Bifunctional Diterpene Synthase Research Articles

Structural and mechanistic insights into the precise product synthesis by a bifunctional miltiradiene synthase.

Selaginella moellendorffii miltiradiene synthase (SmMDS) is a unique bifunctional diterpene synthase (diTPS) that catalyses the successive cyclization of (E,E,E)-geranylgeranyl diphosphate (GGPP) via (+)-copalyl diphosphate (CPP) to miltiradiene, which is a crucial precursor of important medicinal compounds, such as triptolide, ecabet sodium and carnosol. Miltiradiene synthetic processes have been studied in monofunctional diTPSs, while the precise mechanism by which active site amino acids determine product simplicity and the experimental evidence for reaction intermediates remain elusive. In addition, how bifunctional diTPSs work compared to monofunctional enzymes is attractive for detailed research. Here, by mutagenesis studies of SmMDS, we confirmed that pimar-15-en-8-yl+ is an intermediate in miltiradiene synthesis. Moreover, we determined the apo-state and the GGPP-bound state crystal structures of SmMDS. By structure analysis and mutagenesis experiments, possible contributions of key residues both in class I and II active sites were suggested. Based on the structural and functional analyses, we confirmed the copal-15-yl+ intermediate and unveiled more details of the catalysis process in the SmMDS class I active site. Moreover, the structural and experimental results suggest an internal channel for (+)-CPP produced in the class II active site moving towards the class I active site. Our research is a good example for intermediate identification of diTPSs and provides new insights into the product specificity determinants and intermediate transport, which should greatly facilitate the precise controlled synthesis of various diterpenes.

Engineering the Prenyltransferase Domain of a Bifunctional Assembly-Line Terpene Synthase.

Copalyl diphosphate (CPP) synthase from Penicillium verruculosum (PvCPS) is a bifunctional diterpene synthase with both prenyltransferase and class II cyclase activities. The prenyltransferase α domain catalyzes the condensation of C5 dimethylallyl diphosphate with three successively added C5 isopentenyl diphosphates (IPPs) to form C20 geranylgeranyl diphosphate (GGPP), which then undergoes a class II cyclization reaction at the βγ domain interface to generate CPP. The prenyltransferase α domain mediates oligomerization to form a 648-kD (αβγ)6 hexamer. In the current study, we explore prenyltransferase structure-function relationships in this oligomeric assembly-line platform with the goal of generating alternative linear isoprenoid products. Specifically, we report steady-state enzyme kinetics, product analysis, and crystal structures of various site-specific variants of the prenyltransferase α domain. Crystal structures of the H786A, F760A, S723Y, S723F, and S723T variants have been determined at resolutions of 2.80, 3.10, 3.15, 2.65, and 2.00 Å, respectively. The substitution of S723 with bulky aromatic amino acids in the S723Y and S723F variants constricts the active site, thereby directing the formation of the shorter C15 isoprenoid, farnesyl diphosphate. While the S723T substitution only subtly alters enzyme kinetics and does not compromise GGPP biosynthesis, the crystal structure of this variant reveals a nonproductive binding mode for IPP that likely accounts for substrate inhibition at high concentrations. Finally, mutagenesis of the catalytic general acid in the class II cyclase domain, D313A, significantly compromises prenyltransferase activity. This result suggests molecular communication between the prenyltransferase and cyclase domains despite their distant connection by a flexible polypeptide linker.

Elucidation of Terpenoid Biosynthesis in Sclerotinia

Terpenes are a large class of specialized metabolites found in plant and fungus species that have been widely studied for their medicinal and cytotoxic properties. The plant pathogenic fungus Sclerotinia homoeocarpa was previously shown to produce eleven nortetralabdane compounds that exhibit strong antiplasmodial and growth inhibitive properties. Our objective is to elucidate the biosynthesis of these unique terpene compounds in S. homoeocarpa. Genome mining identified two terpene cyclases as well as multiple clustered cytochromes P450 (CYPs). Combinatorial expression in Escherichia coli and GC‐MS analysis of extracts revealed that one cyclase, ShTPS1, is a bifunctional diterpene synthase that produces the 20‐carbon compound pimara‐8(14),15‐diene. This diterpene likely acts as a scaffold to be modified by downstream CYPs into nortetralabdanes. The second cyclase, ShTPS2, produces several 25‐carbon sesterterpenes, one of the least well‐characterized subsets of terpenes and a frontier for pharmaceutical research. The discovery of ShTPS2 is uniquely interesting due to its promiscuity and the rarity of characterized bifunctional sesterterpene synthases ‐ terpene synthases with a chimeric prenyltransferase domain. Homology modeling of ShTPS2 identified targets for site‐directed mutagenesis in the prenyltransferase and terpene cyclase domains. Mutations in these areas reveal both the functional plasticity of the enzyme and that the promiscuity may result from cationic intermediates of a single terpene cyclase pathway. This research contributes to understanding the diversity of terpene biosynthesis in fungi and allows for the biosynthesis of compounds of interest that are naturally produced at low levels, enabling future work to investigate their function.Support or Funding InformationThis work was supported by the Massachusetts Agricultural Experiment Station as well as the University of Massachusetts Amherst Commonwealth Honors College and Department of Biochemistry and Molecular Biology.

