RESUMO
Terpenes comprise the largest class of natural products and are used in applications spanning the areas of medicine, cosmetics, fuels, flavorings, and more. Copalyl diphosphate synthase from the Penicillium genus is the first bifunctional terpene synthase identified to have both prenyltransferase and class II cyclase activities within the same polypeptide chain. Prior studies of bifunctional terpene synthases reveal that these systems achieve greater catalytic efficiency by channeling geranylgeranyl diphosphate between the prenyltransferase and cyclase domains. A molecular-level understanding of substrate transit phenomena in these systems is highly desirable, but a long disordered polypeptide segment connecting the prenyltranferase and cyclase domains thwarts the crystallization of full-length enzymes. Accordingly, these systems are excellent candidates for structural analysis using cryo-electron microscopy (cryo-EM). Notably, these systems form hexameric or octameric oligomers, so the quaternary structure of the full-length enzyme may influence substrate transit between catalytic domains. Here, we describe methods for the preparation of bifunctional hexameric copalyl diphosphate synthase from Penicillium fellutanum (PfCPS). We also outline approaches for the preparation of cryo-EM grids, data collection, and data processing to yield two-dimensional and three-dimensional reconstructions.
Assuntos
Alquil e Aril Transferases , Microscopia Crioeletrônica , Penicillium , Penicillium/enzimologia , Alquil e Aril Transferases/química , Alquil e Aril Transferases/metabolismo , Alquil e Aril Transferases/isolamento & purificação , Microscopia Crioeletrônica/métodos , Diterpenos/metabolismo , Diterpenos/química , Proteínas Fúngicas/química , Proteínas Fúngicas/isolamento & purificação , Proteínas Fúngicas/metabolismo , Dimetilaliltranstransferase/metabolismo , Dimetilaliltranstransferase/química , Dimetilaliltranstransferase/genética , Dimetilaliltranstransferase/isolamento & purificaçãoRESUMO
Prenyltransferases are terpene synthases that combine 5-carbon precursor molecules into linear isoprenoids of varying length that serve as substrates for terpene cyclases, enzymes that catalyze fascinating cyclization reactions to form diverse terpene natural products. Terpenes and their derivatives comprise the largest class of natural products and have myriad functions in nature and diverse commercial uses. An emerging class of bifunctional terpene synthases contains both prenyltransferase and cyclase domains connected by a disordered linker in a single polypeptide chain. Fusicoccadiene synthase from Phomopsis amygdali (PaFS) is one of the most well-characterized members of this subclass and serves as a model system for the exploration of structure-function relationships. PaFS has been structurally characterized using a variety of biophysical techniques. The enzyme oligomerizes to form a stable core of six or eight prenyltransferase domains that produce a 20-carbon linear isoprenoid, geranylgeranyl diphosphate (GGPP), which then transits to the cyclase domains for the generation of fusicoccadiene. Cyclase domains are in dynamic equilibrium between randomly splayed-out and prenyltransferase-associated positions; cluster channeling is implicated for GGPP transit from the prenyltransferase core to the cyclase domains. In this chapter, we outline the methods we are developing to interrogate the nature of cluster channeling in PaFS, including enzyme activity and product analysis assays, approaches for engineering the linker segment connecting the prenyltransferase and cyclase domains, and structural analysis by cryo-EM.
Assuntos
Alquil e Aril Transferases , Alquil e Aril Transferases/metabolismo , Alquil e Aril Transferases/química , Alquil e Aril Transferases/genética , Dimetilaliltranstransferase/metabolismo , Dimetilaliltranstransferase/química , Dimetilaliltranstransferase/genética , Diterpenos/metabolismo , Diterpenos/química , Ensaios Enzimáticos/métodos , Fosfatos de Poli-Isoprenil/metabolismo , Fosfatos de Poli-Isoprenil/química , CiclizaçãoRESUMO
Fusicoccadiene synthase from P. amygdala (PaFS) is a bifunctional assembly-line terpene synthase containing a prenyltransferase domain that generates geranylgeranyl diphosphate (GGPP) from dimethylallyl diphosphate (DMAPP) and three equivalents of isopentenyl diphosphate (IPP), and a cyclase domain that converts GGPP into fusicoccadiene, a precursor of the diterpene glycoside Fusicoccin A. The two catalytic domains are linked by a flexible 69-residue polypeptide segment. The prenyltransferase domain mediates oligomerization to form predominantly octamers, and cyclase domains are randomly splayed out around the prenyltransferase core. Previous studies suggest that substrate channeling is operative in catalysis, since most of the GGPP formed by the prenyltransferase remains on the protein for the cyclization reaction. Here, we demonstrate that the flexible linker is not required for substrate channeling, nor must the prenyltransferase and cyclase domains be covalently linked to sustain substrate channeling. Moreover, substrate competition experiments with other diterpene cyclases indicate that the PaFS prenyltransferase and cyclase domains are preferential partners regardless of whether they are covalently linked or not. The cryo-EM structure of engineered "linkerless" construct PaFSLL, in which the 69-residue linker is spliced out and replaced with the tripeptide PTQ, reveals that cyclase pairs associate with all four sides of the prenyltransferase octamer. Taken together, these results suggest that optimal substrate channeling is achieved when a cyclase domain associates with the side of the prenyltransferase octamer, regardless of whether the two domains are covalently linked and regardless of whether this interaction is transient or locked in place.
