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Eine Diterpensynthase aus Saccharopolyspora spinosa konvertierte Geranylgeranyldiphosphat in die neuen Naturstoffe Spinodien A und B, begleitet von 2,7,18‐Dolabellatrien. Die Strukturen und die Bildungsmechanismen der Enzymprodukte wurden mithilfe von Isotopenmarkierungsexperimenten studiert, wodurch ein ungewöhnlicher verzweigter Isomerisierungsmechanismus zu dem neutralen Intermediat 2,7,18‐Dolabellatrien aufgedeckt wurde. Durch eine Diels‐Alder‐Reaktion wurde das Diterpen‐Hauptprodukt mit seiner seltenen konjugierten Dien‐Einheit in formale Sesterterpenalkohole überführt. Herausforderung angenommen: Studien an einer bakteriellen Diterpen‐Synthase zeigten deren ungewöhnlichen Cyclisierungsmechanismus zu Spinodien A und B über neutrale Intermediate. Spinodien A enthält eine seltene konjugierte Dien‐Einheit, die chemischen Zugang zu künstlichen Sesterterpenalkoholen mittels Diels‐Alder‐Reaktion ermöglicht. [ABSTRACT FROM AUTHOR]

A diterpene synthase from Saccharopolyspora spinosa was found to convert geranylgeranyl diphosphate into the new natural products spinodiene A and B, accompanied by 2,7,18‐dolabellatriene. The structures and the formation mechanism of the enzyme products were investigated by extensive isotopic labelling experiments, which revealed an unusual branched isomerisation mechanism towards the neutral intermediate 2,7,18‐dolabellatriene. A Diels–Alder reaction was used to convert the main diterpene product with its rare conjugated diene moiety into formal sesterterpene alcohols. Taking up the challenge: Investigations on a bacterial diterpene synthase have revealed an unusual cyclisation mechanism to spinodiene A and B via neutral intermediates. Spinodiene A features a rare conjugated diene moiety that provides chemical access to artificial sesterterpene alcohols by Diels–Alder reactions. [ABSTRACT FROM AUTHOR]

Based on a terpenoid overproduction platform in yeast for genome mining, a chimeric diterpene synthase from the endophytic fungus Colletotrichum gloeosporioides ES026 was characterized as the (5R,12R,14S)‐dolasta‐1(15),8‐diene synthase. The absolute configuration was independently verified through the use of enantioselectively deuterated terpene precursors, which unequivocally established the predicted C1‐III‐IV cyclization mode for this first characterized clade II‐D enzyme. Extensive isotopic labeling experiments and isolation of the intermediate (1R)‐δ‐araneosene supported the proposed cyclization mechanism. The missing link: Fungal chimeric diterpene synthases with prenyltransferase and terpene synthase domains fall into two clades (I+II) with six subclades (A–F). The present study reports the characterization of the first clade II‐D enzyme, the structures of its products, and the cyclization mechanism. The observed 12R configuration reveals a uniform stereochemistry at this carbon for all clade II enzyme products. [ABSTRACT FROM AUTHOR]

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Copyright of Angewandte Chemie is the property of Wiley-Blackwell and its content may not be copied or emailed to multiple sites or posted to a listserv without the copyright holder's express written permission. However, users may print, download, or email articles for individual use. This abstract may be abridged. No warranty is given about the accuracy of the copy. Users should refer to the original published version of the material for the full abstract. (Copyright applies to all Abstracts.)

Abstract: Zwei Diterpensynthasen aus Allokutzneria albata wurden auf ihre Produkte untersucht. Das Produkt des ersten Enzyms wurde als die neue Verbindung Bonnadien identifiziert. Obwohl das zweite Enzym phylogenetisch nicht mit der pilzlichen Phomopsen‐Synthase in Verbindung steht, produzierte es eine Mischung aus Phomopsen und einer biosynthetisch verwandten neuen Verbindung, Allokutzneren, sowie Spiroviolen. Beide Enzyme wurden im Detail durch Isotopenmarkierungsexperimente, Variation des Metallcofaktors und ortsgerichtete Mutagenese auf ihren Mechanismus untersucht. Oxidationsprodukte von Phomopsen und Allokutzneren werden ebenfalls diskutiert. [ABSTRACT FROM AUTHOR]

Abstract: Two diterpene synthases from Allokutzneria albata were studied for their products, resulting in the identification of the new compound bonnadiene from the first enzyme. Although phylogenetically unrelated to fungal phomopsene synthase, the second enzyme produced a mixture of phomopsene and a biosynthetically linked new compound, allokutznerene, as well as spiroviolene. Both enzymes were subjected to in‐depth mechanistic studies involving isotopic labelling experiments, metal‐cofactor variation, and site‐directed mutagenesis. Oxidation products of phomopsene and allokutznerene are also discussed. [ABSTRACT FROM AUTHOR]

