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.

Structural basis for halogenation by iron- and 2-oxo-glutarate-dependent enzyme WelO5.

A 2.4-Å-resolution X-ray crystal structure of the carrier-protein-independent halogenase WelO5 in complex with its welwitindolinone precursor substrate, 12-epi-fischerindole U, reveals that the C13 chlorination target is proximal to the anticipated site of the oxo group in a presumptive cis-halo-oxo-iron(IV) (haloferryl) intermediate. Prior study of related halogenases forecasts substrate hydroxylation in this active-site configuration, but X-ray crystallographic verification of C13 halogenation in single crystals mandates that ligand dynamics must reposition the oxygen ligand to enable the observed outcome. S189A WelO5 produces a mixture of halogenation and hydroxylation products, showing that an outer-sphere hydrogen-bonding group orchestrates ligand movements to achieve a configuration that promotes halogen transfer.

Molecular Insight into the Mg2+ -Dependent Allosteric Control of Indole Prenylation by Aromatic Prenyltransferase AmbP1.

AmbP1 is a cyanobacterial aromatic prenyltransferase and a dedicated synthase for (R)-3-geranyl-3-isocyanovinyl indolenine (2), the biogenetic precursor for hapalindole-type alkaloids. The regioselective geranylation of cis-indolyl vinyl isonitrile (1) by the standalone AmbP1 to give 2 has been shown to require a magnesium ion (Mg2+ ) to suppress the formation of cis-2-geranylindolyl vinyl isonitrile (3). Here, we report high-resolution crystal structures of AmbP1 in complex with 1 and geranyl S-thiodiphosphate (GSPP) in the presence and absence of a Mg2+ effector. The comparative study of these structures revealed a unique allosteric binding site for Mg2+ that modulates the conformation of 1 in the active site of AmbP1 for its selective geranylation. This work defines the structural basis for AmbP1 catalysis in the biogenesis of hapalindole-type alkaloids and provides the first atomic-level insight to the allosteric regulation of prenyltransferases.

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.

Aliphatic Halogenase Enables Late-Stage C-H Functionalization: Selective Synthesis of a Brominated Fischerindole Alkaloid with Enhanced Antibacterial Activity.

The anion promiscuity of a newly discovered standalone aliphatic halogenase WelO5 was probed and enabled the selective synthesis of 13R-bromo-12-epi-fischerindole U via late-stage enzymatic functionalization of an unactivated sp(3) C-H bond. Pre-saturating the WelO5 active site with a non-native bromide anion was found to be critical to the highly selective in vitro transfer of bromine, instead of chlorine, to the target carbon center and also allowed the relative binding affinity of bromide and chloride towards the WelO5 enzyme to be assessed. This study further revealed the critical importance of halogen substitution on modulating the antibiotic activity of fischerindole alkaloids and highlights the promise of WelO5-type aliphatic halogenases as enzymatic tools to fine-tune the bioactivity of complex natural products.

A new family of iron-dependent halogenases acts on freestanding substrates.

Regio- and stereospecific incorporation of a halogen atom to an unactivated sp(3) carbon in a freestanding molecule is a challenging transformation that is currently missing in the inventory of enzyme-mediated reactions. Here we report what is to our knowledge the first example of a nonheme iron enzyme (WelO5) in the welwitindolinone biosynthetic pathway that can monochlorinate an aliphatic carbon in 12-epi-fischerindole U and 12-epi-hapalindole C, substrates that are free from peptidyl or acyl carrier protein.

CYP76AH1 catalyzes turnover of miltiradiene in tanshinones biosynthesis and enables heterologous production of ferruginol in yeasts.

Cytochrome P450 enzymes (CYPs) play major roles in generating highly functionalized terpenoids, but identifying the exact biotransformation step(s) catalyzed by plant CYP in terpenoid biosynthesis is extremely challenging. Tanshinones are abietane-type norditerpenoid naphthoquinones that are the main lipophilic bioactive components of the Chinese medicinal herb danshen (Salvia miltiorrhiza). Whereas the diterpene synthases responsible for the conversion of (E,E,E)-geranylgeranyl diphosphate into the abietane miltiradiene, a potential precursor to tanshinones, have been recently described, molecular characterization of further transformation of miltiradiene remains unavailable. Here we report stable-isotope labeling results that demonstrate the intermediacy of miltiradiene in tanshinone biosynthesis. We further use a next-generation sequencing approach to identify six candidate CYP genes being coregulated with the diterpene synthase genes in both the rhizome and danshen hairy roots, and demonstrate that one of these, CYP76AH1, catalyzes a unique four-electron oxidation cascade on miltiradiene to produce ferruginol both in vitro and in vivo. We then build upon the previous establishment of miltiradiene production in Saccharomyces cerevisiae, with incorporation of CYP76AH1 and phyto-CYP reductase genes leading to heterologous production of ferruginol at 10.5 mg/L. As ferruginol has been found in many plants including danshen, the results and the approaches that were described here provide a solid foundation to further elucidate the biosynthesis of tanshinones and related diterpenoids. Moreover, these results should facilitate the construction of microbial cell factories for the production of phytoterpenoids.