RESUMO
The zinc-dependent histone deacetylases (HDACs 1-11) belong to the arginase-deacetylase superfamily of proteins, members of which share a common α/ß fold and catalytic metal binding site. While several HDACs play a role in epigenetic regulation by catalyzing acetyllysine hydrolysis in histone proteins, the biological activities of HDACs extend far beyond histones. HDACs also deacetylate nonhistone proteins in the nucleus as well as the cytosol to regulate myriad cellular processes. The substrate pool is even more diverse in that certain HDACs can hydrolyze other covalent modifications. For example, HDAC6 is also a lysine decrotonylase, and HDAC11 is a lysine-fatty acid deacylase. Surprisingly, HDAC10 is not a lysine deacetylase but instead is a polyamine deacetylase. Thus, the HDACs are biologically and chemically versatile catalysts as they regulate the function of diverse protein and nonprotein substrates throughout the cell.Owing to their critical regulatory functions, HDACs serve as prominent targets for drug design. At present, four HDAC inhibitors are FDA-approved for cancer chemotherapy. However, these inhibitors are active against multiple HDAC isozymes, and a lack of selectivity is thought to contribute to undesirable side effects. Current medicinal chemistry campaigns focus on the development of isozyme-selective inhibitors, and many such studies largely focus on HDAC6 and HDAC10. HDAC6 is a target for therapeutic intervention due to its cellular role as a tubulin deacetylase and tau deacetylase, and selective inhibitors are being studied in cancer chemotherapy and the treatment of peripheral neuropathy. Crystal structures of enzyme-inhibitor complexes reveal how various features of inhibitor design, such as zinc-coordinating groups, bifurcated capping groups, and aromatic fluorination patterns, contribute to affinity and isozyme selectivity. The polyamine deacetylase HDAC10 is also an emerging target for cancer chemotherapy. Crystal structures of intact substrates trapped in the HDAC10 active site reveal the molecular basis of strikingly narrow substrate specificity for N8-acetylspermidine hydrolysis. Active site features responsible for substrate specificity have been successfully exploited in the design of potent and selective inhibitors.In this Account, I review the structural chemistry and inhibition of HDACs, highlighting recent X-ray crystallographic and functional studies of HDAC6 and HDAC10 in my laboratory. These studies have yielded fascinating snapshots of catalysis as well as novel chemical transformations involving bound inhibitors. The zinc-bound water molecule in the HDAC active site is the catalytic nucleophile in the deacetylation reaction, but this activated water molecule can also react with inhibitor CâO or CâN groups to yield unanticipated reaction products that bind exceptionally tightly. Versatile active site chemistry unleashes the full inhibitory potential of such compounds, and X-ray crystallography allows us to view this chemistry in action.
