Sesterterpene Synthase Research Articles

Function Switch of a Fungal Sesterterpene Synthase through Molecular Dynamics Simulation Assisted Alteration of an Aromatic Residue Cluster in the Active Pocket of PfNS

AbstractTerpene synthases (TPSs) play pivotal roles in generating diverse terpenoids through complex cyclization pathways. Protein engineering of TPSs offers a crucial approach to expanding terpene diversity. However, significant potential remains untapped due to limited understanding of the structure‐function relationships of TPSs. In this investigation, using a joint approach of molecular dynamics simulations‐assisted engineering and site‐directed mutagenesis, we manipulated the aromatic residue cluster (ARC) of a bifunctional terpene synthase (BFTPS), Pestalotiopsis fici nigtetraene synthase (PfNS). This led to the discovery of previously unreported catalytic functions yielding different cyclization patterns of sesterterpenes. Specifically, a quadruple variant (F89A/Y113F/W193L/T194W) completely altered PfNS′s function, converting it from producing the bicyclic sesterterpene nigtetraene to the tricyclic ophiobolin F. Additionally, analysis of catalytic profiles by double, triple, and quadruple variants demonstrated that the ARC functions as a switch, unprecedently redirecting the production of 5/11 bicyclic (Type B) sesterterpenes to 5/15 bicyclic (Type A) ones. Molecular dynamics simulations and theozyme calculations further elucidated that, in addition to cation‐π interactions, C−H⋅⋅⋅π interactions also play a key role in the cyclization patterns. This study offers a feasible strategy in protein engineering of TPSs for various industrial applications.

Emerindanols A and B: Two Bipolyhydroindenol Sesterterpenes with 5/6-6/5 Coupled Ring System Discovered by Genome Mining.

Genome mining of Emericella sp. XL-029 achieved a new type E sesterterpene synthase, EmES, which affored a novel bipolyhydroindenol sesterterpene, emerindanol A. Heterologous coexpression with the upstream P450 oxidase revealed C-4 hydroxylated product, emerindanol B. Notably, emerindanols A and B represented the first sesterterpenes featuring a unique 5/6-6/5 coupled ring system. EmES was postulated to initiate through C1-IV-V pathway and convert the fused ring intermediate into the final coupled ring product through a spiro skeleton.

Strukturelle Einblicke in die Terpencyclasedomänen Zweier Pilzlicher Sesterterpensyntasen und Enzymengineering zur Diversifizierung von Sesterterpenen

AbstractÜber die Strukturen und katalytischen Mechanismen von Sesterterpensynthasen (StTSs) ist wenig bekannt, was das strukturbasierte Engineering von StTSs zur Erweiterung der strukturellen Diversität von Sesterterpenen erheblich erschwert. Wir berichten hier über die Kristallstrukturen der Terpencyclisierungsdomänen (TC) von zwei pilzlichen StTSs: der Sesterfisherol‐Synthase (NfSS) und der Sesterbrasiliatrien‐Synthase (PbSS). Beide TC‐Strukturen enthalten Benzyltriethylammoniumchlorid (BTAC), Pyrophosphat (PPi) und Magnesiumionen (Mg2+), wodurch die katalytisch aktiven Zentren klar definiert sind. Eine Kombination aus Theorie und Experimenten, einschließlich der Modellierung carbokationischer Intermediate, ortsgerichteter Mutagenese und Isotopenmarkierungen, lieferte detaillierte Einblicke in die strukturellen Grundlagen der katalytischen Mechanismen. Strukturbasiertes Engineering von NfSS und PbSS führte zur Bildung von 20 Sesterterpenen, darunter 13 neue Verbindungen und vier Epimerpaare mit unterschiedlichen Konfigurationen an C18. Diese Ergebnisse erweitern die strukturelle Vielfalt von Sesterterpenen und liefern wichtige Erkenntnisse für die zukünftige Forschung im Bereich der synthetischen Biologie.

