10-membered Enediyne Research Articles

Jejucarbosides B-E, Chlorinated Cycloaromatized Enediynes, from a Marine Streptomyces sp.

Four new chlorinated cycloaromatized enediyne compounds, jejucarbosides B-E (1-4), were discovered together with previously-identified jejucarboside A from a marine actinomycete strain. Compounds 1-4 were identified as new chlorinated cyclopenta[a]indene glycosides based on 1D and 2D nuclear magnetic resonance, high-resolution mass spectrometry, and circular dichroism (CD) spectra. Jejucarbosides B and E bear a carbonate functional group whereas jejucarbosides C and D are variants possessing 1,2-diol by losing the carbonate functionality. It is proposed that the production of 1-4 occurs via Bergman cycloaromatization capturing Cl- and H+ in the alternative positions of a p-benzyne intermediate derived from a 9-membered enediyne core. Jejucarboside E (4) displayed significant cytotoxicity against human cancer cell lines including SNU-638, SK-HEP-1, A549, HCT116, and MDA-MB-231, with IC50 values of 0.31, 0.40, 0.25, 0.29, and 0.48 μM, respectively, while jejucarbosides B-D (1-3) showed moderate or no cytotoxic effects.

A discrete intermediate for the biosynthesis of both the enediyne core and the anthraquinone moiety of enediyne natural products

The enediynes are structurally characterized by a 1,5-diyne-3-ene motif within a 9- or 10-membered enediyne core. The anthraquinone-fused enediynes (AFEs) are a subclass of 10-membered enediynes that contain an anthraquinone moiety fused to the enediyne core as exemplified by dynemicins and tiancimycins. A conserved iterative type I polyketide synthase (PKSE) is known to initiate the biosynthesis of all enediyne cores, and evidence has recently been reported to suggest that the anthraquinone moiety also originates from the PKSE product. However, the identity of the PKSE product that is converted to the enediyne core or anthraquinone moiety has not been established. Here, we report the utilization of recombinant E. coli coexpressing various combinations of genes that encode a PKSE and a thioesterase (TE) from either 9- or 10-membered enediyne biosynthetic gene clusters to chemically complement ΔPKSE mutant strains of the producers of dynemicins and tiancimycins. Additionally, 13C-labeling experiments were performed to track the fate of the PKSE/TE product in the ΔPKSE mutants. These studies reveal that 1,3,5,7,9,11,13-pentadecaheptaene is the nascent, discrete product of the PKSE/TE that is converted to the enediyne core. Furthermore, a second molecule of 1,3,5,7,9,11,13-pentadecaheptaene is demonstrated to serve as the precursor of the anthraquinone moiety. The results establish a unified biosynthetic paradigm for AFEs, solidify an unprecedented biosynthetic logic for aromatic polyketides, and have implications for the biosynthesis of not only AFEs but all enediynes.

Intramolecular C-C Bond Formation Links Anthraquinone and Enediyne Scaffolds in Tiancimycin Biosynthesis.

First discovered in 1989, the anthraquinone-fused enediynes are a class of DNA-cleaving bacterial natural products composed of a DNA-intercalating anthraquinone moiety and a 10-membered enediyne warhead. However, until recently, there has been a lack of genetically amenable hosts and sequenced biosynthetic gene clusters available for solving the biosynthetic questions surrounding these molecules. Herein, we have identified and biochemically and structurally characterized TnmK1, a member of the α/β-hydrolase fold superfamily responsible for the C-C bond formation linking the anthraquinone moiety and enediyne core together in tiancimycin (TNM) biosynthesis. In doing so, two intermediates, TNM H and TNM I, in anthraquinone-fused enediyne biosynthesis, containing an unprecedented cryptic C16 aldehyde group, were identified. This aldehyde plays a key role in the TnmK1-catalyzed C-C bond formation via a Michael addition, representing the first example of this chemistry for the α/β-hydrolase fold superfamily. Additionally, TNM I shows sub-nanomolar cytotoxicity against selected cancer cell lines, indicating a new mechanism of action compared to previously known anthraquinone-fused enediynes. Together, the findings from this study are expected to impact enzymology, natural product biosynthesis, and future efforts at enediyne discovery and drug development.

Functionalized 10-Membered Aza- and Oxaenediynes through the Nicholas Reaction.

