Diterpene synthases and their responsible cyclic natural products.
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ABSTRACT:
SUBMITTER: Gong HY
PROVIDER: S-EPMC4004862 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:The diversity of terpenoid compounds produced by plants plays an important role in mediating various plant-herbivore, plant-pollinator, and plant-pathogen interactions. This diversity has resulted from gene duplication and neofunctionalization of the enzymes that synthesize and subsequently modify terpenes. Two diterpene synthases in Norway spruce (Picea abies), isopimaradiene synthase and levopimaradiene/abietadiene synthase, provide the hydrocarbon precursors for most of the diterpene resin acids found in the defensive oleoresin of conifers. Although these paralogous enzymes are 91% identical at the amino acid level, one is a single-product enzyme, whereas the other is a multiproduct enzyme that forms completely different products. We used a rational approach of homology modeling, protein sequence comparison, domain swapping, and a series of reciprocal site-directed mutagenesis to identify the specific residues that direct the different product outcomes. A one-amino acid mutation switched the levopimaradiene/abietadiene synthase into producing isopimaradiene and sandaracopimaradiene and none of its normal products. Four mutations were sufficient to reciprocally reverse the product profiles for both of these paralogous enzymes while maintaining catalytic efficiencies similar to the wild-type enzymes. This study illustrates how neofunctionalization can result from relatively minor changes in protein sequence, increasing the diversity of secondary metabolites important for conifer defense.
| S-EPMC2242725 | biostudies-literature
Project description:Two bacterial diterpene synthases (DTSs) from Chryseobacterium were characterised. The first enzyme yielded the new compound chryseodiene that closely resembles the known fusicoccane diterpenes from fungi, but its experimentally and computationally studied cyclisation mechanism is fundamentally different to the mechanism of fusicoccadiene synthase. The second enzyme produced wanjudiene, a diterpene hydrocarbon with a new skeleton, besides traces of the enantiomer of bonnadiene that was recently discovered from Allokutzneria albata.
| S-EPMC7383580 | biostudies-literature
Project description:Arabidopsis thaliana maintains a complex metabolism for the production of secondary or specialized metabolites. Such metabolites include volatile and semivolatile terpenes, which have been associated with direct and indirect defensive activities in flowers and leaves. In comparison, the structural diversity and function of terpenes in Arabidopsis roots has remained largely unexplored despite a substantial number of root-expressed genes in the Arabidopsis terpene synthase (TPS) gene family. We show that five root-expressed TPSs of an expanded subfamily-a type clade in the Arabidopsis TPS family function as class I diterpene synthases that predominantly convert geranylgeranyl diphosphate (GGPP) to different semi-volatile diterpene products, which are in part detectable at low levels in the ecotypes Columbia (Col) and Cape Verde Island (Cvi). The enzyme TPS20 produces a macrocyclic dolabellane diterpene alcohol and a dolabellane-related diterpene olefin named dolathaliatriene with a so far unknown C6-C11 bicyclic scaffold besides several minor olefin products. The TPS20 compounds occur in all tissues of Cvi but are absent in the Col ecotype because of deletion and substitution mutations in the Col TPS20 sequence. The primary TPS20 diterpene products retard the growth of the root rot pathogen Pythium irregulare but only at concentrations exceeding those in planta. Together, our results demonstrate that divergence and pseudogenization in the Arabidopsis TPS gene family allow for structural plasticity in diterpene profiles of above- and belowground tissues.
| S-EPMC5122590 | biostudies-literature
Project description:Fungal highly reducing polyketide synthases (HRPKSs) are highly programmed multidomain enzymes that synthesize reduced polyketide structures. Recent reports indicated salicylaldehydes are synthesized by HRPKS biosynthetic gene clusters, which are unexpected based on known enzymology of HRPKSs. Using genome mining of a Trichoderma virens HRPKS gene cluster that encodes a number of redox enzymes, we uncover the strategy used by HRPKS pathways in the biosynthesis of aromatic products such as salicylaldehyde 4, which can be oxidatively modified to the epoxycyclohexanol natural product trichoxide 1. We show selective β-hydroxyl groups in the linear HRPKS product are individually reoxidized to β-ketones by short-chain dehydrogenase/reductase enzymes, which enabled intramolecular aldol condensation and aromatization. Our work expands the chemical space of natural products accessible through HRPKS pathways.