Structural Insights Into the Terpene Cyclization Domains of Two Fungal Sesterterpene Synthases and Enzymatic Engineering for Sesterterpene Diversification.

Little is known about the structures and catalytic mechanisms of sesterterpene synthases (StTSs), which greatly hinders the structure-based engineering of StTSs for structural diversity expansion of sesterterpenes. We here report on the crystal structures of the terpene cyclization (TC) domains of two fungal StTSs: sesterfisherol synthase (NfSS) and sesterbrasiliatriene synthase (PbSS). Both TC structures contain benzyltriethylammonium chloride (BTAC), pyrophosphate (PPi), and magnesium ions (Mg2+), clearly defining the catalytic active sites. A combination of theory and experiments including carbocationic intermediates modeling, site-directed mutagenesis, and isotope labeling provided detailed insights into the structural basis for their catalytic mechanisms. Structure-based engineering of NfSS and PbSS resulted in the formation of 20 sesterterpenes including 13 new compounds and four pairs of epimers with different configurations at C18. These results expand the structural diversity of sesterterpenes and provide important insights for future synthetic biology research.

Two Sesterterpene Synthases from Lentzea atacamensis Demonstrate the Role of Conformational Variability in Terpene Biosynthesis.

Mining of two multiproduct sesterterpene synthases from Lentzea atacamensis resulted in the identification of the synthases for lentzeadiene (LaLDS) and atacamatriene (LaATS). The main product of LaLDS (lentzeadiene) is a new compound, while one of the side products (lentzeatetraene) is the enantiomer of brassitetraene B and the other side product (sestermobaraene F) is known from a surprisingly distantly related sesterterpene synthase. LaATS produces six new compounds, one of which is the enantiomer of the known sesterterpene Bm1. Notably, for both enzymes the products cannot all be explained from one and the same starting conformation of geranylfarnesyl diphosphate, demonstrating the requirement of conformational flexibility of the substrate in the enzymes' active sites. For lentzeadiene an intriguing thermal [1,5]-sigmatropic rearrangement was discovered, reminiscent of the biosynthesis of vitamin D3. All enzyme reactions and the [1,5]-sigmatropic rearrangement were investigated through isotopic labeling experiments and DFT calculations. The results also emphasize the importance of conformational changes during terpene cyclizations.

Zwei Sesterterpen‐Syntasen aus Lentzea atacamensis demonstrieren die Rolle der Konformationsvariabilität in der Terpenbiosynthese

AbstractStudien an zwei Multiprodukt‐Sesterterpensynthasen aus Lentzea atacamensis führten zur Identifizierung der Synthasen für Lentzeadien (LaLDS) und Atacamatrien (LaATS). Das Hauptprodukt von LaLDS (Lentzeadien) ist eine neue Verbindung, während eines der Nebenprodukte (Lentzeatetraen) das Enantiomer von Brassitetraen B ist und ein anderes Nebenprodukt (Sestermobaraen F) von einer überraschend weit entfernt verwandten Sesterterpensynthase bekannt ist. LaATS stellt sechs neue Verbindungen her, darunter das Enantiomer des bekannten Sesterterpens Bm1. Bemerkenswert ist, dass die Produkte beider Enzyme nicht alle aus ein und derselben Ausgangskonformation von Geranylfarnesyldiphosphat erklärt werden können, was die Notwendigkeit einer Konformationsflexibilität des Substrats in den aktiven Zentren der Enzyme zeigt. Für Lentzeadien wurde eine faszinierende thermische [1,5]‐sigmatrope Umlagerung entdeckt, die an die Biosynthese von Vitamin D3 erinnert. Alle Enzymreaktionen und die [1,5]‐sigmatrope Umlagerung wurden durch Isotopenmarkierungsexperimente und DFT‐Berechnungen untersucht. Die Ergebnisse unterstreichen auch die Bedeutung von Konformationsänderungen während Terpencyclisierungen.