The scope and limitations of the Nicholas-type cyclization for the synthesis of 10-membered benzothiophene-fused heterocyclic enediynes with different functionalities were investigated. Although the Nicholas cyclization through oxygen could be carried out in the presence of an ester group, the final oxaenediyne was unstable under storage. Among the N-type Nicholas reactions, cyclization via an arenesulfonamide functional group followed by mild Co-deprotection was found to be the most promising, yielding 10-membered azaendiynes in high overall yields. By contrast, the Nicholas cyclization through the acylated nitrogen atom did not give the desired 10-membered cycle. It resulted in the formation of a pyrroline ring, whereas cyclization via an alkylated amino group resulted in a poor yield of the target 10-membered enediyne. The acylated 4-aminobenzenesulfonamide nucleophilic group was found to be the most convenient for the synthesis of functionalized 10-membered enediynes bearing a clickable function, such as a terminal triple bond. All the synthesized cyclic enediynes exhibited moderate activity against lung carcinoma NCI-H460 cells and had a minimal effect on lung epithelial-like WI-26 VA4 cells and are therefore promising compounds in the search for novel antitumor agents that can be converted into conjugates with tumor-targeting ligands.

Deciphering the pathway-specific regulatory network for production of ten-membered enediyne Tiancimycins in Streptomyces sp. CB03234-S

BackgroundThe anthraquinone-fused 10-membered enediynes (AFEs), represented by tiancimycins (TNMs), possess a unique structural feature and promising potentials as payloads of antitumor antibody–drug conjugates. Despite many efforts, the insufficient yields remain a practical challenge for development of AFEs. Recent studies have suggested a unified basic biosynthetic route for AFEs, those core genes involved in the formation of essential common AFE intermediates, together with multiple regulatory genes, are highly conserved among the reported biosynthetic gene clusters (BGCs) of AFEs. The extreme cytotoxicities of AFEs have compelled hosts to evolve strict regulations to control their productions, but the exact roles of related regulatory genes are still uncertain.ResultsIn this study, the genetic validations of five putative regulatory genes present in the BGC of TNMs revealed that only three (tnmR1, tnmR3 and tnmR7) of them were involved in the regulation of TNMs biosynthesis. The bioinformatic analysis also revealed that they represented three major but distinct groups of regulatory genes conserved in all BGCs of AFEs. Further transcriptional analyses suggested that TnmR7 could promote the expressions of core enzymes TnmD/G and TnmN/O/P, while TnmR3 may act as a sensor kinase to work with TnmR1 and form a higher class unconventional orphan two-component regulatory system, which dynamically represses the expressions of TnmR7, core enzymes TnmD/G/J/K1/K2 and auxiliary proteins TnmT2/S2/T1/S1. Therefore, the biosynthesis of TNMs was stringently restricted by this cascade regulatory network at early stage to ensure the normal cell growth, and then partially released at the stationary phase for product accumulation.ConclusionThe pathway-specific cascade regulatory network consisting with TnmR3/R1 and TnmR7 was deciphered to orchestrate the production of TNMs. And it could be speculated as a common regulatory mechanism for productions of AFEs, which shall provide us new insights in future titer improvement of AFEs and potential dynamic regulatory applications in synthetic biology.

Discovery of pentaene polyols by the activation of an enediyne gene cluster: biosynthetic implications for 9-membered enediyne core structures.

The identification and characterization of enediyne polyketide synthases (PKSEs) revealed that PKSE-bound polyene is a common intermediate, while its subsequent tailoring steps to enediyne cores remain obscure. Herein, we report pentaene polyols 5-7 and cinnamic acid derivatives 8 and 9 biosynthesized from an activated enediyne biosynthetic gene cluster in Streptomyces sp. CB02130. The C-1027 pksE could partially complement production of these polyene polyols in a CB02130 mutant where the native pksE is inactivated. The yields of 5-7 were improved by increasing the cellular pool of l-Phe through either gene inactivation of a prephenate dehydrogenase WlsPDH or supplementation of l-Phe. A flexible ammonia lyase WlsC4 is responsible for biosynthesis of 8 and 9 from l-Phe. The co-localization of wlsPDH and PKSE gene cassette supports their close evolutionary relationships and an enediyne genome mining strategy using WlsPDH. These findings not only provide a facile approach to activate silent enediyne BGCs, but suggest that a polyene epoxide intermediate may be formed for construction of 9-membered enediyne macrocycles.