Discovery of a Promiscuous Non-Heme Iron Halogenase in Ambiguine Alkaloid Biogenesis: Implication for an Evolvable Enzyme Family for Late-Stage Halogenation of Aliphatic Carbons in Small Molecules.

The elucidation of enigmatic enzymatic chlorination timing in ambiguine indole alkaloid biogenesis led to the discovery and characterization of AmbO5 protein as a promiscuous non-heme iron aliphatic halogenase. AmbO5 was shown capable of selectively modifying seven structurally distinct ambiguine, fischerindole and hapalindole alkaloids with chlorine via late-stage aliphatic C-H group functionalization. Cross-comparison of AmbO5 with a previously characterized aliphatic halogenase homolog WelO5 that has a restricted substrate scope led to the identification of a C-terminal sequence motif important for substrate tolerance and specificity. Mutagenesis of 18 residues of WelO5 within the identified sequence motif led to a functional mutant with an expanded substrate scope identical to AmbO5, but an altered substrate specificity from the wild-type enzymes. These observations collectively provide evidence on the evolvable nature of AmbO5/WelO5 enzyme duo in the context of hapalindole-type alkaloid biogenesis and implicate their promise for the future development of designer biocatalysis for the selective late-stage modification of unactivated aliphatic carbon centers in small molecules with halogens.

Combining metabolomics and transcriptomics to characterize tanshinone biosynthesis in Salvia miltiorrhiza.

BACKGROUND: Plant natural products have been co-opted for millennia by humans for various uses such as flavor, fragrances, and medicines. These compounds often are only produced in relatively low amounts and are difficult to chemically synthesize, limiting access. While elucidation of the underlying biosynthetic processes might help alleviate these issues (e.g., via metabolic engineering), investigation of this is hindered by the low levels of relevant gene expression and expansion of the corresponding enzymatic gene families. However, the often-inducible nature of such metabolic processes enables selection of those genes whose expression pattern indicates a role in production of the targeted natural product. RESULTS: Here, we combine metabolomics and transcriptomics to investigate the inducible biosynthesis of the bioactive diterpenoid tanshinones from the Chinese medicinal herb, Salvia miltiorrhiza (Danshen). Untargeted metabolomics investigation of elicited hairy root cultures indicated that tanshinone production was a dominant component of the metabolic response, increasing at later time points. A transcriptomic approach was applied to not only define a comprehensive transcriptome (comprised of 20,972 non-redundant genes), but also its response to induction, revealing 6,358 genes that exhibited differential expression, with significant enrichment for up-regulation of genes involved in stress, stimulus and immune response processes. Consistent with our metabolomics analysis, there appears to be a slower but more sustained increased in transcript levels of known genes from diterpenoid and, more specifically, tanshinone biosynthesis. Among the co-regulated genes were 70 transcription factors and 8 cytochromes P450, providing targets for future investigation. CONCLUSIONS: Our results indicate a biphasic response of Danshen terpenoid metabolism to elicitation, with early induction of sesqui- and tri- terpenoid biosynthesis, followed by later and more sustained production of the diterpenoid tanshinones. Our data provides a firm foundation for further elucidation of tanshinone and other inducible natural product metabolism in Danshen.

Identification and characterization of a welwitindolinone alkaloid biosynthetic gene cluster in the stigonematalean Cyanobacterium Hapalosiphon welwitschii.

The identification of a 36 kb welwitindolinone (wel) biosynthetic gene cluster in Hapalosiphon welwitschii UTEX B1830 is reported. Characterization of the enzymes responsible for assembling the early biosynthetic intermediates geranyl pyrophosphate and 3-((Z)-2'-isocyanoethenyl)indole as well as a dedicated N-methyltransferase in the maturation of N-methylwelwitindolinone C isothiocyanate solidified the link between the wel pathway and welwitindolinone biosynthesis. Comparative analysis of the ambiguine and welwitindolinone biosynthetic pathways in two different organisms provided insights into the origins of diverse structures within hapalindole-type molecules.