Assuntos
Lisina , Neoplasias , Humanos , Epigênese Genética , Isoenzimas/metabolismo , Histona Desacetilases/metabolismo , Inibidores de Histona Desacetilases/farmacologia , Inibidores de Histona Desacetilases/química , Inibidores de Histona Desacetilases/metabolismo , Poliaminas/química , Catálise , Histonas/metabolismo , Zinco/química , Água/metabolismoRESUMO
The sesquiterpene cyclase epi-isozizaene synthase (EIZS) from Streptomyces coelicolor catalyzes the metal-dependent conversion of farnesyl diphosphate (FPP) into the complex tricyclic product epi-isozizaene. This remarkable transformation is governed by an active site contour that serves as a template for catalysis, directing the conformations of multiple carbocation intermediates leading to the final product. Mutagenesis of residues defining the active site contour remolds its three-dimensional shape and reprograms the cyclization cascade to generate alternative cyclization products. In some cases, mutagenesis enables alternative chemistry to quench carbocation intermediates, e.g., through hydroxylation. Here, we combine structural and biochemical data from previously characterized EIZS mutants to design and prepare F95S-F198S EIZS, which converts EIZS into an α-bisabolol synthase with moderate fidelity (65% at 18 °C, 74% at 4 °C). We report the complete biochemical characterization of this double mutant as well as the 1.47 Å resolution X-ray crystal structure of its complex with three Mg2+ ions, inorganic pyrophosphate, and the benzyltriethylammonium cation, which partially mimics a carbocation intermediate. Most notably, the two mutations together create an active site contour that stabilizes the bisabolyl carbocation intermediate and positions a water molecule for the hydroxylation reaction. Structural comparison with a naturally occurring α-bisabolol synthase reveals common active site features that direct α-bisabolol generation. In showing that EIZS can be redesigned to generate a sesquiterpene alcohol product instead of a sesquiterpene hydrocarbon product, we have expanded the potential of EIZS as a platform for the development of designer cyclases that could be utilized in synthetic biology applications.
Assuntos
Carbono-Carbono Liases , Sesquiterpenos , Sesquiterpenos/metabolismo , Sesquiterpenos MonocíclicosRESUMO
Copalyl diphosphate synthase from Penicillium fellutanum (PfCPS) is an assembly-line terpene synthase that contains both prenyltransferase and class II cyclase activities. The prenyltransferase catalyzes processive chain elongation reactions using dimethylallyl diphosphate and three equivalents of isopentenyl diphosphate to yield geranylgeranyl diphosphate, which is then utilized as a substrate by the class II cyclase domain to generate copalyl diphosphate. Here, we report the 2.81 Å-resolution cryo-EM structure of the hexameric prenyltransferase of full-length PfCPS, which is surrounded by randomly splayed-out class II cyclase domains connected by disordered polypeptide linkers. The hexamer can be described as a trimer of dimers; surprisingly, one of the three dimer-dimer interfaces is separated to yield an open hexamer conformation, thus breaking the D3 symmetry typically observed in crystal structures of other prenyltransferase hexamers such as wild-type human GGPP synthase (hGGPPS). Interestingly, however, an open hexamer conformation was previously observed in the crystal structure of D188Y hGGPPS, apparently facilitated by hexamer-hexamer packing in the crystal lattice. The cryo-EM structure of the PfCPS prenyltransferase hexamer is the first to reveal that an open conformation can be achieved even in the absence of a point mutation or interaction with another hexamer. Even though PfCPS octamers are not detected, we suggest that the open hexamer conformation represents an intermediate in the hexamer-octamer equilibrium for those prenyltransferases that do exhibit oligomeric heterogeneity.
Assuntos
Alquil e Aril Transferases , Dimetilaliltranstransferase , Penicillium , Humanos , Dimetilaliltranstransferase/genética , Penicillium/genética , Proteínas de Plantas/genéticaRESUMO
Histone deacetylase 6 (HDAC6) is an important drug target in oncological and non-oncological diseases. Most available HDAC6 inhibitors (HDAC6i) utilize hydroxamic acids as a zinc-binding group, which limits therapeutic opportunities due to its genotoxic potential. Recently, difluoromethyl-1,3,4-oxadiazoles (DFMOs) were reported as potent and selective HDAC6i but their mode of inhibition remained enigmatic. Herein, we report that DFMOs act as mechanism-based and essentially irreversible HDAC6i. Biochemical data confirm that DFMO 6 is a tight-binding HDAC6i capable of inhibiting HDAC6 via a two-step slow-binding mechanism. Crystallographic and mechanistic experiments suggest that the attack of 6 by the zinc-bound water at the sp2 carbon closest to the difluoromethyl moiety followed by a subsequent ring opening of the oxadiazole yields deprotonated difluoroacetylhydrazide 13 as active species. The strong anionic zinc coordination of 13 and the binding of the difluoromethyl moiety in the P571 pocket finally result in an essentially irreversible inhibition of HDAC6.
