RESUMEN
Ribosomes frequently stall during mRNA translation, resulting in the context-dependent activation of quality control pathways to maintain proteostasis. However, surveillance mechanisms that specifically respond to stalled ribosomes with an occluded A site have not been identified. We discovered that the elongation factor-1α (eEF1A) inhibitor, ternatin-4, triggers the ubiquitination and degradation of eEF1A on stalled ribosomes. Using a chemical genetic approach, we unveiled a signaling network comprising two E3 ligases, RNF14 and RNF25, which are required for eEF1A degradation. Quantitative proteomics revealed the RNF14 and RNF25-dependent ubiquitination of eEF1A and a discrete set of ribosomal proteins. The ribosome collision sensor GCN1 plays an essential role by engaging RNF14, which directly ubiquitinates eEF1A. The site-specific, RNF25-dependent ubiquitination of the ribosomal protein RPS27A/eS31 provides a second essential signaling input. Our findings illuminate a ubiquitin signaling network that monitors the ribosomal A site and promotes the degradation of stalled translation factors, including eEF1A and the termination factor eRF1.
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Proteínas de Unión al ARN , Transactivadores , Proteínas Portadoras/metabolismo , Factores de Elongación de Péptidos/genética , Biosíntesis de Proteínas , Proteínas Ribosómicas/metabolismo , Ribosomas/metabolismo , Ubiquitina-Proteína Ligasas/metabolismo , Ubiquitinación , Humanos , Células HeLa , Células HEK293 , Proteínas de Unión al ARN/metabolismo , Transactivadores/metabolismo , Factor 1 de Elongación Peptídica/metabolismoRESUMEN
The past decade has seen impressive advances in understanding the biosynthesis of ribosomally synthesized and posttranslationally modified peptides (RiPPs). One of the most common modifications found in these natural products is macrocyclization, a strategy also used by medicinal chemists to improve metabolic stability and target affinity and specificity. Another tool of the peptide chemist, modification of the amides in a peptide backbone, has also been observed in RiPPs. This review discusses the molecular mechanisms of biosynthesis of a subset of macrocyclic RiPP families, chosen because of the unusual biochemistry involved: the five classes of lanthipeptides (thioether cyclization by Michael-type addition), sactipeptides and ranthipeptides (thioether cyclization by radical chemistry), thiopeptides (cyclization by [4+2] cycloaddition), and streptide (cyclization by radical C-C bond formation). In addition, the mechanisms of backbone amide methylation, backbone epimerization, and backbone thioamide formation are discussed, as well as an unusual route to small molecules by posttranslational modification.
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Péptidos , Procesamiento Proteico-Postraduccional , Secuencia de Aminoácidos , Humanos , Péptidos/química , Sulfuros/química , Sulfuros/metabolismoRESUMEN
Microbial natural products have provided an important source of therapeutic leads and motivated research and innovation in diverse scientific disciplines. In recent years, it has become evident that bacteria harbor a large, hidden reservoir of potential natural products in the form of silent or cryptic biosynthetic gene clusters (BGCs). These can be readily identified in microbial genome sequences but do not give rise to detectable levels of a natural product. Herein, we provide a useful organizational framework for the various methods that have been implemented for interrogating silent BGCs. We divide all available approaches into four categories. The first three are endogenous strategies that utilize the native host in conjunction with classical genetics, chemical genetics, or different culture modalities. The last category comprises expression of the entire BGC in a heterologous host. For each category, we describe the rationale, recent applications, and associated advantages and limitations.
