RESUMEN
Many microorganisms use both biological and nonbiological molecules as sources of carbon and energy. This resourcefulness means that some microorganisms have mechanisms to assimilate pollutants found in the environment. One such organism is Comamonas testosteroni, which metabolizes 4-methylbenzenesulfonate and 4-methylbenzoate using the TsaMBCD pathway. TsaM is a Rieske oxygenase, which in concert with the reductase TsaB consumes a molar equivalent of NADH. Following this step, the annotated short-chain dehydrogenase/reductase and aldehyde dehydrogenase enzymes TsaC and TsaD each regenerate a molar equivalent of NADH. This co-occurrence ameliorates the need for stoichiometric addition of reducing equivalents and thus represents an attractive strategy for integration of Rieske oxygenase chemistry into biocatalytic applications. Therefore, in this work, to overcome the lack of information regarding NADH recycling enzymes that function in partnership with Rieske non-heme iron oxygenases (Rieske oxygenases), we solved the X-ray crystal structure of TsaC to a resolution of 2.18 Å. Using this structure, a series of substrate analog and protein variant combination reactions, and differential scanning fluorimetry experiments, we identified active site features involved in binding NAD+ and controlling substrate specificity. Further in vitro enzyme cascade experiments demonstrated the efficient TsaC- and TsaD-mediated regeneration of NADH to support Rieske oxygenase chemistry. Finally, through in-depth bioinformatic analyses, we illustrate the widespread co-occurrence of Rieske oxygenases with TsaC-like enzymes. This work thus demonstrates the utility of these NADH recycling enzymes and identifies a library of short-chain dehydrogenase/reductase enzyme prospects that can be used in Rieske oxygenase pathways for in situ regeneration of NADH.
Asunto(s)
Proteínas Bacterianas , Comamonas testosteroni , Oxigenasas , Aldehído Deshidrogenasa/metabolismo , NAD/metabolismo , Oxigenasas/metabolismo , Especificidad por Sustrato , Comamonas testosteroni/enzimología , Comamonas testosteroni/genética , Proteínas Bacterianas/química , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Proteínas de Hierro no Heme/química , Proteínas de Hierro no Heme/genética , Proteínas de Hierro no Heme/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Estructura Terciaria de Proteína , Modelos Moleculares , Estabilidad Proteica , Biología ComputacionalRESUMEN
The inherent structural properties of enzymes are critical in defining catalytic function. Often, studies to evaluate the relationship between structure and function are limited to only one defined structural element. The two-component flavin-dependent desulfonase family of enzymes involved in bacterial sulfur acquisition utilize a comprehensive range of structural features to carry out the desulfonation of organosulfur compounds. These metabolically essential two-component FMN-dependent desulfonase systems have been proposed to utilize oligomeric changes, protein-protein interactions for flavin transfer, and common mechanistic steps for carbon-sulfur bond cleavage. This review is focused on our current functional and structural understanding of two-component FMN-dependent desulfonase systems from multiple bacterial sources. Mechanistic and structural comparisons from recent independent studies provide fresh insights into the overall functional properties of these systems and note areas in need of further investigation. The review acknowledges current studies focused on evaluating the structural properties of these enzymes in relationship to their distinct catalytic function. The role of these enzymes in maintaining adequate sulfur levels, coupled with the conserved nature of these enzymes in diverse bacteria, underscore the importance in understanding the functional and structural nuances of these systems.
Asunto(s)
Proteínas Bacterianas , Proteínas Bacterianas/química , Proteínas Bacterianas/metabolismo , Bacterias/enzimología , Compuestos de Azufre/metabolismo , Compuestos de Azufre/química , Hidrolasas/química , Hidrolasas/metabolismo , Mononucleótido de Flavina/metabolismo , Mononucleótido de Flavina/química , Azufre/metabolismo , Azufre/química , Flavinas/metabolismo , Flavinas/química , Relación Estructura-Actividad , Carbono/metabolismo , Carbono/químicaRESUMEN
The DNAs of bacterial viruses are known to contain diverse, chemically complex modifications to thymidine that protect them from the endonuclease-based defenses of their cellular hosts, but whose biosynthetic origins are enigmatic. Up to half of thymidines in the Pseudomonas phage M6, the Salmonella phage ViI, and others, contain exotic chemical moieties synthesized through the post-replicative modification of 5-hydroxymethyluridine (5-hmdU). We have determined that these thymidine hypermodifications are derived from free amino acids enzymatically installed on 5-hmdU. These appended amino acids are further sculpted by various enzyme classes such as radical SAM isomerases, PLP-dependent decarboxylases, flavin-dependent lyases and acetyltransferases. The combinatorial permutations of thymidine hypermodification genes found in viral metagenomes from geographically widespread sources suggests an untapped reservoir of chemical diversity in DNA hypermodifications.