Assuntos
Inibidores de Histona Desacetilases , Oxidiazóis , Desacetilase 6 de Histona/metabolismo , Oxidiazóis/farmacologia , Oxidiazóis/química , Inibidores de Histona Desacetilases/farmacologia , Inibidores de Histona Desacetilases/química , Zinco/química , Ácidos Hidroxâmicos/farmacologia , Ácidos Hidroxâmicos/químicaRESUMO
Dysregulation of both tubulin deacetylases sirtuin 2 (Sirt2) and the histone deacetylase 6 (HDAC6) has been associated with the pathogenesis of cancer and neurodegeneration, thus making these two enzymes promising targets for pharmaceutical intervention. Herein, we report the design, synthesis, and biological characterization of the first-in-class dual Sirt2/HDAC6 inhibitors as molecular tools for dual inhibition of tubulin deacetylation. Using biochemical in vitro assays and cell-based methods for target engagement, we identified Mz325 (33) as a potent and selective inhibitor of both target enzymes. Inhibition of both targets was further confirmed by X-ray crystal structures of Sirt2 and HDAC6 in complex with building blocks of 33. In ovarian cancer cells, 33 evoked enhanced effects on cell viability compared to single or combination treatment with the unconjugated Sirt2 and HDAC6 inhibitors. Thus, our dual Sirt2/HDAC6 inhibitors are important new tools to study the consequences and the therapeutic potential of dual inhibition of tubulin deacetylation.
Assuntos
Sirtuína 2 , Tubulina (Proteína) , Desacetilase 6 de Histona , Sirtuína 2/metabolismo , Tubulina (Proteína)/metabolismo , Inibidores de Histona Desacetilases/farmacologia , AcetilaçãoRESUMO
The enzyme cofactor (R)-lipoic acid plays a critical role in central carbon metabolism due to its catalytic function in the generation of acetyl-CoA, which links glycolysis with the tricarboxylic acid cycle. This cofactor is also essential for the generation of succinyl CoA within the tricarboxylic acid cycle. However, the biological functions of (R)-lipoic acid extend beyond metabolism owing to its facile redox chemistry. Most recently, the reduced form of (R)-lipoic acid, (R)-dihydrolipoic acid, has been shown to inhibit histone deacetylases (HDACs) with selectivity for the inhibition of HDAC6. Here, we report the 2.4 Å-resolution X-ray crystal structure of the complex between (R)-dihydrolipoic acid and HDAC6 catalytic domain 2 from Danio rerio, and we report a dissociation constant (KD) of 350 nM for this complex as determined by isothermal titration calorimetry. The crystal structure illuminates key affinity determinants in the enzyme active site, including thiolate-Zn2+ coordination and S-π interactions in the F583-F643 aromatic crevice. This study provides the first visualization of the connection between HDAC function and the biological response to oxidative stress: the dithiol moiety of (R)-dihydrolipoic acid can serve as a redox-regulated pharmacophore capable of simultaneously targeting the catalytic Zn2+ ion and the aromatic crevice in the active site of HDAC6.
Assuntos
Ácido Tióctico , Animais , Desacetilase 6 de Histona/metabolismo , Ácido Tióctico/metabolismo , Inibidores de Histona Desacetilases/farmacologia , Histona Desacetilases/metabolismo , Peixe-Zebra/metabolismoRESUMO
Proteomics studies indicate that 10% of proteins in the opportunistic pathogen Acinetobacter baumannii are acetylated, suggesting that lysine acetyltransferases and deacetylases function to maintain and regulate a robust bacterial acetylome. As the first step in exploring these fascinating prokaryotic enzymes, we now report the preparation and characterization of the lysine deacetylase Kdac1. We show that Kdac1 catalyzes the deacetylation of free acetyllysine and acetyllysine tetrapeptide assay substrates, and we also report the X-ray crystal structures of unliganded Kdac1 as well as its complex with the hydroxamate inhibitor Citarinostat. Kdac1 is a tetramer in solution and in the crystal; the crystal structure reveals that the L1 loop functions to stabilize quaternary structure, forming inter-subunit hydrogen bonds and salt bridges around a central arginine residue (R30). Surprisingly, the L1 loop partially blocks entry to the active site, but it is sufficiently flexible to allow for the binding of two Citarinostat molecules in the active site. The L12 loop is also important for maintaining quaternary structure; here, a conserved arginine (R278) accepts hydrogen bonds from the backbone carbonyl groups of residues in an adjacent monomer. Structural comparisons with two other prokaryotic lysine deacetylases reveal conserved residues in the L1 and L12 loops that similarly support tetramer assembly. These studies provide a structural foundation for understanding enzymes that regulate protein function in bacteria through reversible lysine acetylation, serving as a first step in the exploration of these enzymes as possible targets for the development of new antibiotics.