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Productos Biológicos/química , Vías Biosintéticas/genética , Técnicas de Cultivo/métodos , Familia de Multigenes , Genética Inversa/métodos , Bacterias/genética , Bacterias/metabolismo , Productos Biológicos/metabolismo , Regulación de la Expresión GénicaRESUMEN
The human microbiome encodes a second genome that dwarfs the genetic capacity of the host. Microbiota-derived small molecules can directly target human cells and their receptors or indirectly modulate host responses through functional interactions with other microbes in their ecological niche. Their biochemical complexity has profound implications for nutrition, immune system development, disease progression, and drug metabolism, as well as the variation in these processes that exists between individuals. While the species composition of the human microbiome has been deeply explored, detailed mechanistic studies linking specific microbial molecules to host phenotypes are still nascent. In this review, we discuss challenges in decoding these interaction networks, which require interdisciplinary approaches that combine chemical biology, microbiology, immunology, genetics, analytical chemistry, bioinformatics, and synthetic biology. We highlight important classes of microbiota-derived small molecules and notable examples. An understanding of these molecular mechanisms is central to realizing the potential of precision microbiome editing in health, disease, and therapeutic responses.
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Metagenómica/métodos , Microbiota/fisiología , Péptidos/metabolismo , Policétidos/metabolismo , Microbioma Gastrointestinal/genética , Microbioma Gastrointestinal/fisiología , Humanos , Microbiota/genética , FenotipoRESUMEN
In response to biotic stress, plants produce suites of highly modified fatty acids that bear unusual chemical functionalities. Despite their chemical complexity and proposed roles in pathogen defense, little is known about the biosynthesis of decorated fatty acids in plants. Falcarindiol is a prototypical acetylenic lipid present in carrot, tomato, and celery that inhibits growth of fungi and human cancer cell lines. Using a combination of untargeted metabolomics and RNA sequencing, we discovered a biosynthetic gene cluster in tomato (Solanum lycopersicum) required for falcarindiol production. By reconstituting initial biosynthetic steps in a heterologous host and generating transgenic pathway mutants in tomato, we demonstrate a direct role of the cluster in falcarindiol biosynthesis and resistance to fungal and bacterial pathogens in tomato leaves. This work reveals a mechanism by which plants sculpt their lipid pool in response to pathogens and provides critical insight into the complex biochemistry of alkynyl lipid production.
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Diinos/metabolismo , Ácidos Grasos/biosíntesis , Alcoholes Grasos/metabolismo , Solanum lycopersicum/genética , Resistencia a la Enfermedad/genética , Diinos/química , Ácidos Grasos/metabolismo , Alcoholes Grasos/química , Regulación de la Expresión Génica de las Plantas/genética , Metabolómica , Familia de Multigenes/genética , Enfermedades de las Plantas/microbiología , Hojas de la Planta/metabolismo , Proteínas de Plantas/metabolismo , Plantas Modificadas Genéticamente , Estrés Fisiológico/genéticaRESUMEN
Copper-binding metallophores, or chalkophores, play a role in microbial copper homeostasis that is analogous to that of siderophores in iron homeostasis. The best-studied chalkophores are members of the methanobactin (Mbn) family-ribosomally produced, posttranslationally modified natural products first identified as copper chelators responsible for copper uptake in methane-oxidizing bacteria. To date, Mbns have been characterized exclusively in those species, but there is genomic evidence for their production in a much wider range of bacteria. This review addresses the current state of knowledge regarding the function, biosynthesis, transport, and regulation of Mbns. While the roles of several proteins in these processes are supported by substantial genetic and biochemical evidence, key aspects of Mbn manufacture, handling, and regulation remain unclear. In addition, other natural products that have been proposed to mediate copper uptake as well as metallophores that have biologically relevant roles involving copper binding, but not copper uptake, are discussed.