Asunto(s)
Bacteriófagos , Liasas , Aminoácidos/metabolismo , Bacteriófagos/genética , ADN/metabolismo , Timidina/metabolismoRESUMEN
Nonribosomal peptide synthetase heterocyclization (Cy) domains generate biologically important oxazoline/thiazoline groups found in natural products, including pharmaceuticals and virulence factors such as some siderophores. Cy domains catalyze consecutive condensation and cyclodehydration reactions, although the mechanism is unknown. To better understand Cy domain catalysis, here we report the crystal structure of the second Cy domain (Cy2) of yersiniabactin synthetase from the causative agent of the plague, Yersinia pestis. Our high-resolution structure of Cy2 adopts a conformation that enables exploration of interactions with the extended thiazoline-containing cyclodehydration intermediate and the acceptor carrier protein (CP) to which it is tethered. We also report complementary electrostatic interfaces between Cy2 and its donor CP that mediate donor binding. Finally, we explored domain flexibility through normal mode analysis and identified small-molecule fragment-binding sites that may inform future antibiotic design targeting Cy function. Our results suggest how CP binding may influence global Cy conformations, with consequences for active-site remodeling to facilitate the separate condensation and cyclodehydration steps as well as potential inhibitor development.
Asunto(s)
Dominio Catalítico , Péptido Sintasas , Yersinia pestis , Proteínas Bacterianas/química , Proteínas Bacterianas/metabolismo , Proteínas Portadoras/metabolismo , Péptido Sintasas/química , Péptido Sintasas/metabolismo , Sideróforos/metabolismo , Yersinia pestis/química , Yersinia pestis/enzimologíaRESUMEN
Bacterial two-component flavin-dependent monooxygenases cleave the stable C-S bond of environmental and anthropogenic organosulfur compounds. The monooxygenase MsuD converts methanesulfonate (MS-) to sulfite, completing the sulfur assimilation process during sulfate starvation, but the mechanism of this conversion remains unclear. To explore the mechanism of C-S bond cleavage, we report a series of crystal structures of MsuD from Pseudomonas fluorescens in different liganded states. This report provides the first crystal structures of an alkanesulfonate monooxygenase with a bound flavin and alkanesulfonate, elucidating the roles of the active site lid, the protein C terminus, and an active site loop in flavin and/or alkanesulfonate binding. These structures position MS- closest to the flavin N5 position, consistent with an N5-(hydro)peroxyflavin mechanism rather than a classical C4a-(hydro)peroxyflavin mechanism. A fully enclosed active site is observed in the ternary complex, mediated by interchain interaction of the C terminus at the tetramer interface. These structures identify an unexpected function of the protein C terminus in this protein family in stabilizing tetramer formation and the alkanesulfonate-binding site. Spurred by interest from the crystal structures, we conducted biochemical assays and molecular docking that redefine MsuD as a small- to medium-chain alkanesulfonate monooxygenase. Functional mutations verify the sulfonate-binding site and reveal the critical importance of the protein C terminus for monooxygenase function. These findings reveal a deeper understanding of MsuD's functionality at the molecular level and consequently how it operates within its role as part of the sulfur assimilation pathway.