Functional characterisation of twelve terpene synthases from actinobacteria.

Fifteen type I terpene synthase homologs from diverse actinobacteria that were selected based on a phylogenetic analysis of more than 4000 amino acid sequences were investigated for their products. For four enzymes with functions not previously reported from bacterial terpene synthases the products were isolated and their structures were elucidated by NMR spectroscopy, resulting in the discovery of the first terpene synthases for (+)-δ-cadinol and (+)-α-cadinene, besides the first two bacterial (-)-amorpha-4,11-diene synthases. For other terpene synthases with functions reported from bacteria before the products were identified by GC-MS. The characterised enzymes include a new epi-isozizaene synthase with monoterpene synthase side activity, a 7-epi-α-eudesmol synthase that also produces hedycaryol and germacrene A, and four more sesquiterpene synthases that produce mixtures of hedycaryol and germacrene A. Three phylogenetically related enzymes were in one case not expressed and in two cases inactive, suggesting pseudogenisation in the respective branch of the phylogenetic tree. Furthermore, a diterpene synthase for allokutznerene and a sesterterpene synthase for sesterviolene were identified.

Turning a Sesquiterpene Synthase into a Di- and Sesterterpene Synthase

Turning a Sesquiterpene Synthase into a Di- and Sesterterpene Synthase

Genomics-driven derivatization of the bioactive fungal sesterterpenoid variecolin: Creation of an unnatural analogue with improved anticancer properties

A biosynthetic gene cluster for the bioactive fungal sesterterpenoids variecolin (1) and variecolactone (2) was identified in Aspergillus aculeatus ATCC 16872. Heterologous production of 1 and 2 was achieved in Aspergillus oryzae by expressing the sesterterpene synthase VrcA and the cytochrome P450 VrcB. Intriguingly, the replacement of VrcB with homologous P450s from other fungal terpenoid pathways yielded three new variecolin analogues (5–7). Analysis of the compounds' anticancer activity in vitro and in vivo revealed that although 5 and 1 had comparable activities, 5 was associated with significantly reduced toxic side effects in cancer-bearing mice, indicating its potentially broader therapeutic window. Our study describes the first tests of variecolin and its analogues in animals and demonstrates the utility of synthetic biology for creating molecules with improved biological activities.

Bioinformatic Analysis of Secondary Metabolite Biosynthetic Potential in Pathogenic Fusarium.

Fusarium species are among the filamentous fungi with the most pronounced impact on agricultural production and human health. The mycotoxins produced by pathogenic Fusarium not only attack various plants including crops, causing various plant diseases that lead to reduced yields and even death, but also penetrate into the food chain of humans and animals to cause food poisoning and consequent health hazards. Although sporadic studies have revealed some of the biosynthetic pathways of Fusarium toxins, they are insufficient to satisfy the need for a comprehensive understanding of Fusarium toxin production. In this study, we focused on 35 serious pathogenic Fusarium species with available genomes and systematically analyzed the ubiquity of the distribution of identified Fusarium- and non-Fusarium-derived fungal toxin biosynthesis gene clusters (BGCs) in these species through the mining of core genes and the comparative analysis of corresponding BGCs. Additionally, novel sesterterpene synthases and PKS_NRPS clusters were discovered and analyzed. This work is the first to systematically analyze the distribution of related mycotoxin biosynthesis in pathogenic Fusarium species. These findings enhance the knowledge of mycotoxin production and provide a theoretical grounding for the prevention of fungal toxin production using biotechnological approaches.

Ruptenes: A Family of Terpene Analogs Give Insight into Cyclisation Mechanisms by Cascade Disruption.