Characterization of an orphan diterpenoid biosynthetic operon from Salinispora arenicola.

While more commonly associated with plants than microbes, diterpenoid natural products have been reported to have profound effects in marine microbe-microbe interactions. Intriguingly, the genome of the marine bacterium Salinispora arenicola CNS-205 contains a putative diterpenoid biosynthetic operon, terp1. Here recombinant expression studies are reported, indicating that this three-gene operon leads to the production of isopimara-8,15-dien-19-ol (4). Although 4 is not observed in pure cultures of S. arenicola, it is plausible that the terp1 operon is only expressed under certain physiologically relevant conditions such as in the presence of other marine organisms.

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

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-7ß 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 7ß-hydroxy-ent-sandaracopimaradiene, CYP76M6 and CYP76M8 readily react with 3α-hydroxy-ent-sandaracopimaradiene. Notably, their activity yields distinct products, resulting from hydroxylation at C-9ß by CYP76M6 or C-7ß 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,7ß- and 3α,9ß-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.

Characterization of CYP76M5-8 indicates metabolic plasticity within a plant biosynthetic gene cluster.

Recent reports have revealed genomic clustering of enzymatic genes for particular biosynthetic pathways in plant specialized/secondary metabolism. Rice (Oryza sativa) carries two such clusters for production of antimicrobial diterpenoid phytoalexins, with the cluster on chromosome 2 containing four closely related/homologous members of the cytochrome P450 CYP76M subfamily (CYP76M5-8). Notably, the underlying evolutionary expansion of these CYP appears to have occurred after assembly of the ancestral biosynthetic gene cluster, suggesting separate roles. It has been demonstrated that CYP76M7 catalyzes C11α-hydroxylation of ent-cassadiene, and presumably mediates an early step in biosynthesis of the derived phytocassane class of phytoalexins. Here we report biochemical characterization of CYP76M5, -6, and -8. Our results indicate that CYP76M8 is a multifunctional/promiscuous hydroxylase, with CYP76M5 and -7 seeming to provide only redundant activity, while CYP76M6 seems to provide both redundant and novel activity, relative to CYP76M8. RNAi-mediated double knockdown of CYP76M7 and -8 suppresses elicitor inducible phytocassane production, indicating a role for these monooxygenases in phytocassane biosynthesis. In addition, our data suggests that CYP76M5, -6, and -8 may play redundant roles in production of the oryzalexin class of phytoalexins as well. Intriguingly, the preceding diterpene synthase for oryzalexin biosynthesis, unlike that for the phytocassanes, is not found in the chromosome 2 diterpenoid biosynthetic gene cluster. Accordingly, our results not only uncover a complex evolutionary history, but also further suggest some intriguing differences between plant biosynthetic gene clusters and the seemingly similar microbial operons. The implications for the underlying metabolic evolution of plants are then discussed.

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.

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 cytochrome P450 (CYP) mono-oxygenases, CYP99A2 and CYP99A3, with undefined roles; although it has been previously shown that RNA interference double knock-down of this pair of closely related CYPs reduced momilactone accumulation. Here we attempted biochemical characterization of CYP99A2 and CYP99A3, which was ultimately 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 syn-stemod-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.

Domain loss has independently occurred multiple times in plant terpene synthase evolution.

The extensive family of plant terpene synthases (TPSs) generally has a bi-domain structure, yet phylogenetic analyses consistently indicate that these synthases have evolved from larger diterpene synthases. In particular, that duplication of the diterpene synthase genes required for gibberellin phytohormone biosynthesis provided an early predecessor, whose loss of a approximately 220 amino acid 'internal sequence element' (now recognized as the γ domain) gave rise to the precursor of the modern mono- and sesqui-TPSs found in all higher plants. Intriguingly, TPSs are conserved by taxonomic relationships rather than function. This relationship demonstrates that such functional radiation has occurred both repeatedly and relatively recently, yet phylogenetic analyses assume that the 'internal/γ' domain loss represents a single evolutionary event. Here we provide evidence that such a loss was not a singular event, but rather has occurred multiple times. Specifically, we provide an example of a bi-domain diterpene synthase from Salvia miltiorrhiza, along with a sesquiterpene synthase from Triticum aestivum (wheat) that is not only closely related to diterpene synthases, but retains the ent-kaurene synthase activity relevant to the ancestral gibberellin metabolic function. Indeed, while the wheat sesquiterpene synthase clearly no longer contains the 'internal/γ' domain, it is closely related to rice diterpene synthase genes that retain the ancestral tri-domain structure. Thus, these findings provide examples of key evolutionary intermediates that underlie the bi-domain structure observed in the expansive plant TPS gene family, as well as indicating that 'internal/γ' domain loss has occurred independently multiple times, highlighting the complex evolutionary history of this important enzymatic family.