Asunto(s)
Proteínas Bacterianas/química , Proteínas Bacterianas/metabolismo , Oxigenasas de Función Mixta/química , Oxigenasas de Función Mixta/metabolismo , Multimerización de Proteína , Pseudomonas fluorescens/enzimología , Secuencia de Aminoácidos , Sitios de Unión , Dominio Catalítico , Mononucleótido de Flavina/metabolismo , Mesilatos/metabolismo , Modelos Moleculares , Especificidad por Sustrato , Azufre/metabolismoRESUMEN
Methyl sulfur compounds are a rich source of environmental sulfur for microorganisms, but their use requires redox systems. The bacterial sfn and msu operons contain two-component flavin-dependent monooxygenases for dimethylsulfone (DMSO2) assimilation: SfnG converts DMSO2 to methanesulfinate (MSI-), and MsuD converts methanesulfonate (MS-) to sulfite. However, the enzymatic oxidation of MSI- to MS- has not been demonstrated, and the function of the last enzyme of the msu operon (MsuC) is unresolved. We employed crystallographic and biochemical studies to identify the function of MsuC from Pseudomonas fluorescens. The crystal structure of MsuC adopts the acyl-CoA dehydrogenase fold with putative binding sites for flavin and MSI-, and functional assays of MsuC in the presence of its oxidoreductase MsuE, FMN, and NADH confirm the enzymatic generation of MS-. These studies reveal that MsuC converts MSI- to MS- in sulfite biosynthesis from DMSO2.
Asunto(s)
Proteínas Bacterianas/metabolismo , Pseudomonas fluorescens/enzimología , Azufre/química , Acil-CoA Deshidrogenasa/metabolismo , Sitios de Unión , Cristalografía por Rayos X , Dimerización , Dimetilsulfóxido/química , Flavinas/química , Espectroscopía de Resonancia Magnética , Mesilatos/química , Simulación del Acoplamiento Molecular , Oxidorreductasas/metabolismo , Oxígeno/química , Estructura Secundaria de Proteína , Relación Estructura-Actividad , Sulfuros/química , Sulfonas/química , Tiofenos/químicaRESUMEN
Epothilones are thiazole-containing natural products with anticancer activity that are biosynthesized by polyketide synthase (PKS)-nonribosomal peptide synthetase (NRPS) enzymes EpoA-F. A cyclization domain of EpoB (Cy) assembles the thiazole functionality from an acetyl group and l-cysteine via condensation, cyclization, and dehydration. The PKS carrier protein of EpoA contributes the acetyl moiety, guided by a docking domain, whereas an NRPS EpoB carrier protein contributes l-cysteine. To visualize the structure of a cyclization domain with an accompanying docking domain, we solved a 2.03-Å resolution structure of this bidomain EpoB unit, comprising residues M1-Q497 (62 kDa) of the 160-kDa EpoB protein. We find that the N-terminal docking domain is connected to the V-shaped Cy domain by a 20-residue linker but otherwise makes no contacts to Cy. Molecular dynamic simulations and additional crystal structures reveal a high degree of flexibility for this docking domain, emphasizing the modular nature of the components of PKS-NRPS hybrid systems. These structures further reveal two 20-Å-long channels that run from distant sites on the Cy domain to the active site at the core of the enzyme, allowing two carrier proteins to dock with Cy and deliver their substrates simultaneously. Through mutagenesis and activity assays, catalytic residues N335 and D449 have been identified. Surprisingly, these residues do not map to the location of the conserved HHxxxDG motif in the structurally homologous NRPS condensation (C) domain. Thus, although both C and Cy domains have the same basic fold, their active sites appear distinct.
Asunto(s)
Epotilonas/química , Péptido Sintasas/química , Sintasas Poliquetidas/química , Dominios Proteicos , Secuencia de Aminoácidos , Proteínas Bacterianas/química , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Vías Biosintéticas/genética , Dominio Catalítico , Cristalografía por Rayos X , Ciclización , Epotilonas/metabolismo , Modelos Moleculares , Myxococcales/genética , Myxococcales/metabolismo , Péptido Sintasas/genética , Péptido Sintasas/metabolismo , Sintasas Poliquetidas/genética , Sintasas Poliquetidas/metabolismo , Unión Proteica , Homología de Secuencia de Aminoácido , Tiazoles/química , Tiazoles/metabolismoRESUMEN
Queuosine (Q) was discovered in the wobble position of a transfer RNA (tRNA) 47 years ago, yet the final biosynthetic enzyme responsible for Q-maturation, epoxyqueuosine (oQ) reductase (QueG), was only recently identified. QueG is a cobalamin (Cbl)-dependent, [4Fe-4S] cluster-containing protein that produces the hypermodified nucleoside Q in situ on four tRNAs. To understand how QueG is able to perform epoxide reduction, an unprecedented reaction for a Cbl-dependent enzyme, we have determined a series of high resolution structures of QueG from Bacillus subtilis Our structure of QueG bound to a tRNATyr anticodon stem loop shows how this enzyme uses a HEAT-like domain to recognize the appropriate anticodons and position the hypermodified nucleoside into the enzyme active site. We find Q bound directly above the Cbl, consistent with a reaction mechanism that involves the formation of a covalent Cbl-tRNA intermediate. Using protein film electrochemistry, we show that two [4Fe-4S] clusters adjacent to the Cbl have redox potentials in the range expected for Cbl reduction, suggesting how Cbl can be activated for nucleophilic attack on oQ. Together, these structural and electrochemical data inform our understanding of Cbl dependent nucleic acid modification.
