RESUMEN
All known triterpenes are generated by triterpene synthases (TrTSs) from squalene or oxidosqualene1. This approach is fundamentally different from the biosynthesis of short-chain (C10-C25) terpenes that are formed from polyisoprenyl diphosphates2-4. In this study, two fungal chimeric class I TrTSs, Talaromyces verruculosus talaropentaene synthase (TvTS) and Macrophomina phaseolina macrophomene synthase (MpMS), were characterized. Both enzymes use dimethylallyl diphosphate and isopentenyl diphosphate or hexaprenyl diphosphate as substrates, representing the first examples, to our knowledge, of non-squalene-dependent triterpene biosynthesis. The cyclization mechanisms of TvTS and MpMS and the absolute configurations of their products were investigated in isotopic labelling experiments. Structural analyses of the terpene cyclase domain of TvTS and full-length MpMS provide detailed insights into their catalytic mechanisms. An AlphaFold2-based screening platform was developed to mine a third TrTS, Colletotrichum gloeosporioides colleterpenol synthase (CgCS). Our findings identify a new enzymatic mechanism for the biosynthesis of triterpenes and enhance understanding of terpene biosynthesis in nature.
Asunto(s)
Ascomicetos , Talaromyces , Triterpenos , Ascomicetos/enzimología , Colletotrichum/enzimología , Ciclización , Difosfatos/metabolismo , Escualeno/química , Especificidad por Sustrato , Talaromyces/enzimología , Triterpenos/química , Triterpenos/metabolismoRESUMEN
Enediyne natural products are renowned for their potent cytotoxicities but the biosynthesis of their defining 1,5-diyne-3-ene core moiety remains largely enigmatic. Since the discovery of the enediyne polyketide synthase cassette in 2002, genome sequencing has revealed thousands of distinct enediyne biosynthetic gene clusters, each harboring the conserved enediyne polyketide synthase cassette. Here we report that (1) the products of this cassette are an iodoheptaene, a diiodotetrayne and two pentaynes; (2) the diiodotetrayne represents a common biosynthetic intermediate for all known enediynes; and (3) cryptic iodination can be exploited to increase enediyne titers. These findings establish a unified biosynthetic pathway for the enediynes, set the stage to further advance enediyne core biosynthesis and enable fundamental breakthroughs in chemistry, enzymology and translational applications of enediyne natural products.
Asunto(s)
Productos Biológicos , Enediinos , Enediinos/química , Enediinos/metabolismo , Productos Biológicos/química , Productos Biológicos/metabolismo , Familia de Multigenes , Sintasas Poliquetidas/metabolismo , Sintasas Poliquetidas/genética , Vías Biosintéticas , Streptomyces/genética , Streptomyces/metabolismoRESUMEN
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.
RESUMEN
Ectoine is a solute compatible with the physiologies of both prokaryotic and eukaryotic cells and is widely synthesized by bacteria as an osmotic stress protectant. Because it preserves functional attributes of proteins and macromolecular complexes, it is considered a chemical chaperone and has found numerous practical applications. However, the mechanism of its biosynthesis is incompletely understood. The second step in ectoine biosynthesis is catalyzed by l-2,4-diaminobutyrate acetyltransferase (EctA; EC 2.3.1.178), which transfers the acetyl group from acetyl-CoA to EctB-formed l-2,4-diaminobutyrate (DAB), yielding N-γ-acetyl-l-2,4-diaminobutyrate (N-γ-ADABA), the substrate of ectoine synthase (EctC). Here, we report the biochemical and structural characterization of the EctA enzyme from the thermotolerant bacterium Paenibacillus lautus (Pl). We found that (Pl)EctA forms a homodimer whose enzyme activity is highly regiospecific by producing N-γ-ADABA but not the ectoine catabolic intermediate N-α-acetyl-l-2,4-diaminobutyric acid. High-resolution crystal structures of (Pl)EctA (at 1.2-2.2 Å resolution) (i) for its apo-form, (ii) in complex with CoA, (iii) in complex with DAB, (iv) in complex with both CoA and DAB, and (v) in the presence of the product N-γ-ADABA were obtained. To pinpoint residues involved in DAB binding, we probed the structure-function relationship of (Pl)EctA by site-directed mutagenesis. Phylogenomics shows that EctA-type proteins from both Bacteria and Archaea are evolutionarily highly conserved, including catalytically important residues. Collectively, our biochemical and structural findings yielded detailed insights into the catalytic core of the EctA enzyme that laid the foundation for unraveling its reaction mechanism.
