Pristinol,a Sesquiterpene Alcohol with an Unusual Skeleton from Streptomyces pristinaespiralis

A terpene cyclase from Streptomyces pristinaespiralis was characterized as the synthase for (+)‐(2S,3S,9R)‐pristinol. The structure of this sesquiterpene alcohol, which has a new carbon skeleton, was established by NMR spectroscopy and single‐wavelength anomalous‐dispersion X‐ray crystallography. Extensive isotopic labelling experiments were performed to distinguish between various possible cyclization mechanisms of the terpene cyclase and to decipher the EI‐MS fragmentation mechanism for pristinol.     

A Branched Diterpene Cascade: The Mechanism of Spinodiene Synthase from Saccharopolyspora spinosa

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.     

Two Diterpene Synthases from Chryseobacterium: Chryseodiene Synthase and Wanjudiene Synthase

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.     

Two Bacterial Diterpene Synthases from Allokutzneria albata Produce Bonnadiene,Phomopsene, and Allokutznerene

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.     

Diterpene Biosynthesis in Actinomycetes: Studies on Cattleyene Synthase and Phomopsene Synthase

Three diterpene synthases from actinomycetes have been studied. The first enzyme from Streptomyces cattleya produced the novel compound cattleyene. The other two enzymes from Nocardia testacea and Nocardia rhamnosiphila were identified as phomopsene synthases. The cyclisation mechanism of cattleyene synthase and the EIMS fragmentation mechanism of its product were extensively studied by incubation experiments with isotopically labelled precursors. Oxidative transformations expanded the chemical space of these unique diterpenes.     

Mechanistic Characterisation of Two Sesquiterpene Cyclases from the Plant Pathogenic Fungus Fusarium fujikuroi

Two sesquiterpene cyclases from Fusarium fujikuroi were expressed in Escherichia coli and purified. The first enzyme was inactive because of a critical mutation, but activity was restored by sequence correction through site‐directed mutagenesis. The mutated enzyme and two naturally functional homologues from other fusaria converted farnesyl diphosphate into guaia‐6,10(14)‐diene. The second enzyme produced eremophilene. The absolute configuration of guaia‐6,10(14)‐diene was elucidated by enantioselective synthesis, while that of eremophilene was evident from the sign of its optical rotation and is opposite to that in plants but the same as in Sorangium cellulosum. The mechanisms of both terpene cyclases were studied with various ¹³C‐ and ²H‐labelled FPP isotopomers.     

An Unusual Terpene Cyclization Mechanism Involving a Carbon–Carbon Bond Rearrangement

Terpene cyclization reactions are fascinating owing to the precise control of connectivity and stereochemistry during the catalytic process. Cyclooctat‐9‐en‐7‐ol synthase (CotB2) synthesizes an unusual 5‐8‐5 fused‐ring structure with six chiral centers from the universal diterpene precursor, the achiral C₂₀ geranylgeranyl diphosphate substrate. An unusual new mechanism for the exquisite CotB2‐catalyzed cyclization that involves a carbon–carbon backbone rearrangement and three long‐range hydride shifts is proposed, based on a powerful combination of in vivo studies using uniformly ¹³C‐labeled glucose and in vitro reactions of regiospecifically deuterium‐substituted geranylgeranyl diphosphate substrates. This study shows that CotB2 elegantly demonstrates the synthetic virtuosity and stereochemical control that evolution has conferred on terpene synthases.     

Mechanistic Investigations of Two Bacterial Diterpene Cyclases: Spiroviolene Synthase and Tsukubadiene Synthase

The mechanisms of two diterpene cyclases from streptomycetes—one with an unknown product that was identified as the spirocyclic hydrocarbon spiroviolene and one with the known product tsukubadiene—were investigated in detail by isotope labeling experiments. Although the structures of the products were very different, the cyclization mechanisms of both enzymes proceed through the same initial cyclization reactions, before they diverge towards the individual products, which is reflected in the close phylogenetic relationship of the enzymes.     

