ABSTRACT
Spirotetronate and spirotetramate natural products include a multitude of compounds with potent antimicrobial and antitumor activities. Their biosynthesis incorporates many unusual biocatalytic steps, including regio- and stereo-specific modifications, cyclizations promoted by Diels-Alderases, and acetylation-elimination reactions. Here we focus on the acetate elimination catalyzed by AbyA5, implicated in the formation of the key Diels-Alder substrate to give the spirocyclic system of the antibiotic abyssomicinâ C. Using synthetic substrate analogues, it is shown that AbyA5 catalyzes stereospecific acetate elimination, establishing the (R)-tetronate acetate as a biosynthetic intermediate. The X-ray crystal structure of AbyA5, the first of an acetate-eliminating enzyme, reveals a deviant acetyl esterase fold. Molecular dynamics simulations and enzyme assays show the use of a His-Ser dyad to catalyze either elimination or hydrolysis, via disparate mechanisms, under substrate control.
Subject(s)
Acetates/metabolism , Lyases/metabolism , Spiro Compounds/metabolism , Acetates/chemistry , Biocatalysis , Crystallography, X-Ray , Models, Molecular , Molecular Structure , Spiro Compounds/chemistryABSTRACT
Strobilurins from fungi are the inspiration for the creation of the ß-methoxyacrylate class of agricultural fungicides. However, molecular details of the biosynthesis of strobilurins have remained cryptic. Here we report the sequence of genomes of two fungi that produce strobilurins and show that each contains a biosynthetic gene cluster, which encodes a highly reducing polyketide synthase with very unusual C-terminal hydrolase and methyltransferase domains. Expression of stpks1 in Aspergillus oryzae leads to the production of prestrobilurin A when the fermentation is supplemented with a benzoyl coenzyme A (CoA) analogue. This enables the discovery of a previously unobserved route to benzoyl CoA. Reconstruction of the gene cluster in A. oryzae leads to the formation of prestrobilurin A, and addition of the gene str9 encoding an FAD-dependent oxygenase leads to the key oxidative rearrangement responsible for the creation of the ß-methoxyacrylate toxophore. Finally, two methyltransferases are required to complete the synthesis.
Subject(s)
Basidiomycota/enzymology , Polyketide Synthases/metabolism , Strobilurins/metabolism , Aspergillus oryzae , Basidiomycota/genetics , Multigene FamilyABSTRACT
The strobilurins are important antifungal metabolites isolated from a number of basidiomycetes and have been valuable leads for the development of commercially important fungicides. Isotopic labelling studies with early and advanced intermediates confirm for the first time that they are produced via a linear tetraketide, primed with the rare benzoate starter unit, itself derived from phenylalanine via cinnamate. Isolation of a novel biphenyl metabolite, pseudostrobilurin B, provides evidence for the involvement of an epoxide in the key rearrangement to form the ß-methoxyacrylate moiety essential for biological activity. Formation of two bolineol related metabolites, strobilurins Y and Z, also probably involves epoxide intermediates. Time course studies indicate a likely biosynthetic pathway from strobilurin A, with the simplest non-subsubstituted benzoate ring, to strobilurin G with a complex dioxepin terpenoid-derived substituent. Precursor-directed biosynthetic studies allow production of a number of novel ring-halogenated analogues as well as a new pyridyl strobilurin. These studies also provide evidence for a non-linear biosynthetic relationship between strobilurin A and strobilurin B.