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Proteínas Bacterianas/metabolismo , Quelantes/metabolismo , Cobre/metabolismo , Imidazoles/metabolismo , Oligopéptidos/metabolismo , Proteínas Bacterianas/química , Proteínas Bacterianas/genética , Fenómenos Biofísicos , Quelantes/química , Genoma Bacteriano , Homeostasis , Imidazoles/química , Methylosinus trichosporium/genética , Methylosinus trichosporium/metabolismo , Modelos Biológicos , Estructura Molecular , Oligopéptidos/química , Oligopéptidos/genética , Operón , Transporte de ProteínasRESUMEN
Although microbes are routinely grown in monocultures in the laboratory, they are almost never encountered as single species in the wild. Our ability to detect and identify new microorganisms has advanced significantly in recent years, but our understanding of the mechanisms that mediate microbial interactions has lagged behind. What makes this task more challenging is that microbial alliances can be dynamic, consisting of multiple phases. The transitions between phases, and the interactions in general, are often mediated by a chemical language consisting of small molecules, also referred to as secondary metabolites or natural products. In this microbial lexicon, the molecules are like words and through their effects on recipient cells they convey meaning. The current review highlights three dynamic microbial interactions in which some of the words and their meanings have been characterized, especially those that mediate transitions in selected multiphasic associations. These systems provide insights into the principles that govern microbial symbioses and a playbook for interrogating similar associations in diverse ecological niches.
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Productos Biológicos , Interacciones Microbianas , Productos Biológicos/metabolismo , Ecosistema , SimbiosisRESUMEN
Aromatic polyketides are renowned for their wide-ranging pharmaceutical activities. Their structural diversity is mainly produced via modification of limited types of basic frameworks. In this study, we characterized the biosynthesis of a unique basic aromatic framework, phenyldimethylanthrone (PDA) found in (+)/(-)-anthrabenzoxocinones (ABXs) and fasamycin (FAS). Its biosynthesis employs a methyltransferase (Abx(+)M/Abx(-)M/FasT) and an unusual TcmI-like aromatase/cyclase (ARO/CYC, Abx(+)D/Abx(-)D/FasL) as well as a nonessential helper ARO/CYC (Abx(+)C/Abx(-)C/FasD) to catalyze the aromatization/cyclization of polyketide chain, leading to the formation of all four aromatic rings of the PDA framework, including the C9 to C14 ring and a rare angular benzene ring. Biochemical and structural analysis of Abx(+)D reveals a unique loop region, giving rise to its distinct acyl carrier protein-dependent specificity compared to other conventional TcmI-type ARO/CYCs, all of which impose on free molecules. Mutagenic analysis discloses critical residues of Abx(+)D for its catalytic activity and indicates that the size and shape of its interior pocket determine the orientation of aromatization/cyclization. This study unveils the tetracyclic and non-TcmN type C9 to C14 ARO/CYC, significantly expanding our cognition of ARO/CYCs and the biosynthesis of aromatic polyketide framework.
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Aromatasa , Policétidos , Ciclización , Proteína Transportadora de Acilo , CatálisisRESUMEN
The multinuclear nonheme iron-dependent oxidases (MNIOs) are a rapidly growing family of enzymes involved in the biosynthesis of ribosomally synthesized, posttranslationally modified peptide natural products (RiPPs). Recently, a secreted virulence factor from nontypeable Haemophilus influenzae (NTHi) was found to be expressed from an operon, which we designate the hvf operon, that also encodes an MNIO. Here, we show by Mössbauer spectroscopy that the MNIO HvfB contains a triiron cofactor. We demonstrate that HvfB works together with HvfC [a RiPP recognition element (RRE)-containing partner protein] to perform six posttranslational modifications of cysteine residues on the virulence factor precursor peptide HvfA. Structural characterization by tandem mass spectrometry and NMR shows that these six cysteine residues are converted to oxazolone and thioamide pairs, similar to those found in the RiPP methanobactin. Like methanobactin, the mature virulence factor, which we name oxazolin, uses these modified residues to coordinate Cu(I) ions. Considering the necessity of oxazolin for host cell invasion by NTHi, these findings point to a key role for copper during NTHi infection. Furthermore, oxazolin and its biosynthetic pathway represent a potential therapeutic target for NTHi.