Abstract: Zwei bakterielle Diterpensynthasen aus dem Actinomyceten Allokutzneria albata wurden untersucht. Dies führte zur Identifizierung des strukturell neuartigen Produkts Spiroalbaten des ersten und Cembren A des zweiten Enzyms. Beide Enzyme wurden durch Isotopenmarkierungsexperimente, ortsgerichtete Mutagenese, Variation des Metallcofaktors und des pH‐Wertes auf ihre Mechanismen untersucht. Für die Spiroalbaten‐Synthase wurde eine pH‐ und Mn2+‐abhängige Bildung des Nebenproduktes Thunbergol beobachtet, das biosynthetisch mit Spiroalbaten verwandt ist. [ABSTRACT FROM AUTHOR]

Abstract: Two bacterial diterpene synthases from the actinomycete Allokutzneria albata were investigated, resulting in the identification of the structurally unprecedented compound spiroalbatene from the first and cembrene A from the second enzyme. Both enzymes were thoroughly investigated in terms of their mechanisms by isotope labeling experiments, site‐directed mutagenesis, and variation of the metal cofactors and pH value. For spiroalbatene synthase, the pH‐ and Mn2+‐dependent formation of the side product thunbergol was observed, which is biosynthetically linked to spiroalbatene. [ABSTRACT FROM AUTHOR]

Ectoine is a compatible solute widely synthesized by microorganisms as an osmostress protectant. Due to its stabilizing effect on macromolecules, it is adressed in the literature as a chemical chaperone and used for medical and biotechnological applications. The 2,4-diaminobutyrate acetyltransferase (EctA) catalyzes the second step of the ectoine synthesis pathway, the regio-specific acetylation of 2,4-diaminobutyrate (DAB) to N-y-acetyl-2,4-diaminobutyric acid (ADABA). Here we focus on the biochemical and structural characteristics of EctA from the thermo-tolerant bacterium Paenibacillus lautus. A codon-optimized ectA gene of P. lautus was cloned into expression vectors for heterologous expression in Escherichia coli. The produced protein was purified, used for enzymatic assays, and crystallized. The enzyme activity of EctA was assessed by photometry-based assays measuring the formation of the co-product CoA. Biochemical characterization of the wildtype enzyme showed optimal activity conditions at high temperature (>40 °C) and high pH-values (8.5-9.5). Crystal structures of apo-EctA, and EctA in complex with the substrate DAB and CoA were obtained. These revealed a functional dimer and illuminated the architecture of the enzymes active site. Putative DAB-binding amino acids were verified by site-directed mutagenesis studies. [ABSTRACT FROM AUTHOR]

The dauc-8-en-11-ol synthase from Streptomyces venezuelae was investigated for its catalytic activity towards alternative terpene precursors, specifically designed to enable new cyclisation pathways. Exchange of aromatic amino acid residues at the enzyme surface by site-directed mutagenesis led to a 4-fold increase of the yield in preparative scale incubations, which likely results from an increased enzyme stability instead of improved enzyme kinetics., (© 2021 The Authors. Chemistry - A European Journal published by Wiley-VCH GmbH.)

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., (© 2020 The Authors. Published by Wiley-VCH Verlag GmbH & Co. KGaA.)

Methylated analogues of isopentenyl diphosphate were synthesised and enzymatically incorporated into methylated terpenes. A detailed stereochemical analysis of the obtained products is presented. The methylated terpene precursors were also used in conjunction with various isotopic labellings to gain insights into the mechanisms of their enzymatic formation., (© 2020 The Authors. Published by Wiley-VCH Verlag GmbH & Co. KGaA.)

The skeletons of some classes of terpenoids are unusual in that they contain a larger number of Me groups (or their biosynthetic equivalents such as olefinic methylene groups, hydroxymethyl groups, aldehydes, or carboxylic acids and their derivatives) than provided by their oligoprenyl diphosphate precursor. This is sometimes the result of an oxidative ring-opening reaction at a terpene-cyclase-derived molecule containing the regular number of Me group equivalents, as observed for picrotoxan sesquiterpenes. In this study a sesquiterpene cyclase from Trichoderma spp. is described that can convert farnesyl diphosphate (FPP) directly via a remarkable skeletal rearrangement into trichobrasilenol, a new brasilane sesquiterpene with one additional Me group equivalent compared to FPP. A mechanistic hypothesis for the formation of the brasilane skeleton is supported by extensive isotopic labelling studies., (© 2019 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.)