Understanding the biological activity of Hypericum species through metabolomics studies

The Hypericum genus contains plants which biosynthesize various polyketides with various known and unknown biological and medicinal activities. However, many Hypericum species have not been chemically or biochemically characterized. These species represent an opportunity for understanding chemical ecology and for new drug discovery research. Acyl-phloroglucinols are of particular interest since they are the most diverse class of specialized metabolites in Hypericum. We used analytical methods such as HPLC, LC-MS and 2-D NMR to screen Hypericum species for metabolite diversity. One species, H. gentianoides, was investigated further based on its historical use by Native Americans and its unique metabolite fingerprint. This species was found to contain a set of biochemically-related dimeric acyl-phloroglucinols. Furthermore, H. gentianoides extracts revealed these compounds induce various biological responses in mammalian cells including anti-inflammatory responses, TRPV1 pain receptor-dependent calcium influx, and inhibition of two lentiviruses (HIV and EIAV). Extracts were highly cytotoxic to HeLa cancer cells. Bioactivity-guided fractionation of extracts defines specific acyl-phloroglucinols associated with the bioactivities. We propose that the bioactivities of these biochemically related acyl-phloroglucinols represent a coordinated process that alters cellular processes in other organisms via redox changes and protein interactions

CYP99A3: functional identification of a diterpene oxidase from the momilactone biosynthetic gene cluster in rice

Rice (Oryza sativa) produces momilactone diterpenoids as both phytoalexins and allelochemicals. Strikingly, the rice genome contains a biosynthetic gene cluster for momilactone production, located on rice chromosome 4, which contains two cytochromes P450 mono-oxygenases, CYP99A2 and CYP99A3, with undefined roles; although it has been previously shown that RNAi double knock-down of this pair of closely related CYP reduced momilactone accumulation. Here we attempted biochemical characterization of CYP99A2 and CYP99A3, which ultimately was achieved by complete gene recoding, enabling functional recombinant expression in bacteria. With these synthetic gene constructs it was possible to demonstrate that, while CYP99A2 does not exhibit significant activity with diterpene substrates, CYP99A3 catalyzes consecutive oxidations of the C19 methyl group of the momilactone precursor syn-pimara-7,15-diene to form, sequentially, syn-pimaradien-19-ol, syn-pimaradien-19-al and syn-pimaradien-19-oic acid. These are presumably intermediates in momilactone biosynthesis, as a C19 carboxylic acid moiety is required for formation of the core 19,6-γ-lactone ring structure. We further were able to detect syn-pimaradien-19-oic acid in rice plants, which indicates physiological relevance for the observed activity of CYP99A3. In addition, we found that CYP99A3 also oxidized synstemod- 13(17)-ene at C19 to produce, sequentially, syn-stemoden-19-ol, syn-stemoden-19-al and syn-stemoden-19-oic acid, albeit with lower catalytic efficiency than with syn-pimaradiene. Although the CYP99A3 syn-stemodene derived products were not detected in planta, these results nevertheless provide a hint at the currently unknown metabolic fate of this diterpene in rice. Regardless of any wider role, our results strongly indicate that CYP99A3 acts as a multifunctional diterpene oxidase in momilactone biosynthesis

Picking sides: distinct roles for CYP76M6 and CYP76M8 in rice oryzalexin biosynthesis

Natural products biosynthesis often requires the action of multiple cytochromes P450 (CYPs), whose ability to introduce oxygen, increasing solubility, is critical for imparting biological activity. In previous investigations of rice diterpenoid biosynthesis, we have characterized CYPs that catalyze alternative hydroxylation of ent-sandaracopimaradiene, the precursor to the rice oryzalexin antibiotic phytoalexins. In particular, CYP76M5, -6 and -8 were all shown to carry out C7β-hydroxylation, while CYP701A8 catalyzes C3α-hydroxylation, with oxy groups found at both positions in oryzalexins A–D, suggesting that these may act consecutively in oryzalexin biosynthesis. Here we report that, although CYP701A8 only poorly reacts with 7β-hydroxy-entsandaracopimaradiene, CYP76M6 and -8 readily react with 3α-hydroxy-entsandaracopimaradiene. Notably, their activity yields distinct products, resulting from hydroxylation at C9β by CYP76M6 or C7β by CYP76M8, on different sides of the core tricyclic ring structure. Thus, CYP76M6 and -8 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, our results complete the functional identification of the biosynthetic pathway underlying the production of these bioactive phytoalexins. In addition, the altered regiochemistry catalyzed by CYP76M6 following C3α-hydroxylation has some implications for its active site configuration, offering further molecular insight

Parsing a multifunctional biosynthetic gene cluster from rice: Biochemical characterization of CYP71Z6 & 7

Rice (Oryza sativa) contains a biosynthetic gene cluster associated with production of at least two groups of diterpenoid phytoalexins, the antifungal phytocassanes and antibacterial oryzalides. While cytochromes P450 (CYP) from this cluster are known to be involved in phytocassane production, such mono-oxygenase activity relevant to oryzalide biosynthesis was unknown. Here we report biochemical characterization demonstrating that CYP71Z6 from this cluster acts as an ent-isokaurene C2-hydroxylase that is presumably involved in the biosynthesis of oryzalides. Our results further suggest that the closely related and co-clustered CYP71Z7 likely acts as a C2- hydroxylase involved in a latter step of phytocassane biosynthesis. Thus, CYP71Z6 & 7 appear to have evolved distinct roles in rice diterpenoid metabolism, offering insight into plant biosynthetic gene cluster evolution

CYP701A8: A Rice ent-Kaurene Oxidase Paralog Diverted to More Specialized Diterpenoid Metabolism