The terpenoid substrate analogs (7R)-6,7-dihydrogeranylgeranyl diphosphate (6,7-dihydro-GGPP) and (7R)-6,7-dihydrogeranylfarnesyl diphosphate (6,7-dihydro-GFPP) were synthesised from (S)-citronellol and enzymatically converted with nine diterpene and two sesterterpene synthases, respectively. In two cases the substrate analogs were converted into diterpenes in cyclisation reactions corresponding to those observed for the native substrate GGPP, while the cyclisation cascade was disrupted or redirected in the other nine cases, leading to products that were named ruptenes. Several of the isolated ruptenes represent deprotonation products of cationic intermediates that are analogs of the intermediates proposed along the cyclisation cascades for the native substrates GGPP or GFPP, thus giving insights into the complex reaction mechanisms of terpene synthase mediated biosynthesis.

Ruptene – Eine Familie von Terpenanaloga Erlaubt Einblicke in Cyclisierungsmechanismen durch Unterbrechung der Kaskade

AbstractDie terpenoiden Substratanaloga (7R)‐6,7‐Dihydrogeranylgeranyldiphosphat (6,7‐Dihydro‐GGPP) und (7R)‐6,7‐Dihydrogeranylfarnesyldiphosphat (6,7‐Dihydro‐GFPP) wurden ausgehend von (S)‐Citronellol synthetisiert und enzymatisch mit neun Diterpen‐ bzw. zwei Sesterterpen‐Synthasen umgewandelt. In zwei Fällen wurden die Substratanaloga in Cyclisierungsreaktionen, die denen des nativen Substrats GGPP entsprachen, zu Diterpenen umgesetzt, während in den anderen neun Fällen die Cyclisierungskaskade unterbrochen oder umgelenkt wurde, was zu Produkten führte, die als Ruptene bezeichnet wurden. Mehrere der isolierten Ruptene stellen Deprotonierungsprodukte kationischer Intermediate dar, die Analoga der Intermediate sind, die entlang der Cyclisierungskaskaden für die nativen Substrate GGPP oder GFPP vorgeschlagen werden, und geben so Einblicke in die komplexen Reaktionsmechanismen der durch Terpensynthasen vermittelten Biosynthese.

Mechanistic Characterisation of the Bacterial Sesterviridene Synthase from Kitasatospora viridis.

A gene coding for a terpene synthase homolog from Kitasatospora viridis was cloned and expressed in Escherichia coli. The purified recombinant protein possessed sesterterpene synthase activity and efficiently converted geranylfarnesyl diphosphate (GFPP) with 19 % yield into the sesterterpene hydrocarbon sesterviridene A. Large scale enzymatic conversions also allowed for the isolation of two side products that are generated with very low yields of ca. 0.1 %. Several derivatives of sesterviridene A were obtained by chemical transformations, securing the NMR-based structural assignments. The absolute configuration of sesterviridene A was determined by chemical correlation using stereoselectively deuterated precursors and by anomalous dispersion X-ray crystallography. The cyclisation mechanism from GFPP to sesterviridene A was extensively studied through isotopic labelling experiments and DFT calculations.

Mechanistic Characterisation and Engineering of Sesterviolene Synthase from Streptomyces violens.

The sesterviolene synthase from Streptomyces violens was identified and represents the second known sesterterpene synthase from bacteria. Isotopic labelling experiments in conjunction with DFT calculations were performed that provided detailed insight into its complex cyclisation mechanism. Enzyme engineering through site-directed mutagenesis gave access to a high-yielding enzyme variant that provided six additional minor products and the main product in sufficient quantities to study its chemistry.

Characterization of two chimeric sesterterpene synthases from a fungal symbiont isolated from a sesterterpenoid-producing Lamiaceae plant Leucosceptrum canum