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Proteínas Bacterianas , Cobre , Haemophilus influenzae , Oxazolona , Factores de Virulencia , Haemophilus influenzae/metabolismo , Haemophilus influenzae/enzimología , Haemophilus influenzae/genética , Haemophilus influenzae/patogenicidad , Factores de Virulencia/metabolismo , Factores de Virulencia/genética , Cobre/metabolismo , Cobre/química , Proteínas Bacterianas/metabolismo , Proteínas Bacterianas/genética , Oxazolona/metabolismo , Tioamidas/metabolismo , Tioamidas/química , Hierro/metabolismo , Procesamiento Proteico-Postraduccional , Oxidorreductasas/metabolismo , Oxidorreductasas/genética , Operón , Cisteína/metabolismoRESUMEN
Monoterpene indole alkaloids (MIAs) are a large and diverse class of plant natural products, and their biosynthetic construction has been a subject of intensive study for many years. The enzymatic basis for the production of aspidosperma and iboga alkaloids, which are produced exclusively by members of the Apocynaceae plant family, has recently been discovered. Three carboxylesterase (CXE)-like enzymes from Catharanthus roseus and Tabernanthe iboga catalyze regio- and enantiodivergent [4+2] cycloaddition reactions to generate the aspidosperma (tabersonine synthase, TS) and iboga (coronaridine synthase, CorS; catharanthine synthase, CS) scaffolds from a common biosynthetic intermediate. Here, we use a combined phylogenetic and biochemical approach to investigate the evolution and functional diversification of these cyclase enzymes. Through ancestral sequence reconstruction, we provide evidence for initial evolution of TS from an ancestral CXE followed by emergence of CorS in two separate lineages, leading in turn to CS exclusively in the Catharanthus genus. This progression from aspidosperma to iboga alkaloid biosynthesis is consistent with the chemotaxonomic distribution of these MIAs. We subsequently generate and test a panel of chimeras based on the ancestral cyclases to probe the molecular basis for differential cyclization activity. Finally, we show through partial heterologous reconstitution of tabersonine biosynthesis using non-pathway enzymes how aspidosperma alkaloids could have first appeared as "underground metabolites" via recruitment of promiscuous enzymes from common protein families. Our results provide insight into the evolution of biosynthetic enzymes and how new secondary metabolic pathways can emerge through small but important sequence changes following co-option of preexisting enzymatic functions.
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Aspidosperma , Catharanthus , Alcaloides de Triptamina Secologanina , Tabernaemontana , Tabernaemontana/metabolismo , Aspidosperma/metabolismo , Carboxilesterasa/metabolismo , Filogenia , Alcaloides Indólicos/metabolismo , Alcaloides de Triptamina Secologanina/química , Alcaloides de Triptamina Secologanina/metabolismo , Plantas/metabolismo , Catharanthus/metabolismoRESUMEN
How to resolve the metabolic dark matter of microorganisms has long been a challenging problem in discovering active molecules. Diverse omics tools have been developed to guide the discovery and characterization of various microbial metabolites, which make it gradually possible to predict the overall metabolites for individual strains. The combinations of multi-omic analysis tools effectively compensates for the shortcomings of current studies that focus only on single omics or a broad class of metabolites. In this review, we systematically update, categorize and sort out different analysis tools for microbial metabolites prediction in the last five years to appeal for the multi-omic combination on the understanding of the metabolic nature of microbes. First, we provide the general survey on different updated prediction databases, webservers, or software that based on genomics, transcriptomics, proteomics, and metabolomics, respectively. Then, we discuss the essentiality on the integration of multi-omics data to predict metabolites of different microbial strains and communities, as well as stressing the combination of other techniques, such as systems biology methods and data-driven algorithms. Finally, we identify key challenges and trends in developing multi-omic analysis tools for more comprehensive prediction on diverse microbial metabolites that contribute to human health and disease treatment.