Asunto(s)
Procesamiento Postranscripcional del ARN , ARN de Transferencia/química , ARN de Transferencia/genética , Vitamina B 12/química , Anticodón , Bacillus subtilis/genética , Enlace de Hidrógeno , Hierro/química , Modelos Moleculares , Conformación Molecular , Conformación de Ácido Nucleico , Nucleósido Q/análogos & derivados , Nucleósido Q/química , Unión Proteica , Estabilidad del ARN , ARN Bacteriano/química , ARN Bacteriano/genética , ARN Bacteriano/metabolismo , ARN de Transferencia/metabolismo , Ribonucleasas/química , Ribonucleasas/metabolismo , Azufre/química , Vitamina B 12/metabolismoRESUMEN
BACKGROUND: Orthology characterizes genes of different organisms that arose from a single ancestral gene via speciation, in contrast to paralogy, which is assigned to genes that arose via gene duplication. An accurate orthology assignment is a crucial step for comparative genomic studies. Orthologous genes in two organisms can be identified by applying a so-called reciprocal search strategy, given that complete information of the organisms' gene repertoire is available. In many investigations, however, only a fraction of the gene content of the organisms under study is examined (e.g., RNA sequencing). Here, identification of orthologous nucleotide or amino acid sequences can be achieved using a graph-based approach that maps nucleotide sequences to genes of known orthology. Existing implementations of this approach, however, suffer from algorithmic issues that may cause problems in downstream analyses. RESULTS: We present a new software pipeline, Orthograph, that addresses and solves the above problems and implements useful features for a wide range of comparative genomic and transcriptomic analyses. Orthograph applies a best reciprocal hit search strategy using profile hidden Markov models and maps nucleotide sequences to the globally best matching cluster of orthologous genes, thus enabling researchers to conveniently and reliably delineate orthologs and paralogs from transcriptomic and genomic sequence data. We demonstrate the performance of our approach on de novo-sequenced and assembled transcript libraries of 24 species of apoid wasps (Hymenoptera: Aculeata) as well as on published genomic datasets. CONCLUSION: With Orthograph, we implemented a best reciprocal hit approach to reference-based orthology prediction for coding nucleotide sequences such as RNAseq data. Orthograph is flexible, easy to use, open source and freely available at https://mptrsen.github.io/Orthograph . Additionally, we release 24 de novo-sequenced and assembled transcript libraries of apoid wasp species.
Asunto(s)
Genómica/métodos , Familia de Multigenes/genética , Sistemas de Lectura Abierta/genética , Análisis de Secuencia de ADN/métodos , Análisis de Secuencia de Proteína/métodos , Programas Informáticos , Animales , Genoma/genética , Transcriptoma/genética , Avispas/genéticaRESUMEN
Radical S-adenosyl-l-methionine (SAM) enzymes are widely distributed and catalyze diverse reactions. SAM binds to the unique iron atom of a site-differentiated [4Fe-4S] cluster and is reductively cleaved to generate a 5'-deoxyadenosyl radical, which initiates turnover. 7-Carboxy-7-deazaguanine (CDG) synthase (QueE) catalyzes a key step in the biosynthesis of 7-deazapurine containing natural products. 6-Carboxypterin (6-CP), an oxidized analogue of the natural substrate 6-carboxy-5,6,7,8-tetrahydropterin (CPH4), is shown to be an alternate substrate for CDG synthase. Under reducing conditions that would promote the reductive cleavage of SAM, 6-CP is turned over to 6-deoxyadenosylpterin (6-dAP), presumably by radical addition of the 5'-deoxyadenosine followed by oxidative decarboxylation to the product. By contrast, in the absence of the strong reductant, dithionite, the carboxylate of 6-CP is esterified to generate 6-carboxypterin-5'-deoxyadenosyl ester (6-CP-dAdo ester). Structural studies with 6-CP and SAM also reveal electron density consistent with the ester product being formed in crystallo. The differential reactivity of 6-CP under reducing and nonreducing conditions highlights the ability of radical SAM enzymes to carry out both polar and radical transformations in the same active site.
