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Fungal meroterpenoids are a diverse group of hybrid natural products with impressive structural complexity and high potential as drug candidates. In this work, we evaluate the promiscuity of the early structure diversity-generating step in fungal meroterpenoid biosynthetic pathways: the multibond-forming polyene cyclizations catalyzed by the yet poorly understood family of fungal meroterpenoid cyclases. In total, 12 unnatural meroterpenoids were accessed chemoenzymatically using synthetic substrates. Their complex structures were determined by 2D NMR studies as well as crystalline-sponge-based X-ray diffraction analyses. The results obtained revealed a high degree of enzyme promiscuity and experimental results which together with quantum chemical calculations provided a deeper insight into the catalytic activity of this new family of non-canonical, terpene cyclases. The knowledge obtained paves the way to design and engineer artificial pathways towards second generation meroterpenoids with valuable bioactivities based on combinatorial biosynthetic strategies.

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Abstratct [beta]-Nicotinamide adenine dinucleotide ([beta]-NAD) is a pivotal metabolite for all living organisms and functions as a diffusible electron acceptor and carrier in the catabolic arms of metabolism.sup.1,2. Furthermore, [beta]-NAD is involved in diverse epigenetic, immunological and stress-associated processes, where it is known to be sacrificially utilized as an ADP-ribosyl donor for protein and DNA modifications, or the generation of cell-signalling molecules.sup.3,4. Here we report the function of [beta]-NAD in secondary metabolite biosynthetic pathways, in which the nicotinamide dinucleotide framework is heavily decorated and serves as a building block for the assembly of a novel class of natural products. The gatekeeping enzyme of the discovered pathway (SbzP) catalyses a pyridoxal phosphate-dependent [3+2]-annulation reaction between [beta]-NAD and S-adenosylmethionine, generating a 6-azatetrahydroindane scaffold. Members of this novel family of [beta]-NAD-tailoring enzymes are widely distributed in the bacterial kingdom and are encoded in diverse biosynthetic gene clusters. The findings of this work set the stage for the discovery and exploitation of [beta]-NAD-derived natural products. [beta]-Nicotinamide adenine dinucleotide is used as a building block in secondary metabolite biosynthetic pathways for a novel class of natural products., Author(s): Lena Barra [sup.1] , Takayoshi Awakawa [sup.1] [sup.2] , Kohei Shirai [sup.1] , Zhijuan Hu [sup.1] , Ghader Bashiri [sup.3] , Ikuro Abe [sup.1] [sup.2] Author Affiliations: (1) Graduate [...]

Sulfonamides and sulfamates are a group of organosulfur compounds that contain the signature sulfamoyl structural motif. These compounds were initially only known as synthetic antibacterial drugs but were later also discovered as natural products. Eight highly potent examples have been isolated from actinomycetes to date, illustrating the large biosynthetic repertoire of this bacterial genus. For the biosynthesis of these compounds, several distinct and unique biosynthetic machineries have been discovered, capable to generate the unique S-N bond. For the creation of novel, second generation natural products by biosynthetic engineering efforts, a detailed understanding of the underlying enzyme machinery toward potent structural motifs is crucial. In this review, we aim to summarize the current state of knowledge on sulfonamide and sulfamate biosynthesis.A detailed discussion for the secondary sulfamate ascamycin, the tertiary sulfonamide sulfadixiamycin A, and the secondary sulfonamide SB-203208 is provided and their bioactivities and mode of actions are discussed. [ABSTRACT FROM AUTHOR]

The asymmetric synthesis of both enantiomers of tetrasubstituted all-trans dimethyl 3,4-diacetylcyclobutane-1,2-dicarboxylate with high enantiomeric purity (>98 % ee) using a valine-derived chiral auxiliary in a diastereoselective photodimerization is reported. The absolute configuration was assigned by single-crystal X-ray diffraction analysis. Because this cyclobutane is a key intermediate in the total synthesis of (-)-sceptrin and ageliferin, our findings strengthen the recently revised absolute configurations of these pyrrole-imidazole alkaloids. [ABSTRACT FROM AUTHOR]

Eine uncharakterisierte Terpenzyklase aus Streptomyces pratensis wurde als (+)‐(1(10)E,4E,6S,7R)‐Germacradien‐6‐ol‐Synthase identifiziert, deren Produkt als zwei ineinander konvertierbare Konformere existiert, wodurch komplexe NMR‐Spektren resultieren. Für eine komplette Zuordnung der NMR‐Daten wurden alle fünfzehn Isotopomere des (13C1)FPP sowie (13C15)FPP synthetisiert und enzymatisch umgesetzt und die Produkte durch diverse NMR‐Methoden einschließlich 13C,13C‐COSY analysiert. Die (13C)FPP‐Isotopomere wurden weiterhin für die Untersuchung der thermischen Umlagerung und des EI‐Fragmentierungsmechanismus des Enzymprodukts verwendet. Für die Strukturaufklärung, die komplette Zuordnung der NMR‐Daten und eine Untersuchung der Cope‐Umlagerung sowie des EI‐MS‐Fragmentierungsmechanismus wurden alle fünfzehn Isotopomere des (13C1)Farnesyldiphosphats und (13C15)FPP synthetisiert und durch eine neu charakterisierte bakterielle Terpenzyklase aus Streptomyces pratensis enzymatisch zu (+)‐(1(10)E,4E,6S,7R)‐Germacradienol umgesetzt. [ABSTRACT FROM AUTHOR]