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Metabolómica , Programas Informáticos , Metabolómica/métodos , Genómica/métodos , Proteómica/métodos , Humanos , Biología Computacional/métodos , Bacterias/metabolismo , Bacterias/genética , Bacterias/clasificación , Metaboloma , Algoritmos , MultiómicaRESUMEN
Cystargolides are natural products originally isolated from Kitasatospora cystarginea NRRL B16505 as inhibitors of the proteasome. They are composed of a dipeptide backbone linked to a ß-lactone warhead. Recently, we identified the cystargolide biosynthetic gene cluster, but systematic genetic analyses had not been carried out because of the lack of a heterologous expression system. Here, we report the discovery of a homologous cystargolide biosynthetic pathway in Streptomyces durhamensis NRRL-B3309 by genome mining. The gene cluster was cloned via transformation-associated recombination and heterologously expressed in Streptomyces coelicolor M512. We demonstrate that it contains all genes necessary for the production of cystargolide A and B. Single gene deletion experiments reveal that only five of the eight genes from the initially proposed gene cluster are essential for cystargolide synthesis. Additional insights into the cystargolide pathway could be obtained from in vitro assays with CysG and chemical complementation of the respective gene knockout. This could be further supported by the in vitro investigation of the CysG homolog BelI from the belactosin biosynthetic gene cluster. Thereby, we confirm that CysG and BelI catalyze a cryptic SAM-dependent transfer of a methyl group that is critical for the construction of the cystargolide and belactosin ß-lactone warheads.
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Dipéptidos , Metiltransferasas , Streptomycetaceae , Vías Biosintéticas , Dipéptidos/metabolismo , Lactonas/metabolismo , Metiltransferasas/química , Metiltransferasas/genética , Metiltransferasas/metabolismo , Familia de Multigenes , Streptomyces coelicolor/genética , Streptomycetaceae/enzimología , Streptomycetaceae/genéticaRESUMEN
Cystargolides and belactosins are natural products with a distinct dipeptide structure and an electrophilic ß-lactone warhead. They are known to inhibit proteases such as the proteasome or caseinolytic protease P, highlighting their potential in treating cancers and neurodegenerative diseases. Recent genetic analyses have shown homology between the biosynthetic pathways of the two inhibitors. Here, we characterize the O-methyltransferases BelI and CysG, which catalyze the initial step of ß-lactone formation. Employing techniques such as crystallography, computational analysis, mutagenesis, and activity assays, we identified a His-His-Asp (HHD) motif in the active sites of the two enzymes, which is crucial for binding a catalytically active calcium ion. Our findings thus elucidate a conserved divalent metal-dependent mechanism in both biosynthetic pathways that distinguishes BelI and CysG from previously characterized O-methyltransferases.
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Nonribosomal peptide synthetases (NRPSs) are responsible for the production of important biologically active peptides. The large, multidomain NRPSs operate through an assembly line strategy in which the growing peptide is tethered to carrier domains that deliver the intermediates to neighboring catalytic domains. While most NRPS domains catalyze standard chemistry of amino acid activation, peptide bond formation, and product release, some canonical NRPS catalytic domains promote unexpected chemistry. The paradigm monobactam antibiotic sulfazecin is produced through the activity of a terminal thioesterase domain of SulM, which catalyzes an unusual ß-lactam-forming reaction in which the nitrogen of the C-terminal N-sulfo-2,3-diaminopropionate residue attacks its thioester tether to release the monobactam product. We have determined the structure of the thioesterase domain as both a free-standing domain and a didomain complex with the upstream holo peptidyl-carrier domain. The position of variant lid helices results in an active site pocket that is quite constrained, a feature that is likely necessary to orient the substrate properly for ß-lactam formation. Modeling of a sulfazecin tripeptide into the active site identifies a plausible binding mode identifying potential interactions for the sulfamate and the peptide backbone with Arg2849 and Asn2819, respectively. The overall structure is similar to the ß-lactone-forming thioesterase domain that is responsible for similar ring closure in the production of obafluorin. We further use these insights to enable bioinformatic analysis to identify additional, uncharacterized ß-lactam-forming biosynthetic gene clusters by genome mining.