Asunto(s)
Proteínas Bacterianas/metabolismo , Productos Biológicos/metabolismo , Liasas de Carbono-Nitrógeno/metabolismo , Purinas/biosíntesis , S-Adenosilmetionina/metabolismo , Proteínas Bacterianas/química , Biocatálisis , Productos Biológicos/química , Liasas de Carbono-Nitrógeno/química , Cristalografía por Rayos X , Radicales Libres/química , Radicales Libres/metabolismo , Modelos Moleculares , Estructura Molecular , Purinas/química , S-Adenosilmetionina/químicaRESUMEN
BACKGROUND: Body plan development in multi-cellular organisms is largely determined by homeotic genes. Expression of homeotic genes, in turn, is partially regulated by insulator binding proteins (IBPs). While only a few enhancer blocking IBPs have been identified in vertebrates, the common fruit fly Drosophila melanogaster harbors at least twelve different enhancer blocking IBPs. We screened recently compiled insect transcriptomes from the 1KITE project and genomic and transcriptomic data from public databases, aiming to trace the origin of IBPs in insects and other arthropods. RESULTS: Our study shows that the last common ancestor of insects (Hexapoda) already possessed a substantial number of IBPs. Specifically, of the known twelve insect IBPs, at least three (i.e., CP190, Su(Hw), and CTCF) already existed prior to the evolution of insects. Furthermore we found GAF orthologs in early branching insect orders, including Zygentoma (silverfish and firebrats) and Diplura (two-pronged bristletails). Mod(mdg4) is most likely a derived feature of Neoptera, while Pita is likely an evolutionary novelty of holometabolous insects. Zw5 appears to be restricted to schizophoran flies, whereas BEAF-32, ZIPIC and the Elba complex, are probably unique to the genus Drosophila. Selection models indicate that insect IBPs evolved under neutral or purifying selection. CONCLUSIONS: Our results suggest that a substantial number of IBPs either pre-date the evolution of insects or evolved early during insect evolution. This suggests an evolutionary history of insulator binding proteins in insects different to that previously thought. Moreover, our study demonstrates the versatility of the 1KITE transcriptomic data for comparative analyses in insects and other arthropods.
Asunto(s)
Artrópodos/genética , Proteínas de Unión al ADN/genética , Evolución Molecular , Elementos Aisladores , Transcriptoma , Animales , Drosophila melanogaster/genética , Elementos de Facilitación Genéticos , Perfilación de la Expresión Génica , FilogeniaRESUMEN
7-carboxy-7-deazaguanine synthase (QueE) catalyzes a key S-adenosyl-L-methionine (AdoMet)- and Mg(2+)-dependent radical-mediated ring contraction step, which is common to the biosynthetic pathways of all deazapurine-containing compounds. QueE is a member of the AdoMet radical superfamily, which employs the 5'-deoxyadenosyl radical from reductive cleavage of AdoMet to initiate chemistry. To provide a mechanistic rationale for this elaborate transformation, we present the crystal structure of a QueE along with structures of pre- and post-turnover states. We find that substrate binds perpendicular to the [4Fe-4S]-bound AdoMet, exposing its C6 hydrogen atom for abstraction and generating the binding site for Mg(2+), which coordinates directly to the substrate. The Burkholderia multivorans structure reported here varies from all other previously characterized members of the AdoMet radical superfamily in that it contains a hypermodified (ß6/α3) protein core and an expanded cluster-binding motif, CX14CX2C.