All higher plants contain an ent-kaurene oxidase (KO), as such a cytochrome P450 (CYP) 701 family member is required for gibberellin (GA) phytohormone biosynthesis. While gene expansion and functional diversification of GA-biosynthesis-derived diterpene synthases into more specialized metabolism has been demonstrated, no functionally divergent KO/CYP701 homologs have been previously identified. Rice (Oryza sativa) contains five CYP701A subfamily members in its genome, despite the fact that only one (OsKO2/CYP701A6) is required for GA biosynthesis. Here we demonstrate that one of the other rice CYP701A subfamily members, OsKOL4/CYP701A8, does not catalyze the prototypical conversion of the ent-kaurene C4α-methyl to a carboxylic acid, but instead carries out hydroxylation at the nearby C3α position in a number of related diterpenes. In particular, under conditions where OsKO2 catalyzes the expected conversion of ent-kaurene to ent-kaurenoic acid required for GA biosynthesis, OsKOL4 instead efficiently reacts with ent-sandaracopimaradiene and ent-cassadiene to produce the corresponding C3α-hydroxylated diterpenoids. These compounds are expected intermediates in biosynthesis of the oryzalexin and phytocassane families of rice antifungal phytoalexins, respectively, and can be detected in rice plants under the appropriate conditions. Thus, it appears that OsKOL4 plays a role in the more specialized diterpenoid metabolism of rice, and our results provide evidence for divergence of a KO/CYP701 family member from GA biosynthesis. This further expands the range of enzymes recruited from the ancestral GA primary pathway to the more complex and specialized labdane-related diterpenoid metabolic network found in rice

Characterizing the Metabolic Fingerprint and Anti-inflammatory Activity of Hypericum gentianoides

In this paper we characterize the metabolic fingerprint and first reported anti-inflammatory activity of Hypericum gentianoides. H. gentianoides has a history of medical use by Native Americans, but it has been studied very little for biological activity. High-performance liquid chromatography (HPLC) and liquid chromatography−electrospray ionization−mass spectrometry (LC-ESI-MS) analyses of a methanol extract show that H. gentianoides contains a family of over nine related compounds that have retention times, mass spectra, and a distinctive UV absorption spectra characteristic of certain acyl-phloroglucinols. These metabolites are abundant relative to other secondary products present in H. gentianoides, accounting for approximately 0.2 g per gram of dry plant tissue. H. gentianoides methanol extracts and a specific semipreparative HPLC fraction from these extracts containing the putative acyl-phloroglucinols reduce prostaglandin E2 synthesis in mammalian macrophages

Probing Labdane-Related Diterpenoid Biosynthesis in the Fungal Genus Aspergillus

While terpenoid production is generally associated with plants, a variety of fungi contain operons predicted to lead to such biosynthesis. Notably, fungi contain a number of cyclases characteristic of labdane-related diterpenoid metabolism, which have not been much explored. These also are often found near cytochrome P450 (CYP) mono-oxygenases that presumably further decorate the ensuing diterpene, suggesting that these fungi might produce more elaborate diterpenoids. To probe the functional diversity of such biosynthetic capacity, an investigation of the phylogenetically diverse cyclases and associated CYPs from the fungal genus Aspergillus was undertaken, revealing their ability to produce isopimaradiene-derived diterpenoids. Intriguingly, labdane-related diterpenoid biosynthetic genes are largely found in plant-associated fungi, hinting that these natural products may play a role in such interactions. Accordingly, it is hypothesized here that isopimarane production may assist the plant-saprophytic lifestyle of Aspergillus fungi

Evident and latent plasticity across the rice diterpene synthase family with potential implications for the evolution of diterpenoid metabolism in the cereals

The evolution of natural products biosynthetic pathways can be envisioned to occur via a number of mechanisms. Here we provide evidence that latent plasticity plays a role in such metabolic evolution. In particular, rice (Oryza sativa) produces both ent- and syn-copalyl diphosphate (CPP), which are substrates for downstream diterpene synthases. Here we report that several members of this enzymatic family exhibit dual reactivity with some pairing of ent-, syn-, or normal CPP stereochemistry. Evident plasticity was observed, as a previously reported entsandaracopimaradiene synthase also converts syn-CPP to syn-labda-8(17),12E,14-triene, which can be found in planta. Notably, normal CPP is not naturally found in rice. Thus, the presence of diterpene synthases that react with this non-native metabolite reveals latent enzymatic/metabolic plasticity, providing biochemical capacity for utilization of such a novel substrate (i.e., normal CPP) that may arise during evolution, the implications of which are discussed

Probing the promiscuity of ent-kaurene oxidases via combinatorial biosynthesis

The substrate specificity of enzymes from natural products’ metabolism is a topic of considerable interest, with potential biotechnological use implicit in the discovery of promiscuous enzymes. However, such studies are often limited by the availability of substrates and authentic standards for identification of the resulting products. Here, a modular metabolic engineering system is used in a combinatorial biosynthetic approach toward alleviating this restriction. In particular, for studies of the multiply reactive cytochrome P450, ent-kaurene oxidase (KO), which is involved in production of the diterpenoid plant hormone gibberellin. Many, but not all, plants make a variety of related diterpenes, whose structural similarity to ent-kaurene makes them potential substrates for KO. Use of combinatorial biosynthesis enabled analysis of more than 20 such potential substrates, as well as structural characterization of 12 resulting unknown products, providing some insight into the underlying structure–function relationships. These results highlight the utility of this approach for investigating the substrate specificity of enzymes from complex natural products’ biosynthesis