Submerged fermentation of Streptomyces uncialis providing a biotechnology platform for uncialamycin biosynthesis, engineering, and production.

Uncialamycin (UCM) belongs to the anthraquinone-fused subfamily of 10-membered enediyne natural products that exhibits an extraordinary cytotoxicity against a wide spectrum of human cancer cell lines. Antibody-drug conjugates, utilizing synthetic analogues of UCM as payloads, are in preclinical development. UCM is exclusively produced by Streptomyces uncialis DCA2648 on solid agar medium with low titers (∼0.019 mg/l), limiting its supply by microbial fermentation and hampering its biosynthetic and engineering studies by in vivo pathway manipulation. Here, we report cultivation conditions that enable genetic manipulation of UCM biosynthesis in vivo and allow UCM production, with improved titers, by submerged fermentation of the engineered S. uncialis strains. Specifically, the titer of UCM was improved nearly 58-fold to ∼1.1 mg/l through the combination of deletion of biosynthetic gene clusters encoding unrelated metabolites from the S. uncialis wild-type, chemical mutagenesis and manipulation of pathway-specific regulators to generate the engineered S. uncialis strains, and finally medium optimization of the latter for UCM production. Genetic manipulation of UCM biosynthesis was demonstrated by inactivating selected genes in the engineered S. uncialis strains, one of which afforded a mutant strain accumulating tiancimycin B, a common biosynthetic intermediate known for the anthraquinone-fused subfamily of enediyne natural products. These findings highlight a biotechnology platform for UCM biosynthesis, engineering, and production that should facilitate both its fundamental studies and translational applications.

Intramolecular Nicholas Reactions in the Synthesis of Heteroenediynes Fused to Indole, Triazole, and Isocoumarin.

The applicability of an intramolecular Nicholas reaction for the preparation of 10-membered O- and N-enediynes fused to indole, 1,2,3-triazole, and isocoumarin was investigated. The general approach to acyclic enediyne precursors fused to heterocycles includes inter- and intramolecular buta-1,3-diyne cyclizations with the formation of iodoethynylheterocycles, followed by Sonogashira coupling. The nature of both a heterocycle and a nucleophilic group affects the possibility of a 10-membered ring closure by the Nicholas reaction. Among oxacycles, an isocoumarin-fused enediyne was obtained. In the case of O-enediyne annulated with indole, instead of the formation of a 10-membered cycle, BF3-promoted addition of an OH-group to the proximal triple bond at the C3 position afforded dihydrofuryl-substituted indole. For 1,2,3-triazole-fused analogues, using NH-Ts as a nucleophilic functional group allowed obtaining 10-membered azaenediyne, while the substrate with a hydroxyl group gave only traces of the desired 10-membered oxacycle. An improved method for the deprotection of Co-complexes of cyclic enediynes using tetrabutylammonium fluoride in an acetone/water mixture and the investigation of the 10-membered enediynes' reactivity in the Bergman cyclization are also reported. In the solid state, all synthesized iodoethynylheterocycles were found to be involved in halogen bond (XB) formation with either O or N atoms as XB acceptors.

Genome shuffling based on different types of ribosome engineering mutants for enhanced production of 10-membered enediyne tiancimycin-A.

Tiancimycin-A (TNM-A) is an anthraquinone-fused ten-membered enediyne produced by Streptomyces sp. CB03234, which is very promising for the development of anticancer antibody-drug conjugates (ADCs). To improve the titer of TNM-A, we have generated high-producing mutants CB03234-S and CB03234-R through ribosome engineering, but still not sufficient for pilot production of TNM-A. As the follow-up work, gentamycin-induced ribosome engineering was further adopted here to generate the mutant CB03234-G, which produced similar level of TNM-A as in CB03234-S and CB03234-R. Benefiting from the distinct antibiotic resistances of three ribosome engineering mutants, genome shuffling between any two of them was respectively carried out, and finally obtained the recombinant CB03234-GS26. Under optimal conditions, CB03234-GS26 produced 40.6 ± 1.0mg/L TNM-A in shaking flasks and 20.8 ± 0.4mg/L in a scaled-up 30-L fermentor. Comparing with the parental high-producing mutants, the over 1.6-fold titer improvement of CB03234-GS26 in fermentor was more promising for pilot production of TNM-A. Besides the distinctive morphological features, genetic characterization revealed that CB03234-GS26 possessed 1.8kb rsmG related deletion just the same as CB03234-S, but no mutation was found in rpsL. Subsequent knockouts proved that rsmG was unrelated to titer improvement of TNM-A, which implied other genomic variations and mechanisms rather than ribosome engineering to enhance the biosynthesis of TNM-A. Therefore, CB03234-GS26 provided a basis to locate potential novel genetic targets, and explore the interactions between complex metabolic network and TNM biosynthetic pathway, which shall promote future construction of high-yielding systems for TNM-A and other anthraquinone-fused enediynes.Key Points •United genome shuffling and ribosome engineering help further strain improvement. •CB03234-GS26 with improved titer is practical for the pilot production of TNM-A. •Enhanced TNM-A production should attribute to novel genetic features/mechanisms.

