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.     

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.     

Modular Oxime Formation by a trans-AT Polyketide Synthase

Modular trans-acyltransferase polyketide synthases (trans-AT PKSs) are enzymatic assembly lines that biosynthesize complex polyketide natural products. Relative to their better studied cis-AT counterparts, the trans-AT PKSs introduce remarkable chemical diversity into their polyketide products. A notable example is the lobatamide A PKS, which incorporates a methylated oxime. Here we demonstrate biochemically that this functionality is installed on-line by an unusual oxygenase-containing bimodule. Furthermore, analysis of the oxygenase crystal structure coupled with site-directed mutagenesis allows us to propose a model for catalysis, as well as identifying key protein-protein interactions that support this chemistry. Overall, our work adds oxime-forming machinery to the biomolecular toolbox available for trans-AT PKS engineering, opening the way to introducing such masked aldehyde functionalities into diverse polyketides.     

Biosynthetic origins of the natural product,thiolactomycin: a unique and selective inhibitor of type II dissociated fatty acid synthases

Thiolactomycin (TLM), a natural product produced by both Nocardia and Streptomyces spp., is a potent and highly selective inhibitor of the type II dissociated fatty acid synthases of plants and bacteria. The unique mode of action of TLM and its low toxicity make it an attractive compound for development of new antimicrobial agents. In this study, incorporation studies with 13C-labeled precursors demonstrate that TLM is derived from one acetate-derived starter unit and three methylmalonate-derived extender units. The unusual thiolactone represented by TLM represents a novel class of polyketide-derived antibiotics in which an unusual cyclization process, which terminates the biosynthetic pathway, involves incorporation of a sulfur atom from l-cysteine. Manipulation of this pathway through techniques such a combinatorial biosynthesis and mutasynthesis may provide a new route for economically viable production of useful TLM analogues.     

Structure and Mechanism of the Monoterpene Cyclolavandulyl Diphosphate Synthase that Catalyzes Consecutive Condensation and Cyclization

We report the three‐dimensional structure of cyclolavandulyl diphosphate (CLPP) synthase (CLDS), which consecutively catalyzes the condensation of two molecules of dimethylallyl diphosphate (DMAPP) followed by cyclization to form a cyclic monoterpene, CLPP. The structures of apo‐CLDS and CLDS in complex with Tris, pyrophosphate, and Mg²⁺ ion were refined at 2.00 Å resolution and 1.73 Å resolution, respectively. CLDS adopts a typical fold for cis‐prenyl synthases and forms a homo‐dimeric structure. An in vitro reaction using a regiospecifically ²H‐substituted DMAPP substrate revealed the intramolecular proton transfer mechanism of the CLDS reaction. The CLDS structure and structure‐based mutagenesis provide mechanistic insights into this unprecedented terpene synthase. The combination of structural and mechanistic insights advances the knowledge of intricate terpene synthase‐catalyzed reactions.     

Theoretical and Experimental Analysis of the Reaction Mechanism of MrTPS2, a Triquinane‐Forming Sesquiterpene Synthase from Chamomile

Terpene synthases, as key enzymes of terpene biosynthesis, have garnered the attention of chemists and biologists for many years. Their carbocationic reaction mechanisms are responsible for the huge variety of terpene structures in nature. These mechanisms are amenable to study by using classical biochemical approaches as well as computational analysis, and in this study we combine quantum‐chemical calculations and deuterium‐labeling experiments to elucidate the reaction mechanism of a triquinane forming sesquiterpene synthase from chamomile. Our results suggest that the reaction from farnesyl diphosphate to triquinanes proceeds through caryophyllyl and presilphiperfolanyl cations and involves the protonation of a stable (?)‐(E)‐β‐caryophyllene intermediate. A tyrosine residue was identified that appears to be involved in the proton‐transfer process.     