Structures and Biosynthesis of Corvol Ethers—Sesquiterpenes from the Actinomycete Kitasatospora setae

Here we present the functional characterization of a sesquiterpene cyclase from Kitasatospora setae. The enzyme converts the sesquiterpene precursor farnesyl diphosphate (FPP) into two previously unknown and unstable sesquiterpene ethers for which we propose the trivial names corvol ethers A and B. Both compounds were purified and their structures were determined by one‐ and two‐dimensional NMR spectroscopy. A biosynthetic mechanism for the FPP cyclization by the corvol ether synthase was proposed. The results from the incubation experiments of the corvol ether synthase with isotopically labeled precursors were in line with this mechanism, while alternative mechanisms could clearly be ruled out.     

Spata‐13,17‐diene Synthase—An Enzyme with Sesqui‐, Di‐, and Sesterterpene Synthase Activity from Streptomyces xinghaiensis

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.     

Two Diterpene Synthases for Spiroalbatene and Cembrene A from Allokutzneria albata

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.     

Lessons from 1,3‐Hydride Shifts in Sesquiterpene Cyclizations

Stereospecifically labelled precursors were subjected to conversion by seven bacterial sesquiterpene cyclases to investigate the stereochemistry of their initial 1,10‐cyclisation‐1,3‐hydride shift cascades. Enzymes with products of known absolute configuration showed a coherent stereochemical course, except for (?)‐α‐amorphene synthase, for which the obtained results are better explained by an initial 1,6‐cyclisation. The link between the absolute configuration of the product and the stereochemical course of the 1,3‐hydride shifts enabled assignment of the absolute configurations of three enzyme products, which were confirmed independently through the absolute configuration of the common byproduct germacrene D‐4‐ol.     

Biosynthesis of Vitamin B1 (Thiamin): An Instance of Biochemical Diversity

The elucidation of the biosynthetic pathway to thiamin (Vitamin B₁) and its pyrophosphate ester, the important coenzyme “cocarboxylase”, has challenged researchers for many years and continues to do so. The problem of the origin of thiamin can be separated into three parts: the independent pathways to the pyrimidine moiety 4-amino-5-hy-droxymethyl-2-methylpyrimidine and to the thiazole moiety 5-(2-hydroxyethyl)-4-methylthiazole, and the route from these subunits to the vitamin. The steps in the latter process were fully established some twenty years ago, and it was shown that the route in aerobic bacteria and yeast differs to some extent from that in enteric bacteria. The pathways to the subunits, on the other hand, are still not clarified. Significant differences exist in the routes whereby each of the two subunits, the pyrimidine moiety and the thiazole moiety, originate in bacteria and yeast. One difficulty that delayed progress was that the incorporation patterns of labeled precursors, which were observed by different research groups in different microorganisms, could not be reconciled on the basis of a single pathway to each of the two subunits. It is now accepted that in each case different pathways exist in enteric bacteria and yeast, and that the biosynthesis of Vitamin B₁ represents an instance of biochemical diversity. A second factor that added to the difficulties is the minute amount of thiamin synthesized in microbiological cultures (about 15 μg per L culture). This limited the investigations until very recently either to the use of radioactive tracers or to the use of stable isotopes in conjunction with mass spectrometric analysis. It is widely recognized that both methods are associated with pitfalls in the interpretation of results. High-field ¹³C NMR, the most powerful modern method available for the determination of incorporation patterns, has only very recently been successfully employed in investigations of thiamin biosynthesis. As a result of the conceptual and experimental problems, even the primary precursors of each of the two relatively simple heterocyclic subunits of thiamin are still not completely established. A search for committed intermediates, the study of the enzymes, and identification of the genes that are involved are the matter of current research.     