Subject(s)
Antifungal Agents/metabolism , Basidiomycota/metabolism , Fungicides, Industrial/metabolism , Strobilurins/metabolism , Antifungal Agents/chemistry , Basidiomycota/chemistry , Biosynthetic Pathways , Epoxy Compounds/chemistry , Epoxy Compounds/metabolism , Fatty Acids, Unsaturated/chemistry , Fatty Acids, Unsaturated/metabolism , Fungicides, Industrial/chemistry , Halogenation , Strobilurins/chemistryABSTRACT
Six potential diketide substrates for the squalestatin tetraketide synthase (SQTKS) dehydratase (DH) domain were synthesised as N-acetyl cysteamine thiolesters (SNAC) and tested in kinetic assays as substrates with an isolated DH domain. 3R-3-hydroxybutyryl SNAC 3R-16 was turned over by the enzyme, but its enantiomer was not. Of the four 2-methyl substrates only 2R,3R-2-methyl-3-hydroxybutyryl SNAC 2R,3R-8 was a substrate. Combined with stereochemical information from the isolated SQTKS enoyl reductase (ER) domain, our results provide a near complete stereochemical description of the first cycle of beta-modification reactions of a fungal highly reducing polyketide synthase (HR-PKS). The results emphasise the close relationship between fungal HR-PKS and vertebrate fatty acid synthases (vFAS).
Subject(s)
Bridged Bicyclo Compounds, Heterocyclic/chemistry , Cyclohexanones/chemistry , Disaccharides/chemistry , Fungi/enzymology , Hydro-Lyases/metabolism , Polyketide Synthases/metabolism , Tricarboxylic Acids/chemistry , Bridged Bicyclo Compounds, Heterocyclic/metabolism , Cyclohexanones/metabolism , Disaccharides/metabolism , Kinetics , Molecular Structure , Stereoisomerism , Tricarboxylic Acids/metabolismABSTRACT
Natural products with anti-cancer activity play a vital role in lead and target discovery. We report here the synthesis and biological evaluation of the plant-derived alkaloid, piperlongumine and analogues. Using a Horner-Wadsworth-Emmons coupling approach, a selection of piperlongumine-like compounds were prepared in good overall yield from a novel phosphonoacetamide reagent. A number of the compounds displayed potent anti-cancer activity against colorectal (HCT 116) and ovarian (IGROV-1) carcinoma cell lines, via a mechanism of action which may involve ROS generation. Contrary to previous reports, no selective action in cancer cell (MRC-5) was observed for piperlongumine analogues.
Subject(s)
Antineoplastic Agents/pharmacology , Dioxolanes/pharmacology , Antineoplastic Agents/chemical synthesis , Antineoplastic Agents/chemistry , Cell Line, Tumor , Cell Proliferation/drug effects , Dioxolanes/chemical synthesis , Dioxolanes/chemistry , Dose-Response Relationship, Drug , Drug Screening Assays, Antitumor , Humans , Molecular Structure , Reactive Oxygen Species/metabolism , Structure-Activity RelationshipABSTRACT
The Diels-Alder reaction, a [4 + 2] cycloaddition of a conjugated diene to a dienophile, is one of the most powerful reactions in synthetic chemistry. Biocatalysts capable of unlocking new and efficient Diels-Alder reactions would have major impact. Here we present a molecular-level description of the reaction mechanism of the spirotetronate cyclase AbyU, an enzyme shown here to be a bona fide natural Diels-Alderase. Using enzyme assays, X-ray crystal structures, and simulations of the reaction in the enzyme, we reveal how linear substrate chains are contorted within the AbyU active site to facilitate a transannular pericyclic reaction. This study provides compelling evidence for the existence of a natural enzyme evolved to catalyze a Diels-Alder reaction and shows how catalysis is achieved.
Subject(s)
Catalysis , Cycloaddition Reaction , Bridged Bicyclo Compounds, Heterocyclic/chemistry , Bridged Bicyclo Compounds, Heterocyclic/metabolism , Catalytic Domain , Crystallography, X-Ray , Cyclohexenes/chemistry , Enzymes/chemistry , Models, Molecular , Quantum Theory , X-Ray DiffractionABSTRACT
A palladium-catalyzed cyanation of alkenyl halides using acetone cyanohydrin is described. A number of structurally diverse alkenylic nitrile containing compounds was prepared in one step under optimized conditions. The reaction proved to be efficient, chemoselective, easy to perform, and tolerant of a number of functional groups.