Yangpumicins F and G, Enediyne Congeners from Micromonospora yangpuensis DSM 45577.

Enediyne natural products are among the most cytotoxic small molecules and thus excellent payload candidates for the development of antibody-drug conjugates (ADCs). Here we report the isolation and structural elucidation of two new 10-membered anthraquinone-fused enediynes, yangpumicins (YPM) F (6) and G (7), together with five known congeners, YPM A-E (1-5), from Micromonospora yangpuensis DSM 45577. YPM F (6) and G (7) showed strong cytotoxicity against the tested human cancer cell lines, as well as activity against several Gram-positive and Gram-negative pathogens. The 1,2-diols in 6 and 7 promise to enable new linker chemistry for the development of YPM-based ADCs.

Structural interrogation of proteins involved in the biosynthesis of 10-membered enediyne anticancer natural products

Structural interrogation of proteins involved in the biosynthesis of 10-membered enediyne anticancer natural products

Discovery of gas vesicles in Streptomyces sp. CB03234-S and potential effects of gas vesicle gene overexpression on morphological and metabolic changes in streptomycetes.

Gas vesicles are usually found in aquatic microbes to help against adverse growth conditions and also present applied potentials in various biotechnological areas. Although gvp genes encoding gas vesicle proteins are occurred in many bacteria, the formation of gas vesicles in streptomycetes has not been reported yet. During the transcriptome analysis of Streptomyces sp. CB03234-S, a high producer of 10-membered enediynes tiancimycins (TNMs) generated in our previous studies, the notable activation of a gvp gene cluster (gvp3234) was detected, which may be related to the morphological change and yield increase in CB03234-S. Subsequent bioinformatic analysis revealed that gvp3234 possessed some unique features different from other gvp gene clusters, and its relative expression level was much higher than homologues in Streptomyces coelicolor. The overexpression of gvp3234 further improved the total titer of TNM-A and TNM-D from 12.3 ± 0.1mg/L up to 21.4 ± 0.8mg/L in CB03234-S, and TEM analysis clearly showed the emergence of gas vesicles, while the heterologous expression of gvp3234 in Streptomyces sp. CB09001 also resulted in the similar formation of fluffy mycelial pellets with long hyphae like in CB03234-S and enhanced the production of xanthone derivative. Our work reported a functionally expressed gvp gene cluster and the corresponding formation of gas vesicles in streptomycetes for the first time. The revealed potential effects of gas vesicles on morphological and metabolic changes shall provide an alternative strategy for strain improvement of industrial streptomycetes to obtain better performances in liquid cultivation and enhance production of secondary metabolites.

Streptomycin-induced ribosome engineering complemented with fermentation optimization for enhanced production of 10-membered enediynes tiancimycin-A and tiancimycin-D.

Tiancimycins (TNMs) are a group of 10-membered anthraquinone-fused enediynes, newly discovered from Streptomyces sp. CB03234. Among them, TNM-A and TNM-D have exhibited excellent antitumor performances and could be exploited as very promising warheads for the development of anticancer antibody-drug conjugates (ADCs). However, their low titers, especially TNM-D, have severely limited following progress. Therefore, the streptomycin-induced ribosome engineering was adopted in this work for strain improvement of CB03234, and a TNMs high producer S. sp. CB03234-S with the K43N mutation at 30S ribosomal protein S12 was successfully screened out. Subsequent media optimization revealed the essential effects of iodide and copper ion on the production of TNMs, while the substitution of nitrogen source could evidently promote the accumulation of TNM-D, and the ratio of produced TNM-A and TNM-D was responsive to the change of carbon and nitrogen ratio in the medium. Further amelioration of the pH control in scaled up 25 L fermentation increased the average titers of TNM-A and TNM-D up to 13.7 ± 0.3 and 19.2 ± 0.4 mg/L, respectively. The achieved over 45-fold titer improvement of TNM-A, and 109-fold total titer improvement of TNM-A and TNM-D enabled the efficient purification of over 200 mg of each target molecule from 25 L fermentation. Our efforts have demonstrated a practical strategy for titer improvement of anthraquinone-fused enediynes and set up a solid base for the pilot scale production and preclinical studies of TNMs to expedite the future development of anticancer ADC drugs.