An Unusual Chimeric Diterpene Synthase from Emericella variecolor and Its Functional Conversion into a Sesterterpene Synthase by Domain Swapping

Di‐ and sesterterpene synthases produce C₂₀ and C₂₅ isoprenoid scaffolds from geranylgeranyl pyrophosphate (GGPP) and geranylfarnesyl pyrophosphate (GFPP), respectively. By genome mining of the fungus Emericella variecolor, we identified a multitasking chimeric terpene synthase, EvVS, which has terpene cyclase (TC) and prenyltransferase (PT) domains. Heterologous gene expression in Aspergillus oryzae led to the isolation of variediene ( 1 ), a novel tricyclic diterpene hydrocarbon. Intriguingly, in vitro reaction with the enzyme afforded the new macrocyclic sesterterpene 2 as a minor product from dimethylallyl pyrophosphate (DMAPP) and isopentenyl pyrophosphate (IPP). The TC domain thus produces the diterpene 1 and the sesterterpene 2 from GGPP and GFPP, respectively. Notably, a domain swap of the PT domain of EvVS with that of another chimeric sesterterpene synthase, EvSS, successfully resulted in the production of 2 in vivo as well. Cyclization mechanisms for the production of these two compounds are proposed.     

Induced Chemical Defense of a Mushroom by a Double-Bond-Shifting Polyene Synthase

The antilarval mushroom polyenes 18-methyl-19-oxoicosaoctaenoic acid and 20-methyl-21-oxodocosanonaenoic acid appear in response to injury of the mycelium of the stereaceous mushroom BY1. We identified a polyketide synthase (PPS1) which belongs to a hitherto completely uncharacterized clade of polyketide synthases. Expression of the PPS1 gene is massively upregulated following mycelial damage. The synthesis of the above polyenes was reconstituted in the mold Aspergillus niger as a heterologous host. This demonstrates that PPS1 1) synchronously produces branched-chain polyketides of varied lengths, and 2) catalyzes the unprecedented shift of eight or nine double bonds. This study represents the first characterization of a reducing polyketide synthase from a mushroom. We also show that injury-induced de novo synthesis of polyketides is a fungal response strategy.     

Using Terpene Synthase Plasticity in Catalysis: On the Enzymatic Conversion of Synthetic Farnesyl Diphosphate Analogues

Four synthetic farnesyl diphosphate analogues were enzymatically converted with three bacterial sesquiterpene synthases, including β-himachalene synthase (HcS) and (Z)-γ-bisabolene synthase (BbS) from Cryptosporangium arvum, and germacrene A synthase (SmTS6) from Streptomyces mobaraensis. These enzyme reactions not only yielded several previously unknown compounds, showing that this approach opened the door to a new chemical space, but substrates with blocked or altered reactivities also gave interesting insights into the cyclisation mechanisms and the potential to catalyse reactions with different initial cyclisation modes.     

An O-Acetylserine (thiol) Lyase from Leucaena leucocephala Is a Cysteine Synthase But Not a Mimosine Synthase

In plants, the final step of cysteine formation is catalyzed by O-acetylserine (thiol) lyase (OAS-TL). The purpose of this study was to isolate and characterize an OAS-TL from the tree legume Leucaena leucocephala (leucaena). Leucaena contains a toxic, nonprotein amino acid, mimosine, which is also formed by an OAS-TL, and characterization of this enzyme is essential for developing a mimosine-free leucaena for its use as a protein-rich fodder. The cDNA for a cytosolic leucaena OAS-TL isoform was obtained through interspecies suppression subtractive hybridization. A 40-kDa recombinant protein was purified from Escherichia coli and used in enzyme activity assays where it was found to synthesize only cysteine. The enzyme followed Michaelis-Menten kinetics, and the K _m was calculated to be 1,850?±?414 μM sulfide and the V _max was 200.6?±?19.92 μM cysteine min^?1. The N-terminal affinity His-tag was cleaved from the recombinant OAS-TL to eliminate its possible interference in binding with the substrate, 3-hydroxy-4-pyridone, for mimosine formation. The His-tag-cleaved OAS-TL was again observed to catalyze the formation of cysteine but not mimosine. Thus, the cytosolic OAS-TL from leucaena used in this study is specific for only cysteine synthesis and is different from previously reported OAS-TLs that also function as β-substituted alanine synthases.     

A Polyketide Synthase Component for Oxygen Insertion into Polyketide Backbones

Enzymatic core components from trans‐acyltransferase polyketide synthases (trans‐AT PKSs) catalyze exceptionally diverse biosynthetic transformations to generate structurally complex bioactive compounds. Here we focus on a group of oxygenases identified in various trans‐AT PKS pathways, including those for pederin, oocydins, and toblerols. Using the oocydin pathway homologue (OocK) from Serratia plymuthica 4Rx13 and N‐acetylcysteamine (SNAC) thioesters as test surrogates for acyl carrier protein (ACP)‐tethered intermediates, we show that the enzyme inserts oxygen into β‐ketoacyl moieties to yield malonyl ester SNAC products. Based on these data and the identification of a non‐hydrolyzed oocydin congener with retained ester moiety, we propose a unified biosynthetic pathway of oocydins, haterumalides, and biselides. By providing access to internal ester, carboxylate pseudostarter, and terminal hydroxyl functions, oxygen insertion into polyketide backbones greatly expands the biosynthetic scope of PKSs.     