Higher-order oligomerization of a chimeric αβγ bifunctional diterpene synthase with prenyltransferase and class II cyclase activities is concentration-dependent

The unusual diterpene (C20) synthase copalyl diphosphate synthase from Penicillium verruculosum (PvCPS) is the first bifunctional terpene synthase identified with both prenyltransferase and class II cyclase activities in a single polypeptide chain with αβγ domain architecture. The C-terminal prenyltransferase α domain generates geranylgeranyl diphosphate which is then cyclized to form copalyl diphosphate at the N-terminal βγ domain interface. We now demonstrate that PvCPS exists as a hexamer at high concentrations – a unique quaternary structure for known αβγ terpene synthases. Hexamer assembly is corroborated by a 2.41 Å-resolution crystal structure of the α domain prenyltransferase obtained from limited proteolysis of full-length PvCPS, as well as the ab initio model of full-length PvCPS derived from small-angle X-ray scattering data. Hexamerization of the prenyltransferase α domain appears to drive the hexamerization of full-length PvCPS. The PvCPS hexamer dissociates into lower-order species at lower concentrations, as evidenced by size-exclusion chromatography in-line with multiangle light scattering, sedimentation velocity analytical ultracentrifugation, and native polyacrylamide gel electrophoresis experiments, suggesting that oligomerization is concentration dependent. Even so, PvCPS oligomer assembly does not affect prenyltransferase activity in vitro.

Biosynthetic study of conidiation-inducing factor conidiogenone: heterologous production and cyclization mechanism of a key bifunctional diterpene synthase.

Conidiogenone, a diterpene with a unique structure, is known to induce the conidiation of Penicillium cyclopium. The biosynthetic pathway of (-)-conidiogenone has been fully elucidated by the heterologous expression of biosynthetic genes in Aspergillus oryzae and by in vitro enzyme assay with 13C-labeled substrates. After construction of deoxyconidiogenol by the action of bifunctional terpene synthase, one cytochrome P450 catalyzes two rounds of oxidation to furnish conidiogenone. Notably, similar biosynthetic genes are conserved among more than 10 Penicillium sp., suggesting that conidiogenone is a common conidiation inducer in this genus. The cyclization mechanism catalyzed by terpene synthase, which involves successive 1,2-alkyl shifts, was fully elucidated using 13C-labeled geranylgeranyl pyrophosphate (GGPP) as substrate. During the structural analysis of deoxyconidiogenol, we observed broadening of some of the 13C signals measured at room temperature, which has not been observed with other structurally related compounds. Careful examination using techniques including 13C NMR studies at -80 °C, conformational analysis and prediction of the 13C chemical shifts using density functional theory gave insights into this intriguing phenomenon.

Engineering modular diterpene biosynthetic pathways in Physcomitrella patens

Main conclusionModular assembly and heterologous expression in the moss Physcomitrella patens of pairs of diterpene synthases results in accumulation of modern land plant diterpenoids.Physcomitrella patens is a representative of the ancient bryophyte plant lineage with a genome size of 511 Mb, dominant haploid life cycle and limited chemical and metabolic complexity. For these plants, exceptional capacity for genome editing through homologous recombination is met with recently demonstrated in vivo assembly of multiple heterologous DNA fragments. These traits earlier made P. patens an attractive choice as a biotechnological chassis for photosynthesis-driven production of recombinant peptides. The lack of diterpene gibberellic acid phytohormones in P. patens combined with the recent targeted disruption of the single bifunctional diterpene synthase yielded lines devoid of endogenous diterpenoid metabolites and well-suited for engineering of terpenoid production. Here, we mimicked the modular nature of diterpene biosynthetic pathways found in modern land plants by developing a flexible pipeline to install three combinations of class II and class I diterpene synthases in P. patens to access industrially relevant diterpene biomaterials. In addition to a well-established neutral locus for targeted integration, we also explored loci created by a class of Long Terminal Repeat Retrotransposon present at moderate number in the genome of P. patens. Assembly of the pathways and production of the enzymes from the neutral locus led to accumulation of diterpenes matching the reported activities in the angiosperm sources. In contrast, insights gained with the retrotransposon loci indicate their suitability for targeting, but reveal potentially inherent complications which may require adaptation of the experimental design.