Asunto(s)
Magnesio/química , Manganeso/química , Modelos Moleculares , S-Adenosilmetionina/química , S-Adenosilmetionina/metabolismo , Sodio/química , Sitios de Unión , Burkholderia/enzimología , Radicales Libres/química , Radicales Libres/metabolismo , Enlace de Hidrógeno/efectos de los fármacos , Magnesio/farmacología , Manganeso/farmacología , Estructura Molecular , Estructura Terciaria de Proteína , Sodio/farmacologíaRESUMEN
Cleptoparasitic wasps and bees smuggle their eggs into the nest of a host organism. Here the larvae of the cleptoparasite feed upon the food provision intended for the offspring of the host. As cleptoparasitism incurs a loss of fitness for the host organism (offspring of the host fail to develop), hosts of cleptoparasites are expected to exploit cues that alert them to potential cleptoparasite infestation. Cuticular hydrocarbons (CHCs) could serve as such cues, as insects inevitably leave traces of them behind when entering a nest. By mimicking the host's CHC profile, cleptoparasites can conceal their presence and evade detection by their host. Previous studies have provided evidence of cleptoparasites mimicking their host's CHC profile. However, the impact of this strategy on the evolution of the host's CHC profile has remained unexplored. Here, we present results from our investigation of a host-cleptoparasite system consisting of a single mason wasp species that serves syntopically as the host to three cuckoo wasp species. We found that the spiny mason wasp (Odynerus spinipes) is able to express two substantially different CHC profiles, each of which is seemingly mimicked by a cleptoparasitic cuckoo wasp (i.e. Chrysis mediata and Pseudospinolia neglecta). The CHC profile of the third cuckoo wasp (Chrysis viridula), a species not expected to benefit from mimicking its host's CHC profile because of its particular oviposition strategy, differs from the two CHC profiles of its host. Our results corroborate the idea that the similarity of the CHC profiles between cleptoparasitic cuckoo wasps and their hosts are the result of chemical mimicry. They further suggest that cleptoparasites may represent a hitherto unappreciated force that drives the evolution of their hosts' CHCs.
Asunto(s)
Hidrocarburos/química , Avispas/química , Avispas/parasitología , Comunicación Animal , Animales , Evolución Biológica , Señales (Psicología) , Interacciones Huésped-Parásitos , Odorantes , Análisis de Secuencia de ADN , Especificidad de la Especie , Avispas/fisiologíaRESUMEN
AdoMet radical enzymes are involved in processes such as cofactor biosynthesis, anaerobic metabolism, and natural product biosynthesis. These enzymes utilize the reductive cleavage of S-adenosylmethionine (AdoMet) to afford l-methionine and a transient 5'-deoxyadenosyl radical, which subsequently generates a substrate radical species. By harnessing radical reactivity, the AdoMet radical enzyme superfamily is responsible for an incredible diversity of chemical transformations. Structural analysis reveals that family members adopt a full or partial Triose-phosphate Isomerase Mutase (TIM) barrel protein fold, containing core motifs responsible for binding a catalytic [4Fe-4S] cluster and AdoMet. Here we evaluate over twenty structures of AdoMet radical enzymes and classify them into two categories: 'traditional' and 'ThiC-like' (named for the structure of 4-amino-5-hydroxymethyl-2-methylpyrimidine phosphate synthase (ThiC)). In light of new structural data, we reexamine the 'traditional' structural motifs responsible for binding the [4Fe-4S] cluster and AdoMet, and compare and contrast these motifs with the ThiC case. We also review how structural data combine with biochemical, spectroscopic, and computational data to help us understand key features of this enzyme superfamily, such as the energetics, the triggering, and the molecular mechanisms of AdoMet reductive cleavage. This article is part of a Special Issue entitled: Radical SAM Enzymes and Radical Enzymology.
Asunto(s)
Proteínas Bacterianas/química , Coenzimas/metabolismo , Transferasas Intramoleculares/química , Proteínas Hierro-Azufre/química , S-Adenosilmetionina/metabolismo , Triosa-Fosfato Isomerasa/química , Secuencias de Aminoácidos , Proteínas Bacterianas/metabolismo , Coenzimas/química , Radicales Libres/química , Radicales Libres/metabolismo , Humanos , Transferasas Intramoleculares/metabolismo , Proteínas Hierro-Azufre/metabolismo , Cinética , Modelos Moleculares , Datos de Secuencia Molecular , Estructura Terciaria de Proteína , S-Adenosilmetionina/química , Termodinámica , Triosa-Fosfato Isomerasa/metabolismoRESUMEN
Ox-/thiazoline groups in nonribosomal peptides are formed by a variant of peptide-forming condensation domains called heterocyclization (Cy) domains and appear in a range of pharmaceutically important natural products and virulence factors. Recent cryo-EM, crystallographic, and NMR studies of Cy domains make it opportune to revisit outstanding questions regarding their molecular mechanisms. This review covers structural and dynamical findings about Cy domains that will inform future bioengineering efforts and our understanding of natural product synthesis.