Asunto(s)
Acetiltransferasas/química , Aminoácidos Diaminos/biosíntesis , Proteínas Bacterianas/química , Dominio Catalítico , Paenibacillus/química , Cristalografía por Rayos X , Dimerización , Mutagénesis Sitio-Dirigida , Relación Estructura-ActividadRESUMEN
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.
Asunto(s)
Streptomyces , Ciclización , Mutagénesis Sitio-Dirigida , Streptomyces/genética , TerpenosRESUMEN
The flavobacterium Chryseobacterium polytrichastri was investigated for its volatile profile by use of a closed-loop stripping apparatus (CLSA) and subsequent GC-MS analysis. The analyses revealed a rich headspace extract with 71 identified compounds. Compound identification was based on a comparison to library mass spectra for known compounds and on a synthesis of authentic standards for unknowns. Important classes were phenylethyl amides and a series of corresponding imines and pyrroles.
Asunto(s)
Chryseobacterium/química , Compuestos Orgánicos Volátiles/análisis , Cromatografía de Gases y Espectrometría de Masas , Estructura MolecularRESUMEN
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.
Asunto(s)
Transferasas Alquil y Aril/metabolismo , Bacterias/enzimología , Proteínas Bacterianas/metabolismo , Terpenos/metabolismo , Chloroflexi/enzimología , Chryseobacterium/enzimología , Hemiterpenos/síntesis química , Hemiterpenos/química , Hemiterpenos/metabolismo , Metilación , Compuestos Organofosforados/síntesis química , Compuestos Organofosforados/química , Compuestos Organofosforados/metabolismo , Estereoisomerismo , Terpenos/químicaRESUMEN
A sesquiterpene synthase from Streptomyces bungoensis was characterised and produces the new compound bungoene. The enzyme mechanism was deeply investigated using isotopically labelled substrates. Two other enzymes from S. bungoensis made epi-isozizaene and pentalenene. Synthetic oxidative chemistry towards structurally related fusagramineol and pentalenal was explored.
Asunto(s)
Ciclopentanos/metabolismo , Ligasas/metabolismo , Sesquiterpenos/metabolismo , Streptomyces/metabolismo , Ciclopentanos/química , Estructura Molecular , Oxidación-Reducción , Sesquiterpenos/químicaRESUMEN
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.
Asunto(s)
Transferasas Alquil y Aril/metabolismo , Chryseobacterium/enzimología , Diterpenos/metabolismo , Actinobacteria , Modelos Moleculares , Conformación Molecular , EstereoisomerismoRESUMEN
A diterpene synthase from Saccharopolyspora spinosa was found to convert geranylgeranyl diphosphate into the new natural products spinodieneâ 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.
Asunto(s)
Diterpenos/química , Espectroscopía de Resonancia Magnética/métodos , Saccharopolyspora/química , Estructura MolecularRESUMEN
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.
Asunto(s)
Liasas de Carbono-Carbono/metabolismo , Fosfatos de Poliisoprenilo/metabolismo , Sesquiterpenos/metabolismo , Trichoderma/metabolismo , Liasas de Carbono-Carbono/química , Liasas de Carbono-Carbono/genética , Estructura Molecular , Fosfatos de Poliisoprenilo/química , Sesquiterpenos/química , Estereoisomerismo , Trichoderma/enzimología , Trichoderma/genéticaRESUMEN
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.
RESUMEN
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 cembreneâ 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 pHâ value. For spiroalbatene synthase, the pH- and Mn2+ -dependent formation of the side product thunbergol was observed, which is biosynthetically linked to spiroalbatene.
Asunto(s)
Actinomycetales/enzimología , Transferasas Alquil y Aril/metabolismo , Diterpenos/metabolismo , Transferasas Alquil y Aril/química , Diterpenos/química , Concentración de Iones de Hidrógeno , Estructura MolecularRESUMEN
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.