Assuntos
Acinetobacter baumannii , Lisina , Acetilação , Antibacterianos/farmacologia , ArgininaRESUMO
The enzyme cofactor ( R )-lipoic acid plays a critical role in central carbon metabolism due to its catalytic function in the generation of acetyl-CoA, which links glycolysis with the tricarboxylic acid cycle. This cofactor is also essential for the generation of succinyl CoA within the tricarboxylic acid cycle. However, the biological functions of ( R )-lipoic acid extend beyond metabolism owing to its facile redox chemistry. Most recently, the reduced form of ( R )-lipoic acid, ( R )-dihydrolipoic acid, has been shown to inhibit histone deacetylases (HDACs) with selectivity for the inhibition of HDAC6. Here, we report the 2.4 Å-resolution X-ray crystal structure of the HDAC6-( R )-dihydrolipoic acid complex, and we report a dissociation constant (K D ) of 350 nM for this complex as determined by isothermal titration calorimetry. The crystal structure illuminates key affinity determinants in the enzyme active site, including thiolate-Zn 2+ coordination and S-π interactions in the F583-F643 aromatic crevice. This study provides the first visualization of the connection between HDAC function and the biological response to oxidative stress: the dithiol moiety of ( R )-dihydrolipoic acid can serve as a redox-regulated pharmacophore capable of simultaneously targeting the catalytic Zn 2+ ion and the aromatic crevice in the active site of HDAC6.
RESUMO
The class I sesquiterpene cyclase epi-isozizaene synthase from Streptomyces coelicolor (EIZS) catalyzes the transformation of linear farnesyl diphosphate (FPP) into the tricyclic hydrocarbon epi-isozizaene in the biosynthesis of albaflavenone antibiotics. The active site cavity of EIZS is largely framed by four aromatic residues - F95, F96, F198, and W203 - that form a product-shaped contour, serving as a template to chaperone conformations of the flexible substrate and multiple carbocation intermediates leading to epi-isozizaene. Remolding the active site contour by mutagenesis can redirect the cyclization cascade away from epi-isozizaene biosynthesis to generate alternative sesquiterpene products. Here, we present the biochemical and structural characterization of four EIZS mutants in which aromatic residues have been substituted with polar residues (F95S, F96H, F198S, and F198T) to generate alternative cyclization products. Most notably, F95S EIZS generates a mixture of monocyclic sesquiterpene precursors of bisabolane, a D2 diesel fuel substitute. X-ray crystal structures of the characterized mutants reveal subtle changes in the active site contour showing how each aromatic residue influences the chemistry of a different carbocation intermediate in the cyclization cascade. We advance that EIZS may serve as a robust platform for the development of designer cyclases for the generation of high-value sesquiterpene products ranging from pharmaceuticals to biofuels in synthetic biology approaches.
Assuntos
Alquil e Aril Transferases , Sesquiterpenos , Streptomyces coelicolor , Terpenos/química , Ciclização , Sesquiterpenos/química , Streptomyces coelicolor/genética , Sesquiterpenos Monocíclicos , Alquil e Aril Transferases/genéticaRESUMO
Histone deacetylases (HDACs) are essential for the regulation of myriad biological processes, and their aberrant function is implicated in cancer, neurodegeneration, and other diseases. The cytosolic isozyme HDAC6 is unique among the greater family of deacetylases in that it contains two catalytic domains, CD1 and CD2. HDAC6 CD2 is responsible for tubulin deacetylase and tau deacetylase activities, inhibition of which is a key goal as new therapeutic approaches are explored. Of particular interest as HDAC inhibitors are naturally occurring cyclic tetrapeptides such as Trapoxin A or HC Toxin, or the cyclic depsipeptides Largazole and Romidepsin. Even more intriguing are larger, computationally designed macrocyclic peptide inhibitors. Here, we report the 2.0 Å resolution crystal structure of HDAC6 CD2 complexed with macrocyclic octapeptide 1. Comparison with the previously reported structure of the complex with macrocyclic octapeptide 2 reveals that a potent thiolate-zinc interaction made by the unnatural amino acid (S)-2-amino-7-sulfanylheptanoic acid contributes to nanomolar inhibitory potency for each inhibitor. Apart from this zinc-binding residue, octapeptides adopt strikingly different overall conformations and make few direct hydrogen bonds with the protein. Intermolecular interactions are dominated by water-mediated hydrogen bonds; in essence, water molecules appear to cushion the enzyme-octapeptide interface. In view of the broad specificity observed for protein substrates of HDAC6 CD2, we suggest that the binding of macrocyclic octapeptides may mimic certain features of the binding of macromolecular protein substrates.