An Unusual Type II Polyketide Synthase System Involved in Cinnamoyl Lipid Biosynthesis

As a unique structural moiety in natural products, cinnamoyl lipids (CLs), are proposed to be assembled by unusual type II polyketide synthases (PKSs). Herein, we demonstrate that the assembly of the CL compounds youssoufenes is accomplished by a PKS system that uniquely harbors three phylogenetically different ketosynthase/chain length factor (KS/CLF) complexes (YsfB/C, YsfD/E, and YsfJ/K). Through in vivo gene inactivation and in vitro reconstitution, as well as an intracellular tagged carrier‐protein tracking (ITCT) strategy developed in this study, we successfully elucidated the isomerase‐dependent ACP‐tethered polyunsaturated chain elongation process. The three KS/CLFs were revealed to modularly assemble different parts of the youssoufene skeleton, during which benzene ring closure happens right after the formation of an ACP‐tethered C18 polyene. Of note, the ITCT strategy could significantly contribute to the elucidation of other carrier‐protein‐dependent biosynthetic machineries.     

Mechanistic Characterisation of the Bacterial Sesterviridene Synthase from Kitasatospora viridis

A gene coding for a terpene synthase homolog from Kitasatospora viridis was cloned and expressed in Escherichia coli. The purified recombinant protein possessed sesterterpene synthase activity and efficiently converted geranylfarnesyl diphosphate (GFPP) with 19 % yield into the sesterterpene hydrocarbon sesterviridene A. Large scale enzymatic conversions also allowed for the isolation of two side products that are generated with very low yields of ca. 0.1 %. Several derivatives of sesterviridene A were obtained by chemical transformations, securing the NMR-based structural assignments. The absolute configuration of sesterviridene A was determined by chemical correlation using stereoselectively deuterated precursors and by anomalous dispersion X-ray crystallography. The cyclisation mechanism from GFPP to sesterviridene A was extensively studied through isotopic labelling experiments and DFT calculations.     

Prostacyclin and Thromboxane Synthase: New Aspects of Hemethiolate Catalysis

The arachidonic acid metabolites thromboxane A₂ and prostacyclin are highly potent regulators of cell physiology. They are both formed by enzymatic rearrangement of the 9,11-epidioxy prostaglandin H₂ catalyzed, however, by thromboxane and prostacyclin synthase, respectively. The two enzymes have been isolated, sequenced, and characterized as hemethiolate (“P450”) enzymes. The different isomerization products can be explained on the same catalytic principle by a different ligation of the heme centers with the two epidioxy oxygens atoms. This requires different conformations for substrate binding at the active site, which is substantiated by the different inhibitors and amino acid sequences of the enzymes. In a hypothesis which has mechanistic principles in common with the P450-monooxygenases and the allene oxide synthases, oxy radicals are formed first and rearrange to carbon radicals. These could then rapidly be converted into carbocations by the ferrylthiolate or iron(III )thiyl structures formed as intermediates.     

The pseudouridine synthases: revisiting a mechanism that seemed settled

RNA containing 5-fluorouridine, [f 5U]RNA, has been used as a mechanistic probe for the pseudouridine synthases, which convert uridine in RNA to its C-glycoside isomer, pseudouridine. Hydrated products of f 5U were attributed to ester hydrolysis of a covalent complex between an essential aspartic acid residue and f 5U, and the results were construed as strong support for a mechanism involving Michael addition by the aspartic acid residue. Labeling studies with [18O]water are now reported that rule out such ester hydrolysis in one pseudouridine synthase, TruB. The aspartic acid residue does not become labeled, and the hydroxyl group in the hydrated product of f 5U derives directly from solvent. The hydrated product, therefore, cannot be construed to support Michael addition during the conversion of uridine to pseudouridine, but the results do not rule out such a mechanism. A hypothesis is offered for the seemingly disparate behavior of different pseudouridine synthases toward [f 5U]RNA.     