Cr(II)-promoted internal cyclization of acyclic enediynes fused to benzo[b]thiophene core: Macrocycles versus 2-methylenecycloalkan-1-ols formation

The utility of the intramolecular Nozaki-type coupling for the synthesis of macrocyclic benzo[b]thiophene-fused enediynes has been explored. The starting acyclic enediynes were prepared by the iodocyclization of 2-(buta-1,3-diynyl)thioanisoles followed by the Sonogashira cross-coupling of the resulting iodo-substituted benzo[b]thiophene with corresponding acetylenes. We found that Cr(II)-promoted intramolecular cyclization of 7-[2-(iodoethynyl)benzo[b]thiophen-3-yl]hept-6-ynal and 7-[3-(iodoethynyl)benzo-[b]thiophen-2-yl]hept-6-ynal resulted in the formation of 11-membered macrocyclic enediynes, while both expected 10-membered enediynes cannot be produced under the Nozaki-type reaction from corresponding 6-[3-(iodoethynyl)benzo[b]thiophen-2-yl]hex-5-ynal and 6-[2-(iodoethynyl)benzo[b]thiophen-3-yl]hex-5-ynal. In the case the reaction was catalyzed by Ni(II), the attack on a proximal triple bond led to the formation of 2-methylenecycloalkane-1-ol fragments, instead of macrocyclization. The DFT analysis of the ring strain in the benzo[b]thiophene-fused 10- and 11-membered enediyne-containing cycle provides the plausible explanation of the observed regioselectivity.

An Extended Multireference Study of the Singlet and Triplet States of the 9,10-didehydroanthracene Diradical

The 9,10-didehydroanthracene is an aromatic diradical produced by the Bergman cyclization of a benzannulated 10-membered enediyne. It is a 1,4 diradical, similar to p-benzyne. Here we study the spin state occupancy of the ground state of 9,10-didehydroanthracene by employing multireference methods (MR-CISD and MR-AQCC) with different basis sets (cc-pVDZ and cc-pVTZ) and active space sizes (CAS (2,2) through CAS (8,8)). At the CAS (8,8) MR-AQCC/cc-pVDZ level of theory, we find a two-configurational singlet ground state with an adiabatic Δ EST of 6.13 kcal/mol. Unpaired electron density populations and dominant electronic configuration interactions were used to analyze the features of the 9,10-didehydroanthracene diradical.

Relative Reactivity of Benzothiophene-Fused Enediynes in the Bergman Cyclization.

To find promising analogues of naturally occurring enediyne antibiotics with a sufficient reactivity in the Bergman cyclization and moderately stable under isolation and storage, a scale of relative enediynes reactivity was created on the basis of calculated free activation energies for the Bergman cyclization within 12 known and new benozothiophene, benzene, and cinnoline annulated 9- and 10-membered enediynes. To verify the predicted reactivity/stability balance, three new carbocyclic enediynes fused to a benzothiophene core bearing 3,4,5-trimethoxybenzene, fluoroisopropyl, and isopropenyl substituents were synthesized using the Nicholas-type macrocyclization. It was confirmed that annulation of a 3,4,5-trimethoxybenzene moiety to a 10-membered enediyne macrocycle imparts high reactivity to an enediyne while also conferring instability under ambient temperature. Fluoroisopropyl-substituted 10-membered enediyne from the opposite end of the scale was found to be stable while moderately reactive in the Bergman cyclization. Along with the experimentally confirmed moderate reactivity (DSC kinetic studies), (fluoroisopropyl)enediyne showed a significant DNA damaging activity in plasmid cleavage assays comparable with the known anticancer drug Zeocin.