Two chimeric sesterterpene synthases (AaTPS1 and AaTPS2) were functionally characterized from Alternaria alternata MB-30 isolated from the leaves of a sesterterpenoid-producing Lamiaceae plant Leucosceptrum canum. AaTPS1 generated a 5/8/6/5 tetracyclic sesteraltererol (1) and its absolute stereochemistry was determined by X-ray crystallographic analysis of its derivative 10,11-epoxysesteraltererol (2), which enabled revision of the absolute configuration of C7 of sesterfisherol produced by NfSS and PTTS014 characterized previously and its derivative 10,11-epoxysesterfisherol. AaTPS2 produced a 5/15 bicyclic preterpestacin I (3). Site-directed mutagenesis suggested that F192 in AaTPS1 was likely involved in controlling of the hydroxylation of C12, and eight amino acids were important for the enzyme activity of AaTPS1 and AaTPS2. The engineered Escherichia coli and Saccharomyces cerevisiae strains were constructed for the productions of compounds 1 and 3, and the highest titer of compound 1 reached 62.3 mg/L in shake-flask culture. Both compounds 1 and 2 showed anti-adipogenic activity.

Mechanism of the Bifunctional Multiple Product Sesterterpene Synthase AcAS from Aspergillus calidoustus.

The multiproduct chimeric sesterterpene synthase AcAS from Aspergillus calidoustus yielded spirocyclic calidoustene, which exhibits a novel skeleton, besides five known sesterterpenes. The complex cyclisation mechanism to all six compounds was investigated by isotopic labelling experiments in combination with DFT calculations. Chemically synthesised 8‐hydroxyfarnesyl diphosphate was converted with isopentenyl diphosphate and AcAS into four oxygenated sesterterpenoids that structurally resemble cytochrome P450 oxidation products of the sesterterpene hydrocarbons. Protein engineering of AcAS broadened the substrate scope and gave significantly improved enzyme yields.

Mechanismus der bifunktionellen Multiprodukt‐Sesterterpensynthase AcAS aus Aspergillus calidoustus

AbstractDie chimäre Multiprodukt‐Sesterterpensynthase AcAS aus Aspergillus calidoustus lieferte neben fünf bekannten Sesterterpenen das spirocyclische Calidousten, welches ein neues Gerüst aufweist. Der komplexe Cyclisierungsmechanismus zu allen sechs Verbindungen wurde durch Isotopenmarkierungsexperimente in Kombination mit DFT‐Rechnungen untersucht. Chemisch synthetisiertes 8‐Hydroxyfarnesyldiphosphat wurde mit Isopentenyldiphosphat und AcAS in vier oxygenierte Sesterterpenoide umgewandelt, die strukturell den Cytochrom‐P450‐Oxidationsprodukten der Sesterterpen‐Kohlenwasserstoffe ähneln. Das Proteinengineering von AcAS erweiterte dessen Substratspektrum und führte zu signifikant verbesserten Enzymausbeuten.

Genome mining reveals a new cyclopentane-forming sesterterpene synthase with unprecedented stereo-control

Fungal bifunctional terpene synthase PstA catalyzes the formation of 5/15cis-fused ring system.

Targeting active site residues and structural anchoring positions in terpene synthases.

The sesterterpene synthase SmTS1 from Streptomyces mobaraensis contains several unusual residues in positions that are otherwise highly conserved. Site-directed mutagenesis experiments for these residues are reported that showed different effects, resulting in some cases in an improved catalytic activity, but in other cases in a loss of enzyme function. For other enzyme variants a functional switch was observed, turning SmTS1 from a sesterterpene into a diterpene synthase. This article gives rational explanations for these findings that may generally allow for protein engineering of other terpene synthases to improve their catalytic efficiency or to change their functions.

Biosynthetic Gene Cluster for Asperterpenols A and B and the Cyclization Mechanism of Asperterpenol A Synthase.

The biosynthetic gene cluster for the sesterterpenoids asperterpenols A and B from Aspergillus calidoustus CBS121601 is described. The cluster contains two genes, acldAS for a chimeric sesterterpene synthase and acldA-P450 for a cytochrome P450 monooxygenase. The cyclization mechanism of the asperterpenol A synthase, catalyzing the formation of a unique sesterterpene with a 6/6/8/5 tetracyclic ring system, was studied using isotopically labeled terpene precursors.