Reconstitution of a Type II Polyketide Synthase that Catalyzes Polyene Formation

While type II polyketide synthases (PKSs) are known for producing aromatic compounds, a phylogenetically new subfamily of type II PKSs have been recently proposed to synthesize polyene structures. Here we report in vitro analysis of such a type II PKS, IgaPKS for ishigamide biosynthesis. The ketoreductase (Iga13) and dehydratase (Iga16) were shown to catalyze the reduction of a β‐keto group and dehydration of a β‐hydroxy group, respectively, to form a trans double bond. Incubation of the acyl carrier protein (Iga10), the ketosynthase/chain length factor complex (Iga11–Iga12), Iga13 and Iga16 with malonyl and hexanoyl‐CoAs and NADPH followed by KOH hydrolysis resulted in the formation of four unsaturated carboxylic acids (C₈, C₁₀, C₁₂, and C₁₄), indicating that IgaPKS catalyzes tetraene formation by repeating the cycle of condensation, keto‐reduction and dehydration with strict stereo‐specificity. We propose “highly reducing type II PKS subfamily” for the polyene‐producing type II PKSs.     

Rational domain swaps decipher programming in fungal highly reducing polyketide synthases and resurrect an extinct metabolite

The mechanism of programming of iterative highly reducing polyketide synthases remains one of the key unsolved problems of secondary metabolism. We conducted rational domain swaps between the polyketide synthases encoding the biosynthesis of the closely related compounds tenellin and desmethylbassianin. Expression of the hybrid synthetases in Aspergillus oryzae led to the production of reprogrammed compounds in which the changes to the methylation pattern and chain length could be mapped to the domain swaps. These experiments reveal for the first time the origin of programming in these systems. Domain swaps combined with coexpression of two cytochrome P450 encoding genes from the tenellin biosynthetic gene cluster led to the resurrection of the extinct metabolite bassianin.     

Blocking Deprotonation with Retention of Aromaticity in a Plant ent‐Copalyl Diphosphate Synthase Leads to Product Rearrangement

Substitution of a histidine, comprising part of the catalytic base group in the ent‐copalyl diphosphate synthases found in all seed plants for gibberellin phytohormone metabolism, by a larger aromatic residue leads to rearrangements. Through a series of 1,2‐hydride and methyl shifts of the initially formed bicycle predominant formation of (?)‐kolavenyl diphosphate is observed. Further mutational analysis and quantum chemical calculations provide mechanistic insight into the basis for this profound effect on product outcome.     

Didepside Formation by the Nonreducing Polyketide Synthase Preu6 of Preussia isomera Requires Interaction of Starter Acyl Transferase and Thioesterase Domains

Orsellinic acid (OA) derivatives are produced by filamentous fungi using nonreducing polyketide synthases (nrPKSs). The chain-releasing thioesterase (TE) domains of such nrPKSs were proposed to also catalyze dimerization to yield didepsides, such as lecanoric acid. Here, we use combinatorial domain exchanges, domain dissections and reconstitutions to reveal that the TE domain of the lecanoric acid synthase Preu6 of Preussia isomera must collaborate with the starter acyl transferase (SAT) domain from the same nrPKS. We show that artificial SAT-TE fusion proteins are highly effective catalysts and reprogram the ketide homologation chassis to form didepsides. We also demonstrate that dissected SAT and TE domains of Preu6 physically interact, and SAT and TE domains of OA-synthesizing nrPKSs may co-evolve. Our work highlights an unexpected domain–domain interaction in nrPKSs that must be considered for the combinatorial biosynthesis of unnatural didepsides, depsidones, and diphenyl ethers.     

Reversible Product Release and Recapture by a Fungal Polyketide Synthase Using a Carnitine Acyltransferase Domain

Fungal polyketides have significant biological activities, yet the biosynthesis by highly reducing polyketide synthases (HRPKSs) remains enigmatic. An uncharacterized group of HRPKSs was found to contain a C‐terminal domain with significant homology to carnitine O ‐acyltransferase (cAT). Characterization of one such HRPKS (Tv6‐931) from Trichoderma virens showed that the cAT domain is capable of esterifying the polyketide product with polyalcohol nucleophiles. This process is readily reversible, as confirmed through the holo ACP‐dependent transesterification of the released product. The methyltransferase (MT) domain of Tv6‐931 can perform two consecutive α‐methylation steps on the last β‐keto intermediate to yield an α,α‐gem ‐dimethyl product, a new programing feature among HRPKSs. Recapturing of the released product by cAT domain is suggested to facilitate complete gem ‐dimethylation by the MT.