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 product biosynthetic pathways can be envisioned to occur via a number of mechanisms. In the present study we provide evidence that latent plasticity plays a role in such metabolic evolution. In particular, rice (Oryza sativa) produces both ent- and syn-CPP (copalyl diphosphate), which are substrates for downstream diterpene synthases. In the present paper 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 ent-sandaracopimaradiene synthase also converts syn-CPP into 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) which may arise during evolution, the implications of which are discussed.

Hypericum in infection: Identification of anti-viral and anti-inflammatory constituents.

The Iowa Center for Research on Botanical Dietary Supplements seeks to optimize Echinacea, Hypericum, and Prunella botanical supplements for human-health benefit, emphasizing antiviral, anti-inflammatory and anti-pain activities. This mini-review reports on ongoing studies on Hypericum. The Center uses the genetically diverse, well-documented Hypericum populations collected and maintained at the USDA-ARS North Central Regional Plant Introduction Station (NCRPIS), and the strength of research in synthetic chemistry at Iowa State University to tap natural diversity, to help discover key constituents and interactions among constituents that impact bioactivity and toxicity. The NCRPIS has acquired more than 180 distinct populations of Hypericum, with a focus on Hypericum perforatum L. (Hypericaceae), representing about 13% of currently recognized taxa. Center chemists have developed novel synthetic pathways for key flavones, acyl phloroglucinols, hyperolactones and a tetralin that have been found in Hypericum, and these compounds are used as standards and for bioactivity studies. Both light-dependent and light-independent anti-viral activities have been identified by using bioactivity-guided fractionation of H. perforatum and a HIV-1 infection test system. Our Center has focused on light-independent activity, potentially due to novel chemicals, and polar fractions are undergoing further fractionation. Anti-inflammatory activity has been found to be light-independent, and fractionation of a flavonoid-rich extract revealed four compounds (amentoflavone, chlorogenic acid, pseudohypericin and quercetin) that interacted in the light to inhibit lipopolysaccharide-induced prostaglandin E(2) activity. The Center continues to explore novel populations of H. perforatum and related species to identify constituents and interactions of constituents that contribute to potential health benefits related to infection.

Pseudohypericin is necessary for the light-activated inhibition of prostaglandin E2 pathways by a 4 component system mimicking an Hypericum perforatum fraction.

Hypericum perforatum (Hp) has been used medicinally to treat a variety of conditions including mild-to-moderate depression. Recently, several anti-inflammatory activities of Hp have been reported. An ethanol extract of Hp was fractionated with the guidance of an anti-inflammatory bioassay (lipopolysaccharide (LPS)-induced prostaglandin E2 production (PGE2)), and four constituents were identified. When combined together at concentrations detected in the Hp fraction to make a 4 component system, these constituents (0.1microM chlorogenic acid (compound 1), 0.08microM amentoflavone (compound 2), 0.07microM quercetin (compound 3), and 0.03microM pseudohypericin (compound 4)) explained the majority of the activity of the fraction when activated by light, but only partially explained the activity of this Hp fraction in dark conditions. One of the constituents, light-activated pseudohypericin, was necessary, but not sufficient to explain the reduction in LPS-induced PGE2 of the 4 component system. The Hp fraction and the 4 component system inhibited lipoxygenase and cytosolic phospholipase A2, two enzymes in the PGE2-mediated inflammatory response. The 4 component system inhibited the production of the pro-inflammatory cytokine tumor necrosis factor-alpha (TNF-alpha), and the Hp fraction inhibited the anti-inflammatory cytokine interleukin-10 (IL-10). Thus, the Hp fraction and selected constituents from this fraction showed evidence of blocking pro-inflammatory mediators but not enhancing inflammation-suppressing mediators.