| S-EPMC6924165 | biostudies-literature
Project description:Diterpenoid biosynthesis has been extensively studied in plants and fungi, yet cloning and engineering diterpenoid pathways in these organisms remain challenging. Bacteria are emerging as prolific producers of diterpenoid natural products, and bacterial diterpene synthases are poised to make significant contributions to our understanding of terpenoid biosynthesis. Here we will first survey diterpenoid natural products of bacterial origin and briefly review their biosynthesis with emphasis on diterpene synthases (DTSs) that channel geranylgeranyl diphosphate to various diterpenoid scaffolds. We will then highlight differences of DTSs of bacterial and higher organism origins and discuss the challenges in discovering novel bacterial DTSs. We will conclude by discussing new opportunities for DTS mechanistic enzymology and applications of bacterial DTS in biocatalysis and metabolic pathway engineering.
| S-EPMC3328645 | biostudies-literature
Project description:Plants produce an immense diversity of natural products (i.e. secondary or specialized metabolites) that offer a rich source of known and potentially new pharmaceuticals and other desirable bioproducts. The Traditional Chinese Medicinal plant Isodon rubescens (Lamiaceae) contains an array of bioactive labdane-related diterpenoid natural products. Of these, the ent-kauranoid oridonin is the most prominent specialized metabolite that has been extensively studied for its potent antimicrobial and anticancer efficacy. Mining of a previously established transcriptome of I. rubescens leaf tissue identified seven diterpene synthase (diTPSs) candidates. Here we report the functional characterization of four I. rubescens diTPSs. IrTPS5 and IrTPS3 were identified as an ent-copalyl diphosphate (CPP) synthase and a (+)-CPP synthase, respectively. Distinct transcript abundance of IrTPS5 and the predicted ent-CPP synthase IrTPS1 suggested a role of IrTPS5 in specialized ent-kaurene metabolism possibly en route to oridonin. Nicotiana benthamiana co-expression assays demonstrated that IrTPS4 functions sequentially with IrTPS3 to form miltiradiene. In addition, IrTPS2 converted the IrTPS3 product (+)-CPP into the hydroxylated tricyclic diterpene nezukol not previously identified in I. rubescens. Metabolite profiling verified the presence of nezukol in I. rubescens leaf tissue. The proposed IrTPS2-catalyzed reaction mechanism proceeds via the common ionization of the diphosphate group of (+)-CPP, followed by formation of an intermediary pimar-15-en-8-yl+ carbocation and neutralization of the carbocation by water capture at C-8 to yield nezukol, as confirmed by nuclear magnetic resonance (NMR) analysis. Oxygenation activity is rare for the family of class I diTPSs and offers new catalysts for developing metabolic engineering platforms to produce a broader spectrum of bioactive diterpenoid natural products.
| S-EPMC5405970 | biostudies-literature
Project description:Many cyclic peptide natural products are larger and structurally more complex than conventional small molecule drugs. Although some molecules in this class are known to possess favorable pharmacokinetic properties, there have been few reports on the membrane permeabilities of cyclic peptide natural products. Here, we present the passive membrane permeabilities of 39 cyclic peptide natural products, and interpret the results using a computational permeability prediction algorithm based on their known or calculated 3D conformations. We found that the permeabilities of these compounds, measured in a parallel artificial membrane permeability assay, spanned a wide range and demonstrated the important influence of conformation on membrane permeability. These results will aid in the development of these compounds as a viable drug paradigm.