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Deoxypodophyllotoxin contains a core of four fused rings (A to D) with three consecutive chiral centers, the last being created by the attachment of a peripheral trimethoxyphenyl ring (E) to ring C. Previous studies have suggested that the iron(II)- and 2-oxoglutarate-dependent (Fe/2OG) oxygenase, deoxypodophyllotoxin synthase (DPS), catalyzes the oxidative coupling of ring B and ring E to form ring C and complete the tetracyclic core. Despite recent efforts to deploy DPS in the preparation of deoxypodophyllotoxin analogs, the mechanism underlying the regio- and stereoselectivity of this cyclization event has not been elucidated. Herein, we report 1) two structures of DPS in complex with 2OG and (±)-yatein, 2) in vitro analysis of enzymatic reactivity with substrate analogs, and 3) model reactions addressing DPS's catalytic mechanism. The results disfavor a prior proposal of on-pathway benzylic hydroxylation. Rather, the DPS-catalyzed cyclization likely proceeds by hydrogen atom abstraction from C7', oxidation of the benzylic radical to a carbocation, Friedel-Crafts-like ring closure, and rearomatization of ring B by C6 deprotonation. This mechanism adds to the known pathways for transformation of the carbon-centered radical in Fe/2OG enzymes and suggests what types of substrate modification are likely tolerable in DPS-catalyzed production of deoxypodophyllotoxin analogs.
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Berberidaceae/enzimología , Medicamentos Herbarios Chinos/química , Ligasas/química , Proteínas de Plantas/química , Podofilotoxina/análogos & derivados , Oxidación-Reducción , Podofilotoxina/químicaRESUMEN
Xylomyrocins, a unique group of nonribosomal peptide secondary metabolites, were discovered in Paramyrothecium and Colletotrichum spp. fungi by employing a combination of high-resolution tandem mass spectrometry (HRMS/MS)-based chemometrics, comparative genome mining, gene disruption, stable isotope feeding, and chemical complementation techniques. These polyol cyclodepsipeptides all feature an unprecedented d-xylonic acid moiety as part of their macrocyclic scaffold. This biosynthon is derived from d-xylose supplied by xylooligosaccharide catabolic enzymes encoded in the xylomyrocin biosynthetic gene cluster, revealing a novel link between carbohydrate catabolism and nonribosomal peptide biosynthesis. Xylomyrocins from different fungal isolates differ in the number and nature of their amino acid building blocks that are nevertheless incorporated by orthologous nonribosomal peptide synthetase (NRPS) enzymes. Another source of structural diversity is the variable choice of the nucleophile for intramolecular macrocyclic ester formation during xylomyrocin chain termination. This nucleophile is selected from the multiple available alcohol functionalities of the polyol moiety, revealing a surprising polyspecificity for the NRPS terminal condensation domain. Some xylomyrocin congeners also feature N-methylated amino acid residues in positions where the corresponding NRPS modules lack N-methyltransferase (M) domains, providing a rare example of promiscuous methylation in the context of an NRPS with an otherwise canonical, collinear biosynthetic program.
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Depsipéptidos , Proteínas Fúngicas , Hongos , Aminoácidos/química , Metabolismo de los Hidratos de Carbono , Quimiometría , Depsipéptidos/química , Depsipéptidos/genética , Proteínas Fúngicas/química , Proteínas Fúngicas/genética , Hongos/genética , Hongos/metabolismo , Familia de Multigenes , Biosíntesis de Péptidos Independientes de Ácidos Nucleicos , Péptido Sintasas/química , AzúcaresRESUMEN
5-(3'-Indolyl)oxazole moiety is a privileged heterocyclic scaffold, embedded in many biologically interesting natural products and potential therapeutic agents. Compounds containing this scaffold, whether from natural sources or synthesized, have demonstrated a wide array of biological activities. This has piqued the interest of synthetic chemists, leading to a large number of reported synthetic approaches to 5-(3'-indolyl)oxazole scaffold in recent years. In this review, we comprehensively overviewed the different biological activities and chemical synthetic methods for the 5-(3'-indolyl)oxazole scaffold reported in the literatures from 1963 to 2024. The focus of this study is to highlight the significance of 5-(3'-indolyl)oxazole derivatives as the lead compounds for the lead discovery of anticancer, pesticidal, antimicrobial, antiviral, antioxidant and anti-inflammatory agents, to summarize the synthetic methods for the 5-(3'-indolyl)oxazole scaffold. In addition, the reported mechanism of action of 5-(3'-indolyl)oxazoles and advanced molecules studied in animal models are also reviewed. Furthermore, this review offers perspectives on how 5-(3'-indolyl)oxazole scaffold as a privileged structure might be exploited in the future.