Natural‐Product Sugar Biosynthesis and Enzymatic Glycodiversification

Many biologically active small‐molecule natural products produced by microorganisms derive their activities from sugar substituents. Changing the structures of these sugars can have a profound impact on the biological properties of the parent compounds. This realization has inspired attempts to derivatize the sugar moieties of these natural products through exploitation of the sugar biosynthetic machinery. This approach requires an understanding of the biosynthetic pathway of each target sugar and detailed mechanistic knowledge of the key enzymes. Scientists have begun to unravel the biosynthetic logic behind the assembly of many glycosylated natural products and have found that a core set of enzyme activities is mixed and matched to synthesize the diverse sugar structures observed in nature. Remarkably, many of these sugar biosynthetic enzymes and glycosyltransferases also exhibit relaxed substrate specificity. The promiscuity of these enzymes has prompted efforts to modify the sugar structures and alter the glycosylation patterns of natural products through metabolic pathway engineering and enzymatic glycodiversification. In applied biomedical research, these studies will enable the development of new glycosylation tools and generate novel glycoforms of secondary metabolites with useful biological activity.     

Expanding the Landscape of Diterpene Structural Diversity through Stereochemically Controlled Combinatorial Biosynthesis

Plant‐derived diterpenoids serve as important pharmaceuticals, food additives, and fragrances, yet their low natural abundance and high structural complexity limits their broader industrial utilization. By mimicking the modularity of diterpene biosynthesis in plants, we constructed 51 functional combinations of class I and II diterpene synthases, 41 of which are “new‐to‐nature”. Stereoselective biosynthesis of over 50 diterpene skeletons was demonstrated, including natural variants and novel enantiomeric or diastereomeric counterparts. Scalable biotechnological production for four industrially relevant targets was accomplished in engineered strains of Saccharomyces cerevisiae.     

Identification of Intermediates in the Biosynthesis of PR Toxin by Penicillium roqueforti

The sesquiterpenoid 7‐epi‐neopetasone was synthesized via the Wieland–Miescher ketone. The compound was identical to a previously tentatively identified headspace constituent of Penicillium roqueforti. Feeding experiments with ¹³C‐labeled mevalonolactone isotopomers demonstrated that oxidation at C12 and an isomerization of the C11?C12 to a C7?C11 double bond must occur independently and not via a C7‐C11‐C12 allyl radical in one step. Feeding with (11,12,13‐¹³C₃)‐7‐epi‐neopetasone resulted in labelling of the PR toxin, thus establishing this compound as a newly identified pathway intermediate.     

Terpene Cyclases from Social Amoebae

Genome sequences of social amoebae reveal the presence of terpene cyclases (TCs) in these organisms. Two TCs from Dictyostelium discoideum converted farnesyl diphosphate into (2S,3R,6S,9S)‐(?)‐protoillud‐7‐ene and (3S)‐(+)‐asterisca‐2(9),6‐diene. The enzyme mechanisms and EI‐MS fragmentations of the products were studied by labeling experiments.     

Moenomycin Biosynthesis: Structure and Mechanism of Action of the Prenyltransferase MoeN5

The structure of MoeN5, a unique prenyltransferase involved in the biosynthesis of the antibiotic moenomycin, is reported. MoeN5 catalyzes the reaction of geranyl diphosphate (GPP) with the cis‐farnesyl group in phosphoglycolipid 5 to form the (C₂₅) moenocinyl‐sidechain‐containing lipid 7 . GPP binds to an allylic site (S1) and aligns well with known S1 inhibitors. Alkyl glycosides, glycolipids, can bind to both S1 and a second site, S2. Long sidechains in S2 are “bent” and co‐locate with the homoallylic substrate isopentenyl diphosphate in other prenyltransferases. These observations support a MoeN5 mechanism in which 5 binds to S2 with its C6–C11 group poised to attack C1 in GPP to form the moenocinyl sidechain, with the more distal regions of 5 aligning with the distal glucose in decyl maltoside. The results are of general interest because they provide the first structures of MoeN5 and a structural basis for its mechanism of action, results that will facilitate the design of new antibiotics.