| S-EPMC5186412 | biostudies-literature
Project description:Terpenoid synthases catalyze isoprenoid cyclization reactions underlying the generation of more than 80,000 natural products. Such dramatic chemodiversity belies the fact that these enzymes generally consist of only three domain folds designated as ?, ?, and ?. Catalysis by class I terpenoid synthases occurs exclusively in the ? domain, which is found with ?, ??, ??, and ??? domain architectures. Here, we explore the influence of domain architecture on catalysis by taxadiene synthase from Taxus brevifolia (TbTS, ???), fusicoccadiene synthase from Phomopsis amygdali (PaFS, (??)6), and ophiobolin F synthase from Aspergillus clavatus (AcOS, ??). We show that the cyclization fidelity and catalytic efficiency of the ? domain of TbTS are severely compromised by deletion of the ?? domains; however, retention of the ? domain preserves significant cyclization fidelity. In PaFS, we previously demonstrated that one ? domain similarly influences catalysis by the other ? domain [ Chen , M. , Chou , W. K. W. , Toyomasu , T. , Cane , D. E. , and Christianson , D. W. ( 2016 ) ACS Chem. Biol. 11 , 889 - 899 ]. Here, we show that the hexameric quaternary structure of PaFS enables cluster channeling. We also show that the ? domains of PaFS and AcOS can be swapped so as to make functional chimeric ?? synthases. Notably, both cyclization fidelity and catalytic efficiency are altered in all chimeric synthases. Twelve newly formed and uncharacterized C20 diterpene products and three C25 sesterterpene products are generated by these chimeras. Thus, engineered ??? and ?? terpenoid cyclases promise to generate chemodiversity in the greater family of terpenoid natural products.
| S-EPMC5474940 | biostudies-literature
Project description:Tripterygium wilfordii, which has long been used as a medicinal plant, exhibits impressive and effective anti-inflammatory, immunosuppressive and anti-tumor activities. The main active ingredients are diterpenoids and triterpenoids, such as triptolide and celastrol, respectively. A major challenge to harnessing these natural products is that they are found in very low amounts in planta. Access has been further limited by the lack of knowledge regarding their underlying biosynthetic pathways, particularly for the abeo-abietane tri-epoxide lactone triptolide. Here suspension cell cultures of T. wilfordii were found to produce triptolide in an inducible fashion, with feeding studies indicating that miltiradiene is the relevant abietane olefin precursor. Subsequently, transcriptome data were used to identify eight putative (di)terpene synthases that were then characterized for their potential involvement in triptolide biosynthesis. This included not only biochemical studies which revealed the expected presence of class II diterpene cyclases that produce the intermediate copalyl diphosphate (CPP), along with the more surprising finding of an atypical class I (di)terpene synthase that acts on CPP to produce the abietane olefin miltiradiene, but also their subcellular localization and, critically, genetic analysis. In particular, RNA interference targeting either both of the CPP synthases, TwTPS7v2 and TwTPS9v2, or the subsequently acting miltiradiene synthase, TwTPS27v2, led to decreased production of triptolide. Importantly, these results then both confirm that miltiradiene is the relevant precursor and the relevance of the identified diterpene synthases, enabling future studies of the biosynthesis of this important bioactive natural product.
| S-EPMC5848467 | biostudies-literature
Project description:Terpene synthases are capable of mediating complex reactions, but fundamentally simply catalyze lysis of allylic diphosphate esters with subsequent deprotonation. Even with the initially generated tertiary carbocation this offers a variety of product outcomes, and deprotonation further can be preceded by the addition of water. This is particularly evident with labdane-related diterpenes (LRDs) where such lysis follows bicyclization catalyzed by class II diterpene cyclases (DTCs) that generates preceding structural variation. Previous investigation revealed that two diterpene synthases (DTSs), one bacterial and the other plant-derived, exhibit extreme substrate promiscuity, but yet still typically produce exo-ene or tertiary alcohol LRD derivatives, respectively (i.e., demonstrating high catalytic specificity), enabling rational combinatorial biosynthesis. Here two DTSs that produce either cis or trans endo-ene LRD derivatives, also plant and bacterial (respectively), were examined for their potential analogous utility. Only the bacterial trans-endo-ene forming DTS was found to exhibit significant substrate promiscuity (with moderate catalytic specificity). This further led to investigation of the basis for substrate promiscuity, which was found to be more closely correlated with phylogenetic origin than reaction complexity. Specifically, bacterial DTSs exhibited significantly more substrate promiscuity than those from plants, presumably reflecting their distinct evolutionary context. In particular, plants typically have heavily elaborated LRD metabolism, in contrast to the rarity of such natural products in bacteria, and the lack of potential substrates presumably alleviates selective pressure against such promiscuity. Regardless of such speculation, this work provides novel biosynthetic access to almost 19 LRDs, demonstrating the power of the combinatorial approach taken here.
| S-EPMC6717049 | biostudies-literature