Asunto(s)
Péptido Sintasas , Péptidos , Ciclización , Péptido Sintasas/metabolismo , Dominios ProteicosRESUMEN
AIMS: To measure inflammatory markers in offspring of pregestational type 1 diabetic mothers. METHODS: Type 1 diabetes was induced in female C57BL6/J mice using streptozotocin. Offspring from control C57BL6/J and type 1 diabetic mothers were followed up to adulthood and blood was collected at 6 and 12 weeks of age, representing adolescent and adult stages respectively. Five well-established inflammatory markers; Matrix metalloproteinase 9, soluble E-selectin, sICAM-1, sVCAM-1, and total plasminogen activator inhibitor-1 (PAI-1) were measured on an inflammatory multiplex assay in plasma. RESULTS: Blood plasma from adolescent offspring from diabetic mothers displayed an increase in all five inflammatory markers when compared to controls, and there was a highly significant increase in sVCAM-1 (64.56 ± 20.1 vs. 33.8 ± 20.75; p < 0.01) and tPAI-1 (0.05 ± 0.02 vs. 0.02 ± 0.01; p < 0.01) expression. CONCLUSION: Our findings show that inflammatory markers are increased in offspring of pregestational diabetic mothers. This may represent a mechanism for increased risk of cardiovascular disease evident in these offspring.
Asunto(s)
Envejecimiento/sangre , Envejecimiento/patología , Diabetes Mellitus Tipo 1/sangre , Diabetes Mellitus Tipo 1/patología , Mediadores de Inflamación/sangre , Inflamación/sangre , Inflamación/patología , Animales , Biomarcadores/sangre , Femenino , Ratones , Ratones Endogámicos C57BLRESUMEN
Biological activity is governed by the timely redistribution of molecular interactions, and static structural snapshots often appear insufficient to provide the molecular determinants that choreograph communication. This conundrum applies to multidomain enzymatic systems called nonribosomal peptide synthetases (NRPSs), which assemble simple substrates into complex metabolites, where a dynamic domain organization challenges rational design to produce new pharmaceuticals. Using a nuclear magnetic resonance (NMR) atomic-level readout of biochemical transformations, we demonstrate that global structural fluctuations help promote substrate-dependent communication and allosteric responses, and impeding these global dynamics by a point-site mutation hampers allostery and molecular recognition. Our results establish global structural dynamics as sensors of molecular events that can remodel domain interactions, and they provide new perspectives on mechanisms of allostery, protein communication, and NRPS synthesis.
RESUMEN
Largazole is a macrocyclic depsipeptide originally isolated from the marine cyanobacterium Symploca sp., which is indigenous to the warm, blue-green waters of Key Largo, Florida (whence largazole derives its name). Largazole contains an unusual thiazoline-thiazole ring system that rigidifies its macrocyclic skeleton, and it also contains a lipophilic thioester side chain. Hydrolysis of the thioester in vivo yields largazole thiol, which exhibits remarkable antiproliferative effects and is believed to be the most potent inhibitor of the metal-dependent histone deacetylases (HDACs). Here, the 2.14 Å-resolution crystal structure of the HDAC8-largazole thiol complex is the first of an HDAC complexed with a macrocyclic inhibitor and reveals that ideal thiolate-zinc coordination geometry is the key chemical feature responsible for its exceptional affinity and biological activity. Notably, the core structure of largazole is conserved in romidepsin, a depsipeptide natural product formulated as the drug Istodax recently approved for cancer chemotherapy. Accordingly, the structure of the HDAC8-largazole thiol complex is the first to illustrate the mode of action of a new class of therapeutically important HDAC inhibitors.