Assuntos
Desacetilase 6 de Histona , Inibidores de Histona Desacetilases , Histona Desacetilases , Peptídeos Cíclicos , Desacetilase 6 de Histona/química , Inibidores de Histona Desacetilases/farmacologia , Inibidores de Histona Desacetilases/química , Histona Desacetilases/metabolismo , Ligação Proteica , Zinco/metabolismo , Peptídeos Cíclicos/química , Peptídeos Cíclicos/farmacologiaRESUMO
Using a microwave-assisted protocol, we synthesized 16 peptoid-capped HDAC inhibitors (HDACi) with fluorinated linkers and identified two hit compounds. In biochemical and cellular assays, 10h stood out as a potent unselective HDACi with remarkable cytotoxic potential against different therapy-resistant leukemia cell lines. 10h demonstrated prominent antileukemic activity with low cytotoxic activity toward healthy cells. Moreover, 10h exhibited synergistic interactions with the DNA methyltransferase inhibitor decitabine in AML cell lines. The comparison of crystal structures of HDAC6 complexes with 10h and its nonfluorinated counterpart revealed a similar occupation of the L1 loop pocket but slight differences in zinc coordination. The substitution pattern of the acyl residue turned out to be crucial in terms of isoform selectivity. The introduction of an isopropyl group onto the phenyl ring provided the highly HDAC6-selective inhibitor 10p, which demonstrated moderate synergy with decitabine and exceeded the HDAC6 selectivity of tubastatin A.
Assuntos
Antineoplásicos , Leucemia Mieloide Aguda , Peptoides , Humanos , Inibidores de Histona Desacetilases/farmacologia , Inibidores de Histona Desacetilases/uso terapêutico , Inibidores de Histona Desacetilases/química , Desacetilase 6 de Histona , Peptoides/farmacologia , Peptoides/química , Decitabina , Antineoplásicos/farmacologia , Antineoplásicos/uso terapêutico , Antineoplásicos/química , Leucemia Mieloide Aguda/tratamento farmacológico , Linhagem Celular Tumoral , Histona Desacetilase 1 , Ácidos Hidroxâmicos/farmacologia , Ácidos Hidroxâmicos/uso terapêutico , Ácidos Hidroxâmicos/químicaRESUMO
We report the first well-characterized selective chemical probe for histone deacetylase 10 (HDAC10) with unprecedented selectivity over other HDAC isozymes. HDAC10 deacetylates polyamines and has a distinct substrate specificity, making it unique among the 11 zinc-dependent HDAC hydrolases. Taking inspiration from HDAC10 polyamine substrates, we systematically inserted an amino group ("aza-scan") into the hexyl linker moiety of the approved drug Vorinostat (SAHA). This one-atom replacement (CâN) transformed SAHA from an unselective pan-HDAC inhibitor into a specific HDAC10 inhibitor. Optimization of the aza-SAHA structure yielded the HDAC10 chemical probe DKFZ-748, with potency and selectivity demonstrated by cellular and biochemical target engagement, as well as thermal shift assays. Cocrystal structures of our aza-SAHA derivatives with HDAC10 provide a structural rationale for potency, and chemoproteomic profiling confirmed exquisite cellular HDAC10-selectivity of DKFZ-748 across the target landscape of HDAC drugs. Treatment of cells with DKFZ-748, followed by quantification of selected polyamines, validated for the first time the suspected cellular function of HDAC10 as a polyamine deacetylase. Finally, in a polyamine-limiting in vitro tumor model, DKFZ-748 showed dose-dependent growth inhibition of HeLa cells. We expect DKFZ-748 and related probes to enable further studies on the enigmatic biology of HDAC10 and acetylated polyamines in both physiological and pathological settings.
