent-sandaracopimaradiene and, in the case of CYP76M8, also with the derived C-3α-hydroxylated compound. Accordingly, our results clarify the biosynthesis of not only oryzalexin D (i.e. the originally expected 3α,7β-diol), but that of oryzalexin E (the 3α,9β-diol) as well (Figure 3).
The change in regiochemistry of the CYP76M6 catalysed reactions prompted us to examine more closely the underlying alteration in substrate orientation. Although C-7 and C-9 are on opposite ‘sides’, the common β position/hydrogen target does fall on same ‘face’ of the middle ‘B’ ring. Examination of 3D models for the alternative substrates highlights the proximity of these positions, indicating that the catalysed insertion of oxygen occurs via very similar binding modes, with only a slight perturbation in orientation in the CYP76M6 active site imposed by the presence of a C-3α-hydroxyl group. We hypothesize that this is mediated via the presence of a polar group in the CYP76M6 active site that either hydrogen bonds to the hydroxy group, leading to reaction at C-9, or binds a water molecule that then drives the observed alternative regiospecificity for C-7 with ent-sandaracopimaradiene (Figure 6).
We suggest in the present study that oryzalexin biosynthesis proceeds via initial C-3α hydroxylation. This has been suggested by previous isolation of 3α-hydroxy-ent-sandaracopimaradiene and rice plant-derived cell-free assays demonstrating its conversion to oryzalexins D and E , along with the relative abundance of this intermediate compared with the alternative 7β-hydroxy-ent-sandaracopimaradiene in planta reported in the present paper. However, although feeding experiments are consistent with this suggestion, kinetic analysis indicates that such a reaction order is not driven by intrinsic substrate preference, at least in the case of orzyalexin D (3α,7β-diol) biosynthesis. Intriguingly, it has been suggested previously that members of the CYP701A subfamily, which generally act as ent-kaurene oxidases in gibberellin phytohormone biosynthesis, are localized to plastids , where diterpene olefin intermediates are produced . Bioinformatic analysis of CYP701A8 indicates that this is similarly targeted to the plastid, whereas CYP76M6 and CYP76M8 are not (Table 2). Thus we speculate that initial C-3α hydroxylation by CYP701A8 occurs, at least in part, due to preferential access through its co-localization to the plastid, where ent-sandaracopimaradiene is initially produced. CYP76M6 and CYP76M8 then have only secondary access due to their more typical endoplasmic reticulum localization. Accordingly, these catalyse subsequent alternative hydroxylations, CYP76M6 at C-9β and CYP76M8 at C-7β, yielding oryzalexins E (3α,9β-diol) or D (3α,7β-diol) respectively (Figure 5).
In any case, rather than the previously suggested redundancy , it now appears that CYP76M6 and CYP76M8 play separate roles in rice LRD metabolism, particularly orzyalexin biosynthesis, by reacting with distinct sides of the middle ‘B’ ring in 3α-hydroxy-ent-sandaracopimaradiene. Moreover, our results then elucidate the complete biosynthetic pathways leading to these bioactive phytoalexins, adding to the rather few plant natural products for which such knowledge has been accumulated.
The work described was largely carried out by Yisheng Wu, building on preliminary results obtained by Qiang Wang, while Matthew Hillwig provided advice and carried out the described NMR structural analysis, all of which was under the guidance of Reuben Peters.
This work was supported by grants from the USDA (U.S. Department of Agriculture)-NIFA (National Institute of Food and Agriculture)-NRI (National Research Initiative)/AFRI (Agriculture and Food Research Initiative) [grant number 2008-35318-05027] and NIH (National Institutes of Health) [grant number GM086281] to R.J.P.
We thank Meimei Xu for construction of the pETDuet/DEST vector, and Professor Robert M. Coates (University of Illinois, Urbana-Champaign, IL, U.S.A.) for kindly providing oryzalexin E.
Abbreviations: CPP, copalyl diphosphate; CPR, CYP reductase; CPS, CPP synthase; CYP, cytochrome P450; DEPT-135, distortionless enhancement by polarization transfer 135; DQF-COSY, double-quantum-filtered correlation spectroscopy; FID, flame-ionization detection; GGPP, (E,E,E)-geranylgeranyl diphosphate; HMBC, heteronuclear multiple bond correlation; HSQC, heteronuclear single-quantum coherence; KSL, ent-kaurene synthase-like; LRD, labdane-related diterpenoid; MCS, multiple cloning site; Os, Oryza sativa
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