An uncharacterized terpene cyclase from Streptomyces pratensis was identified as (+)-(1(10) E,4 E,6 S,7 R)-germacradien-6-ol synthase. The enzyme product exists as two interconvertible conformers, resulting in complex NMR spectra. For the complete assignment of NMR data, all fifteen (13C1)FPP isotopomers (FPP=farnesyl diphosphate) and (13C15)FPP were synthesized and enzymatically converted. The products were analyzed using various NMR techniques, including 13C, 13C COSY experiments. The (13C)FPP isotopomers were also used to investigate the thermal rearrangement and EI fragmentation of the enzyme product. [ABSTRACT FROM AUTHOR]

Die Fütterung von (2,3,4,5,6-13C5)Mevalonolacton an den Pilz Hypomyces odoratus lieferte einen komplett markierten Sesquiterpenether. Die Konnektivität der Kohlenstoffatome wurde aus einem 13C,13C-COSY-Spektrum erschlossen und ergab eine Struktur, die sich von der für Hypodoratoxid zuvor berichteten unterschied, obwohl die 13C-NMR-spektroskopischen Daten übereinstimmten. Die Strukturrevision für Hypodoratoxid wird präsentiert. Dessen absolute Konfiguration wurde vorläufig aus derjenigen des Cometaboliten cis-Dihydroagarofuran abgeleitet. Die Biosynthese wurde durch Fütterung von (3-13C)- und (4,6-13C2)Mevalonolacton untersucht, die Einblicke in komplexe Gerüstumlagerungen während der Terpencyclisierung durch 13C,13C-Kopplungen gaben. [ABSTRACT FROM AUTHOR]

Feeding of (2,3,4,5,6-13C5)mevalonolactone to the fungus Hypomyces odoratus resulted in a completely labeled sesquiterpene ether. The connectivity of the carbon atoms was easily deduced from a 13C,13C COSY spectrum, revealing a structure that was different from the previously reported structure of hypodoratoxide, even though the reported 13C NMR data matched. A structural revision of hypodoratoxide is thus presented. Its absolute configuration was tentatively assigned from its co-metabolite cis-dihydroagarofuran. Its biosynthesis was investigated by feeding of (3-13C)- and (4,6-13C2)mevalonolactone, which gave insights into the complex rearrangement of the carbon skeleton during terpene cyclization by analysis of the 13C,13C couplings. [ABSTRACT FROM AUTHOR]

A flexible, efficient and robust method for the synthesis of isotopically labelled oligoprenyl diphosphates was developed. The method makes use of just a few building blocks (acetone, triethyl phosphonoacetate, and ethyl acetoacetate) from which several isotopomers with deuterium or 13C-labelling are commercially available or can be easily obtained by synthesis. Besides these building blocks, a few deuterated reagents were used for the introduction of deuterium labelling. Furthermore, the synthesis of [14-2H]geranylgeranyl diphosphate is reported. The material was used for a stereochemical analysis of the cyclisation reaction catalysed by tuberculosinyl diphosphate synthase from Mycobacterium tuberculosis. [ABSTRACT FROM AUTHOR]

Fungal meroterpenoids are a diverse group of hybrid natural products with impressive structural complexity and high potential as drug candidates. In this work, we evaluate the promiscuity of the early structure diversity-generating step in fungal meroterpenoid biosynthetic pathways: the multibond-forming polyene cyclizations catalyzed by the yet poorly understood family of fungal meroterpenoid cyclases. In total, 12 unnatural meroterpenoids were accessed chemoenzymatically using synthetic substrates. Their complex structures were determined by 2D NMR studies as well as crystalline-sponge-based X-ray diffraction analyses. The results obtained revealed a high degree of enzyme promiscuity and experimental results which together with quantum chemical calculations provided a deeper insight into the catalytic activity of this new family of non-canonical, terpene cyclases. The knowledge obtained paves the way to design and engineer artificial pathways towards second generation meroterpenoids with valuable bioactivities based on combinatorial biosynthetic strategies., (© 2020 Wiley-VCH GmbH.)

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.)

The biosynthetic gene cluster for the antiplasmodial natural product siphonazole was identified by using a combination of genome mining, imaging, and expression studies in the natural producer Herpetosiphon sp. B060. The siphonazole backbone is assembled from an unusual starter unit from the shikimate pathway that is extended by the action of polyketide synthases and non-ribosomal peptide synthetases with unusual domain structures, including several split modules and a large number of duplicated domains and domains predicted to be inactive. Product release proceeds through decarboxylation and dehydration independent of the thioesterase SphJ and yields the diene terminus of siphonazole. High variation in terms of codon-usage within the gene cluster, together with the dislocated domain organization, suggest a recent emergence in evolutionary terms., (© 2016 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Feeding of (2,3,4,5,6-(13) C5 )mevalonolactone to the fungus Hypomyces odoratus resulted in a completely labeled sesquiterpene ether. The connectivity of the carbon atoms was easily deduced from a (13) C,(13) C COSY spectrum, revealing a structure that was different from the previously reported structure of hypodoratoxide, even though the reported (13) C NMR data matched. A structural revision of hypodoratoxide is thus presented. Its absolute configuration was tentatively assigned from its co-metabolite cis-dihydroagarofuran. Its biosynthesis was investigated by feeding of (3-(13) C)- and (4,6-(13) C2 )mevalonolactone, which gave insights into the complex rearrangement of the carbon skeleton during terpene cyclization by analysis of the (13) C,(13) C couplings., (© 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)