Asunto(s)
Depsipéptidos/química , Depsipéptidos/farmacología , Inhibidores de Histona Desacetilasas/química , Inhibidores de Histona Desacetilasas/farmacología , Histona Desacetilasas/metabolismo , Proteínas Represoras/metabolismo , Tiazoles/química , Tiazoles/farmacología , Cristalografía por Rayos X , Cianobacterias/química , Histona Desacetilasas/química , Humanos , Modelos Moleculares , Proteínas Represoras/químicaRESUMEN
As part of our continuing search for new amino acid inhibitors of metalloenzymes, we now report the synthesis and biological evaluation of the trifluoromethylketone analogue of L-arginine, (S)-2-amino-8,8,8-trifluoro-7-oxo-octanoic acid (10). While this novel amino acid was initially designed as a potential inhibitor of human arginase I, it exhibits no measurable inhibitory activity against this enzyme. Surprisingly, however, 10 is a potent inhibitor of human histone deacetylase 8, with IC(50)=1.5 ± 0.2 µM. Additionally, 10 weakly inhibits the related bacterial enzyme, acetylpolyamine amidohydrolase, with IC(50)=110 ± 30 µM. The lack of inhibitory activity against human arginase I may result from unfavorable interactions of the bulky trifluoromethyl group of 10 in the constricted active site. Since the active site of histone deacetylase 8 is less constricted, we hypothesize that it accommodates 10 as the gem-diol, which mimics the tetrahedral intermediate and its flanking transition states in catalysis. Therefore, we suggest that 10 represents a new lead in the design of an amino acid or peptide-based inhibitor of histone deacetylases with simpler structure than previously studied trifluoromethylketones.
Asunto(s)
Aminoácidos/síntesis química , Aminoácidos/farmacología , Arginasa/antagonistas & inhibidores , Diseño de Fármacos , Inhibidores Enzimáticos/síntesis química , Inhibidores Enzimáticos/farmacología , Proteínas Represoras/antagonistas & inhibidores , Secuencia de Aminoácidos , Aminoácidos/química , Aminoácidos/toxicidad , Dominio Catalítico , Evaluación Preclínica de Medicamentos , Inhibidores Enzimáticos/química , Inhibidores Enzimáticos/toxicidad , Histona Desacetilasas , Humanos , Concentración 50 Inhibidora , Isoenzimas/metabolismo , Cetonas/química , Metales/química , Estructura Molecular , Terapia Molecular Dirigida , Neoplasias/tratamiento farmacológico , Neoplasias/enzimología , Neoplasias/prevención & control , Proteínas Recombinantes , Relación Estructura-ActividadRESUMEN
The metal-dependent histone deacetylases (HDACs) adopt an alpha/beta protein fold first identified in rat liver arginase. Despite insignificant overall amino acid sequence identity, these enzymes share a strictly conserved metal binding site with divergent metal specificity and stoichiometry. HDAC8, originally thought to be a Zn(2+)-metallohydrolase, exhibits increased activity with Co(2+) and Fe(2+) cofactors based on k(cat)/K(M) (Gantt, S. L., Gattis, S. G., and Fierke, C. A. (2006) Biochemistry 45, 6170-6178). Here, we report the first X-ray crystal structures of metallo-substituted HDAC8, Co(2+)-HDAC8, D101L Co(2+)-HDAC8, D101L Mn(2+)-HDAC8, and D101L Fe(2+)-HDAC8, each complexed with the inhibitor M344. Metal content of protein samples in solution is confirmed by inductively coupled plasma mass spectrometry. For the crystalline enzymes, peaks in Bijvoet difference Fourier maps calculated from X-ray diffraction data collected near the respective elemental absorption edges confirm metal substitution. Additional solution studies confirm incorporation of Cu(2+); Fe(3+) and Ni(2+) do not bind under conditions tested. The metal dependence of the substrate K(M) values and the K(i) values of hydroxamate inhibitors that chelate the active site metal are consistent with substrate-metal coordination in the precatalytic Michaelis complex that enhances catalysis. Additionally, although HDAC8 binds Zn(2+) nearly 10(6)-fold more tightly than Fe(2+), the affinities for both metal ions are comparable to the readily exchangeable metal concentrations estimated in living cells, suggesting that HDAC8 could bind either or both Fe(2+) or Zn(2+) in vivo.