Assuntos
Inibidores de Histona Desacetilases , Isoenzimas , Humanos , Vorinostat , Inibidores de Histona Desacetilases/farmacologia , Inibidores de Histona Desacetilases/química , Células HeLa , Histona Desacetilases/química , Poliaminas/farmacologia , Zinco , Ácidos Hidroxâmicos/farmacologia , Ácidos Hidroxâmicos/químicaRESUMO
Fusicoccadiene synthase from the fungus Phomopsis amygdali (PaFS) is an assembly-line terpene synthase that catalyzes the first two steps in the biosynthesis of Fusiccocin A, a diterpene glycoside. The C-terminal prenyltransferase domain of PaFS catalyzes the condensation of one molecule of C5 dimethylallyl diphosphate and three molecules of C5 isopentenyl diphosphate to form C20 geranylgeranyl diphosphate, which then transits to the cyclase domain for cyclization to form fusicoccadiene. Previous structural studies of PaFS using electron microscopy (EM) revealed a central octameric prenyltransferase core with eight cyclase domains tethered in random distal positions through flexible 70-residue linkers. However, proximal prenyltransferase-cyclase configurations could be captured by covalent cross-linking and observed by cryo-EM and mass spectrometry. Here, we use cryo-EM to show that proximally configured prenyltransferase-cyclase complexes are observable even in the absence of covalent cross-linking; moreover, such complexes can involve multiple cyclase domains. A conserved basic patch on the prenyltransferase domain comprises the primary touchpoint with the cyclase domain. These results support a model for transient prenyltransferase-cyclase association in which the cyclase domains of PaFS are in facile equilibrium between proximal associated and random distal positions relative to the central prenyltransferase octamer. The results of biophysical measurements using small-angle X-ray scattering, analytical ultracentrifugation, dynamic light scattering, and size-exclusion chromatography in-line with multi-angle light scattering are consistent with this model. This model accordingly provides a framework for understanding substrate transit between the prenyltransferase and cyclase domains as well as the cooperativity observed for geranylgeranyl diphosphate cyclization.
Assuntos
Alquil e Aril Transferases , Dimetilaliltranstransferase , Diterpenos , Diterpenos/químicaRESUMO
The regiospecific prenylation of an aromatic amino acid catalyzed by a dimethylallyl-l-tryptophan synthase (DMATS) is a key step in the biosynthesis of many fungal and bacterial natural products. DMATS enzymes share a common "ABBA" fold with divergent active site contours that direct alternative C-C, C-N, and C-O bond-forming trajectories. DMATS1 from Fusarium fujikuroi catalyzes the reverse N-prenylation of l-Trp by generating an allylic carbocation from dimethylallyl diphosphate (DMAPP) that then alkylates the indole nitrogen of l-Trp. DMATS1 stands out among the greater DMATS family because it exhibits unusually broad substrate specificity: it can utilize geranyl diphosphate (GPP) or l-Tyr as an alternative prenyl donor or acceptor, respectively; it can catalyze both forward and reverse prenylation, i.e., at C1 or C3 of DMAPP; and it can catalyze C-N and C-O bond-forming reactions. Here, we report the crystal structures of DMATS1 and its complexes with l-Trp or l-Tyr and unreactive thiolodiphosphate analogues of the prenyl donors DMAPP and GPP. Structures of ternary complexes mimic Michaelis complexes with actual substrates and illuminate active site features that govern prenylation regiochemistry. Comparison with CymD, a bacterial enzyme that catalyzes the reverse N-prenylation of l-Trp with DMAPP, indicates that bacterial and fungal DMATS enzymes share a conserved reaction mechanism. However, the narrower active site contour of CymD enforces narrower substrate specificity. Structure-function relationships established for DMATS enzymes will ultimately inform protein engineering experiments that will broaden the utility of these enzymes as useful tools for synthetic biology.
Assuntos
Produtos Biológicos , Dimetilaliltranstransferase , Triptofano Sintase , Catálise , Dimetilaliltranstransferase/química , Fusarium , Hemiterpenos , Indóis , Neopreno , Nitrogênio , Compostos Organofosforados , Prenilação , Especificidade por Substrato , Triptofano/química , Triptofano Sintase/metabolismoRESUMO
Bavarostat (EKZ-001) is a selective inhibitor of histone deacetylase 6 (HDAC6) that contains a meta-fluorophenylhydroxamate Zn2+-binding group. The recently determined crystal structure of its complex with HDAC6 from Danio rerio (zebrafish) revealed that the meta-fluoro substituent binds exclusively in an aromatic crevice defined by F583 and F643 rather than being oriented out toward solvent. To explore the binding of inhibitor C-F groups in this fluorophilic crevice, we now report a series of 10 simple fluorophenylhydroxamates bearing one or more fluorine atoms with different substitution patterns. Inhibitory potencies against human and zebrafish HDAC6 range widely from 121 to >30,000 nM. The best inhibitory potency is measured for meta-difluorophenylhydroxamate (5) with IC50 = 121 nM against human HDAC6; the worst inhibitory potencies are measured for ortho-fluorophenylhydroxamate (1) as well as fluorophenylhydroxamates 4, 7, 9, and 10, although there are some variations in activity trends against human and zebrafish HDAC6. These studies show that aromatic ring fluorination at the meta position(s) does not improve inhibitory activity against human HDAC6 relative to the nonfluorinated parent compound phenylhydroxamate (IC50 = 120 nM), but meta-fluorination does not seriously compromise inhibitory activity either. Crystal structures of selected zebrafish HDAC6-fluorophenylhydroxamate complexes reveal that the fluoroaromatic ring is uniformly accommodated in the F583-F643 aromatic crevice, so ring fluorination does not perturb the inhibitor binding conformation. However, hydroxamate-Zn2+ coordination is bidentate for some inhibitors and monodentate for others. These studies will inform design strategies underlying the design of 18F-labeled HDAC6 inhibitors intended for positron emission tomography.
