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 biosynthesis of terpenoid natural products begins with a carbocation-based cyclization or prenylation reaction. While these reactions are mechanistically similar, there are several families of enzymes, namely terpene synthases and prenyltransferases, that have evolved to specifically catalyze terpene cyclization or prenylation reactions. Here, we report that bacterial diterpene synthases, enzymes that are traditionally considered to be specific for cyclization, are capable of efficiently catalyzing both diterpene cyclization and the prenylation of small molecules. We investigated this unique dual reactivity of terpene synthases through a series of kinetic, biocatalytic, structural, and bioinformatics studies. Overall, this study unveils the ability of terpene synthases to catalyze C-, N-, O-, and S-prenylation on small molecules, proposes a substrate decoy mechanism for prenylation by terpene synthases, supports the physiological relevance of terpene synthase-catalyzed prenylation in vivo, and addresses questions regarding the evolution of prenylation function and its potential role in natural products biosynthesis.
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Project description:Terpene synthases (TPSs), known gatekeepers of terpenoid diversity, are the main targets for enzyme engineering attempts. To this end, we have determined the crystal structure of Agrocybe pediades linalool synthase (Ap.LS), which has been recently reported to be 44-fold and 287-fold more efficient than bacterial and plant counterparts, respectively. Structure-based molecular modeling followed by in vivo as well as in vitro tests confirmed that the region of 60-69aa and Tyr299 (adjacent to the motif "WxxxxxRY") are essential for maintaining Ap.LS specificity toward a short-chain (C10) acyclic product. Ap.LS Y299 mutants (Y299A, Y299C, Y299G, Y299Q, and Y299S) yielded long-chain (C15) linear or cyclic products. Molecular modeling based on the Ap.LS crystal structure indicated that farnesyl pyrophosphate in the binding pocket of Ap.LS Y299A has less torsion strain energy compared to the wild-type Ap.LS, which can be partially attributed to the larger space in Ap.LS Y299A for better accommodation of the longer chain (C15). Linalool/nerolidol synthase Y298 and humulene synthase Y302 mutations also produced C15 cyclic products similar to Ap.LS Y299 mutants. Beyond the three enzymes, our analysis confirmed that most microbial TPSs have asparagine at the position and produce mainly cyclized products (δ-cadinene, 1,8-cineole, epi-cubebol, germacrene D, β-barbatene, etc.). In contrast, those producing linear products (linalool and nerolidol) typically have a bulky tyrosine. The structural and functional analysis of an exceptionally selective linalool synthase, Ap.LS, presented in this work provides insights into factors that govern chain length (C10 or C15), water incorporation, and cyclization (cyclic vs acyclic) of terpenoid biosynthesis.
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Project description:Pimarane-type diterpenoids are widely distributed in all domains of life, but no structures or catalytic mechanisms of pimarane-type diterpene synthases (DTSs) have been characterized. Here, we report that two class I DTSs, Sat1646 and Stt4548, each accept copalyl diphosphate (CPP) as the substrate to produce isopimara-8,15-diene (1). Sat1646 can also accept syn-CPP and produce syn-isopimaradiene/pimaradiene analogues (2-7), among which 2 possesses a previously unreported "6/6/7" ring skeleton. We solve the crystal structures of Sat1646, Sat1646 complexed with magnesium ions, and Stt4548, thereby revealing the active sites of these pimarane-type DTSs. Substrate modeling and subsequent site-directed mutagenesis experiments demonstrate different structural bases of Sat1646 and Stt4548 for 1 production. Comparisons with previously reported DTSs reveal their distinct carbocation intermediate stabilization mechanisms, which control the conversion of a single substrate CPP into structurally diverse diterpene products. These results illustrate the structural bases for enzymatic catalyses of pimarane-type DTSs, potentially facilitating future DTS engineering and combinatorial biosynthesis.
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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.
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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: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: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