Crystal Structure of Thioesterase SgcE10 Supporting Common Polyene Intermediates in 9- and 10-Membered Enediyne Core Biosynthesis

Enediynes are potent natural product anticancer antibiotics, and are classified as 9- or 10-membered according to the size of their enediyne core carbon skeleton. Both 9- and 10-membered enediyne cores are biosynthesized by the enediyne polyketide synthase (PKSE), thioesterase (TE), and PKSE-associated enzymes. Although the divergence between 9- and 10-membered enediyne core biosynthesis remains unclear, it has been observed that nascent polyketide intermediates, tethered to the acyl carrier protein (ACP) domain of PKSE, could be released by TE in the absence of the PKSE-associated enzymes. In this study, we determined the crystal structure of SgcE10, the TE that participates in the biosynthesis of the 9-membered enediyne C-1027. Structural comparison of SgcE10 with CalE7 and DynE7, two TEs that participate in the biosynthesis of the 10-membered enediynes calicheamicin and dynemicin, respectively, revealed that they share a common α/β hot-dog fold. The amino acids involved in both substrate binding and catalysis are conserved among SgcE10, CalE7, and DynE7. The volume and the shape of the substrate-binding channel and active site in SgcE10, CalE7, and DynE7 confirm that TEs from both 9- and 10-membered enediyne biosynthetic machineries bind the linear form of similar ACP-tethered polyene intermediates. Taken together, these findings further support the proposal that the divergence between 9- and 10-membered enediyne core biosynthesis occurs beyond PKSE and TE catalysis.

Genome neighborhood network reveals insights into enediyne biosynthesis and facilitates prediction and prioritization for discovery.

The enediynes are one of the most fascinating families of bacterial natural products given their unprecedented molecular architecture and extraordinary cytotoxicity. Enediynes are rare with only 11 structurally characterized members and four additional members isolated in their cycloaromatized form. Recent advances in DNA sequencing have resulted in an explosion of microbial genomes. A virtual survey of the GenBank and JGI genome databases revealed 87 enediyne biosynthetic gene clusters from 78 bacteria strains, implying that enediynes are more common than previously thought. Here we report the construction and analysis of an enediyne genome neighborhood network (GNN) as a high-throughput approach to analyze secondary metabolite gene clusters. Analysis of the enediyne GNN facilitated rapid gene cluster annotation, revealed genetic trends in enediyne biosynthetic gene clusters resulting in a simple prediction scheme to determine 9- versus 10-membered enediyne gene clusters, and supported a genomic-based strain prioritization method for enediyne discovery.

Synthesis and Reactivity of Cinnoline-Fused Cyclic Enediyne

A short and efficient synthesis of cinnoline-fused cyclic enediyne is reported. Richter cyclization of o-(1,3-butadiynyl)phenyltriazene produced 3-alkynyl-4-bromocinnoline. The Sonogashira coupling of the latter with 5-hexyn-1-ol was employed for the introduction of a second acetylenic moiety. The crucial cyclization step was achieved under Nozaki-Hiyama-Kishi conditions. Cinnoline-fused 10-membered ring enediyne is more reactive than corresponding carbocyclic analog and produces good yield of the Bergman cyclization product upon mild heating. This enediyne induces single-strand dDNA scissions upon incubation at 40 °C.

Induced-fit upon Ligand Binding Revealed by Crystal Structures of the Hot-dog Fold Thioesterase in Dynemicin Biosynthesis

Dynemicins are structurally related 10-membered enediyne natural products isolated from Micromonospora chernisa with potent antitumor and antibiotic activity. The early biosynthetic steps of the enediyne moiety of dynemicins are catalyzed by an iterative polyketide synthase (DynE8) and a thioesterase (DynE7). Recent studies indicate that the function of DynE7 is to off-load the linear biosynthetic intermediate assembled on DynE8. Here, we report crystal structures of DynE7 in its free form at 2.7 Å resolution and of DynE7 in complex with the DynE8-produced all- trans pentadecen-2-one at 2.1 Å resolution. These crystal structures reveal that upon ligand binding, significant conformational changes throughout the substrate-binding tunnel result in an expanded tunnel that traverses an entire monomer of the tetrameric DynE7 protein. The enlarged inner segment of the channel binds the carbonyl-conjugated polyene mainly through hydrophobic interactions, whereas the putative catalytic residues are located in the outer segment of the channel. The crystallographic information reinforces an unusual catalytic mechanism that involves a strictly conserved arginine residue for this subfamily of hot-dog fold thioesterases, distinct from the typical mechanism for hot-dog fold thioesterases that utilizes an acidic residue for catalysis.