Pilot-scale cultivation of wall-deficient transgenic Chlamydomonas reinhardtii strains expressing recombinant proteins in the chloroplast.

Microalgae have emerged as potentially powerful platforms for the production of recombinant proteins and high-value products. Chlamydomonas reinhardtii is a potentially important host species due to the range of genetic tools that have been developed for this unicellular green alga. Transformation of the chloroplast genome offers important advantages over nuclear transformation, and a wide range of recombinant proteins have now been expressed in the chloroplasts of C. reinhardtii strains. This is often done in cell wall-deficient mutants that are easier to transform. However, only a single study has reported growth data for C. reinhardtii grown at pilot scale, and the growth of cell wall-deficient strains has not been reported at all. Here, we report the first pilot-scale growth study for transgenic, cell wall-deficient C. reinhardtii strains. Strains expressing a cytochrome P450 (CYP79A1) or bifunctional diterpene synthase (cis-abienol synthase, TPS4) were grown for 7days under mixotrophic conditions in a Tris-acetate-phosphate medium. The strains reached dry cell weights of 0.3g/L within 3-4days with stable expression levels of the recombinant proteins during the whole upscaling process. The strains proved to be generally robust, despite the cell wall-deficient phenotype, but grew poorly under phototrophic conditions. The data indicate that cell wall-deficient strains may be highly amenable for transformation and suitable for commercial-scale operations under mixotrophic growth regimes.

Structure and Function of Fusicoccadiene Synthase, a Hexameric Bifunctional Diterpene Synthase.

Fusicoccin A is a diterpene glucoside phytotoxin generated by the fungal pathogen Phomopsis amygdali that causes the plant disease constriction canker, first discovered in New Jersey peach orchards in the 1930s. Fusicoccin A is also an emerging new lead in cancer chemotherapy. The hydrocarbon precursor of fusicoccin A is the tricyclic diterpene fusicoccadiene, which is generated by a bifunctional terpenoid synthase. Here, we report X-ray crystal structures of the individual catalytic domains of fusicoccadiene synthase: the C-terminal domain is a chain elongation enzyme that generates geranylgeranyl diphosphate, and the N-terminal domain catalyzes the cyclization of geranylgeranyl diphosphate to form fusicoccadiene. Crystal structures of each domain complexed with bisphosphonate substrate analogues suggest that three metal ions and three positively charged amino acid side chains trigger substrate ionization in each active site. While in vitro incubations reveal that the cyclase domain can utilize farnesyl diphosphate and geranyl diphosphate as surrogate substrates, these shorter isoprenoid diphosphates are mainly converted into acyclic alcohol or hydrocarbon products. Gel filtration chromatography and analytical ultracentrifugation experiments indicate that full-length fusicoccadiene synthase adopts hexameric quaternary structure, and small-angle X-ray scattering data yield a well-defined molecular envelope illustrating a plausible model for hexamer assembly.

Additional diterpenes from Physcomitrella patens synthesized by copalyl diphosphate/kaurene synthase (PpCPS/KS)

The bifunctional diterpene synthase, copalyl diphosphate/kaurene synthase from the moss Physcomitrella patens (PpCPS/KS), catalyses the formation of at least four diterpenes, including ent-beyerene, ent-sandaracopimaradiene, ent-kaur-16-ene, and 16-hydroxy-ent-kaurene. The enzymatic activity has been confirmed through generation of a targeted PpCPS/KS knock-out mutant in P. patens via homologous recombination, through transient expression of PpCPS/KS in Nicotiana benthamiana, and expression of PpCPS/KS in E. coli. GC-MS analysis of the knock-out mutant shows that it lacks the diterpenoids, supporting that all are products of PpCPS/KS as observed in N. benthamiana and E. coli. These results provide additional knowledge of the mechanism of this bifunctional diterpene synthase, and are in line with proposed reaction mechanisms in kaurene biosynthesis.