Asunto(s)
Transferasas Alquil y Aril/metabolismo , Colletotrichum/enzimología , Transferasas Alquil y Aril/química , Ciclización , Estructura Molecular , EstereoisomerismoRESUMEN
The substrate flexibility of eight purified sesquiterpene cyclases was evaluated using six new heteroatom-modified farnesyl pyrophosphates, and the formation of six new heteroatom-modified macrocyclic and tricyclic sesquiterpenoids is described. GC-O analysis revealed that tricyclic tetrahydrofuran exhibits an ethereal, peppery, and camphor-like olfactoric scent.
RESUMEN
Dimethylsulfoniopropionate (DMSP) catabolism of marine bacteria plays an important role in marine and global ecology. The genome of Ruegeria pomeroyi DSS-3, a model organism from the Roseobacter group, harbours no less than three genes for different DMSP lyases (DddW, DddP and DddQ) that catalyse the degradation of DMSP to dimethyl sulfide (DMS) and acrylate. Despite their apparent similar function these enzymes show no significant overall sequence identity. In this work DddQ and DddW from R. pomeroyi and the DddP homolog from Phaeobacter inhibens DSM 17395 were functionally characterised and their substrate scope was tested using several synthetic DMSP analogues. Comparative kinetic assays revealed differences in the conversion of DMSP and its analogues in terms of selectivity and overall velocity, giving additional insights into the molecular mechanisms of DMSP lyases and into their putatively different biological functions.
Asunto(s)
Liasas de Carbono-Azufre/química , Roseobacter/enzimología , Acrilatos/química , Acrilatos/metabolismo , Liasas de Carbono-Azufre/metabolismo , Estructura Molecular , Sulfuros/química , Sulfuros/metabolismo , Compuestos de Sulfonio/química , Compuestos de Sulfonio/metabolismoRESUMEN
A terpene synthase from the marine bacterium Streptomyces xinghaiensis has been characterised, including a full structure elucidation of its products from various substrates and an in-depth investigation of the enzyme mechanism by isotope labelling experiments, metal cofactor variations, and mutation experiments. The results revealed an interesting dependency of Mn2+ catalysis on the presence of Asp-217, a residue that is occupied by a highly conserved Glu in most other bacterial terpene synthases.
Asunto(s)
Transferasas Alquil y Aril/metabolismo , Streptomyces/enzimología , Manganeso/química , Manganeso/metabolismo , Estructura MolecularRESUMEN
Actinobacteria, the bacterial phylum most renowned for natural product discovery, has been established as a valuable source for drug discovery and biotechnology but is underrepresented within accessible genome and strain collections. Herein, we introduce the Natural Products Discovery Center (NPDC), featuring 122,449 strains assembled over eight decades, the genomes of the first 8490 NPDC strains (7142 Actinobacteria), and the online NPDC Portal making both strains and genomes publicly available. A comparative survey of RefSeq and NPDC Actinobacteria highlights the taxonomic and biosynthetic diversity within the NPDC collection, including three new genera, hundreds of new species, and ~7000 new gene cluster families. Selected examples demonstrate how the NPDC Portal's strain metadata, genomes, and biosynthetic gene clusters can be leveraged using genome mining approaches. Our findings underscore the ongoing significance of Actinobacteria in natural product discovery, and the NPDC serves as an unparalleled resource for both Actinobacteria strains and genomes.
RESUMEN
Terpenes constitute the largest class of natural products. Their skeletons are formed by terpene cyclases (TCs) from acyclic oligoprenyl diphosphates through sophisticated enzymatic conversions. These enzyme reactions start with substrate ionization through diphosphate abstraction, followed by a cascade reaction via cationic intermediates. Based on isotopic-labelling experiments in combination with a computational study, the cyclization mechanism for sodorifen, a highly methylated sesquiterpene from the soil bacterium Serratia plymuthica, was resolved. A peculiar problem in its biosynthesis lies in the formation of several methyl groups from chain methylene carbons. The underlying mechanism involves a methyltransferase-mediated cyclization and unprecedented ring contraction with carbon extrusion from the chain to form a methyl group. A terpene cyclase subsequently catalyses a fragmentation into two reactive intermediates, followed by hydrogen transfers between them and recombination of the fragments by [4 + 3] cycloaddition. This study solves the intricate mechanistic problem of extra methyl group formation in sodorifen biosynthesis.