Assuntos
Inibidores de Histona Desacetilases , Peixe-Zebra , Animais , Flúor/metabolismo , Halogenação , Desacetilase 6 de Histona/química , Inibidores de Histona Desacetilases/química , Histona Desacetilases/metabolismo , Humanos , Solventes/metabolismo , Relação Estrutura-Atividade , Peixe-Zebra/metabolismoRESUMO
The fungal species Aspergillus flavus produces an alkaloid terpenoid, flavunoidine, through a hybrid biosynthetic pathway combining both terpene cyclase and nonribosomal peptide synthetase enzymes. Flavunoidine consists of a tetracyclic, oxygenated sesquiterpene core decorated with dimethyl cadaverine and 5,5-dimethyl-l-pipecolate moieties. Unique to the flavunoidine biosynthetic pathway is FlvF, a putative enzyme implicated in stereospecific C-N bond formation as dimethyl cadaverine is linked to the sesquiterpene core to generate pre-flavunoidine. Here, we report the 2.6 Å resolution crystal structure of FlvF, which adopts the α-helical fold of a class I terpene synthase. However, FlvF is not a terpene synthase, as indicated by its lack of enzymatic activity with farnesyl diphosphate and its lack of signature metal ion binding motifs that would coordinate to catalytic Mg2+ ions. Thus, FlvF is the first example of a protein that adopts a terpene synthase fold but is not a terpene synthase. Two Bis-Tris molecules bind in the active site of FlvF, and the binding of these ligands guided the docking of pre-flavunoidine to generate a model of the enzyme-product complex. Phylogenetic analysis of FlvF and related fungal homologues reveals conservation of residues that interact with the tetracyclic sesquiterpene in this model, but less conservation of residues interacting with the pendant amino moiety. This may hint toward the possibility that alternative amino substrates can be linked to a common sesquiterpene core by FlvF homologues to generate flavunoidine congeners, such as the phospholipase C inhibitor hispidospermidin.
Assuntos
Alquil e Aril Transferases , Sesquiterpenos , Alquil e Aril Transferases/genética , Cadaverina , Filogenia , Sesquiterpenos/metabolismo , Terpenos , Fosfolipases Tipo CRESUMO
Histone deacetylases (HDACs) are important epigenetic regulators involved in many diseases, especially cancer. Five HDAC inhibitors have been approved for anticancer therapy and many are in clinical trials. Among the 11 zinc-dependent HDACs, HDAC10 has received relatively little attention by drug discovery campaigns, despite its involvement, e. g., in the pathogenesis of neuroblastoma. This is due in part to a lack of robust enzymatic conversion assays. In contrast to the protein lysine deacetylase and deacylase activity of most other HDAC subtypes, it has recently been shown that HDAC10 has strong preferences for deacetylation of oligoamine substrates like acetyl-putrescine or -spermidine. Hence, it is also termed a polyamine deacetylase (PDAC). Here, we present the first fluorescent enzymatic conversion assay for HDAC10 using an aminocoumarin-labelled acetyl-spermidine derivative to measure its PDAC activity, which is suitable for high-throughput screening. Using this assay, we identified potent inhibitors of HDAC10-mediated spermidine deacetylation inâ vitro. Based on the oligoamine preference of HDAC10, we also designed inhibitors with a basic moiety in appropriate distance to the zinc binding hydroxamate that showed potent inhibition of HDAC10 with high selectivity, and we solved a HDAC10-inhibitor structure using X-ray crystallography. We could demonstrate selective cellular target engagement for HDAC10 but a lysosomal phenotype in neuroblastoma cells that was previously associated with HDAC10 inhibition was not observed. Thus, we have developed new chemical probes for HDAC10 that allow further clarification of the biological role of this enzyme.