Stable expression of a bifunctional diterpene synthase in the chloroplast of Chlamydomonas reinhardtii

Chlamydomonas reinhardtii has been shown to hold significant promise as a production platform for recombinant proteins, but transformation of the nuclear genome is still a non-trivial process due to random gene insertion and frequent silencing. Insertion of transgenes into the chloroplasts is an alternative strategy, and we report here the stable expression of a large (91 kDa) protein in the chloroplast using a recently developed low-cost transformation protocol. Moreover, selection of transformants is based on restoration of prototrophy using an endogenous gene (psbH) as the marker, thereby allowing the generation of transgenic lines without the use of antibiotic-resistance genes. Here, we have expressed a bifunctional diterpene synthase in C. reinhardtii chloroplasts. Homoplasmic transformants were obtained with the expressed enzyme accounting for 3.7 % of total soluble protein. The enzyme was purified to homogeneity and expression was shown to have a small but reproducible effect on growth rate at the end of log phase growth. These results demonstrate that large recombinant enzymes can be synthesised in the algal chloroplast, and serve to underline its potential as a platform for the biosynthesis of novel metabolites.

A Novel Labda‐7,13E‐dien‐15‐ol‐Producing Bifunctional Diterpene Synthase from Selaginella moellendorffii

Two-in-one enzyme: A bifunctional diterpene synthase from the lycophyte Selaginella moellendorffii has been characterized. The structure of its product, labda-7,13E-dien-15-ol, demonstrates that this enzyme catalyzes a novel class II diterpene cyclization reaction, and clarifies the biosynthetic origins of the family of derived natural products.

Identification and Functional Characterization of Monofunctionalent-Copalyl Diphosphate andent-Kaurene Synthases in White Spruce Reveal Different Patterns for Diterpene Synthase Evolution for Primary and Secondary Metabolism in Gymnosperms

The biosynthesis of the tetracyclic diterpene ent-kaurene is a critical step in the general (primary) metabolism of gibberellin hormones. ent-Kaurene is formed by a two-step cyclization of geranylgeranyl diphosphate via the intermediate ent-copalyl diphosphate. In a lower land plant, the moss Physcomitrella patens, a single bifunctional diterpene synthase (diTPS) catalyzes both steps. In contrast, in angiosperms, the two consecutive cyclizations are catalyzed by two distinct monofunctional enzymes, ent-copalyl diphosphate synthase (CPS) and ent-kaurene synthase (KS). The enzyme, or enzymes, responsible for ent-kaurene biosynthesis in gymnosperms has been elusive. However, several bifunctional diTPS of specialized (secondary) metabolism have previously been characterized in gymnosperms, and all known diTPSs for resin acid biosynthesis in conifers are bifunctional. To further understand the evolution of ent-kaurene biosynthesis as well as the evolution of general and specialized diterpenoid metabolisms in gymnosperms, we set out to determine whether conifers use a single bifunctional diTPS or two monofunctional diTPSs in the ent-kaurene pathway. Using a combination of expressed sequence tag, full-length cDNA, genomic DNA, and targeted bacterial artificial chromosome sequencing, we identified two candidate CPS and KS genes from white spruce (Picea glauca) and their orthologs in Sitka spruce (Picea sitchensis). Functional characterization of the recombinant enzymes established that ent-kaurene biosynthesis in white spruce is catalyzed by two monofunctional diTPSs, PgCPS and PgKS. Comparative analysis of gene structures and enzyme functions highlights the molecular evolution of these diTPSs as conserved between gymnosperms and angiosperms. In contrast, diTPSs for specialized metabolism have evolved differently in angiosperms and gymnosperms.

Gibberellin biosynthesis in bacteria: Separate ent-copalyl diphosphate and ent-kaurene synthases in Bradyrhizobium japonicum

Gibberellins are ent-kaurene-derived diterpenoid phytohormones produced by plants, fungi, and bacteria. The distinct gibberellin biosynthetic pathways in plants and fungi are known, but not that in bacteria. Plants typically use two diterpene synthases to form ent-kaurene, while fungi use only a single bifunctional diterpene synthase. We demonstrate here that Bradyrhizobium japonicum encodes separate ent-copalyl diphosphate and ent-kaurene synthases. These are found in an operon whose enzymatic composition indicates that gibberellin biosynthesis in bacteria represents a third independently assembled pathway relative to plants and fungi. Nevertheless, sequence comparisons also suggest potential homology between diterpene synthases from bacteria, plants, and fungi.