RESUMEN
Berberine bridge enzyme-like oxidases are often involved in natural product biosynthesis and are seen as essential enzymes for the generation of intricate pharmacophores. These oxidases have the ability to transfer a hydride atom to the FAD cofactor, which enables complex substrate modifications and rearrangements including (intramolecular) cyclizations, carbon-carbon bond formations, and nucleophilic additions. Despite the diverse range of activities, the mechanistic details of these reactions often remain incompletely understood. In this Review, we delve into the complexity that BBE-like oxidases from bacteria, fungal, and plant origins exhibit by providing an overview of the shared catalytic features and emphasizing the different reactivities. We propose four generalized modes of action by which BBE-like oxidases enable the synthesis of natural products, ranging from the classic alcohol oxidation reactions to less common amine and amide oxidation reactions. Exploring the mechanisms utilized by nature to produce its vast array of natural products is a subject of considerable interest and can lead to the discovery of unique biochemical activities.
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Nicotianamine synthase (NAS) catalyzes the biosynthesis of the low-molecular-mass metal chelator nicotianamine (NA) from the 2-aminobutyrate moieties of three SAM molecules. NA has central roles in metal nutrition and metal homeostasis of flowering plants. The enzymatic function of NAS remains poorly understood. Crystal structures are available for archaeal and bacterial NAS-like proteins that carry out simpler aminobutanoyl transferase reactions. Here, we report amino acids essential for the activity of AtNAS1 based on structural modeling and site-directed mutagenesis. Using a newly developed enzyme-coupled continuous activity assay, we compare differing NAS proteins identified through multiple sequence alignments and phylogenetic analyses. In most NAS of dicotyledonous and monocotyledonous plants (class Ia and Ib), the core-NAS domain is fused to a variable C-terminal domain. Compared to fungal and moss NAS that comprise merely a core-NAS domain (class III), NA biosynthetic activities of the four paralogous Arabidopsis thaliana NAS proteins were far lower. C-terminally trimmed core-AtNAS variants exhibited strongly elevated activities. Of 320 amino acids of AtNAS1, twelve, 287-TRGCMFMPCNCS-298, accounted for the autoinhibitory effect of the C terminus, of which approximately one-third was attributed to N296 within a CNCS motif that is fully conserved in Arabidopsis. No detectable NA biosynthesis was mediated by two representative plant NAS proteins that naturally lack the C-terminal domain, class Ia Arabidopsis halleri NAS5 and Medicago truncatula NAS2 of class II which is found in dicots and diverged early during the evolution of flowering plants. Next, we will address a possible posttranslational release of autoinhibition in class I NAS proteins.
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Transferasas Alquil y Aril , Proteínas de Arabidopsis , Arabidopsis , Arabidopsis/enzimología , Arabidopsis/genética , Proteínas de Arabidopsis/química , Proteínas de Arabidopsis/genética , Filogenia , Transferasas Alquil y Aril/química , Transferasas Alquil y Aril/genéticaRESUMEN
The everninomicins are bacterially produced antibiotic octasaccharides characterized by the presence of two interglycosidic spirocyclic ortho-δ-lactone (orthoester) moieties. The terminating G- and H-ring sugars, L-lyxose and C-4 branched sugar ß-D-eurekanate, are proposed to be biosynthetically derived from nucleotide diphosphate pentose sugar pyranosides; however, the identity of these precursors and their biosynthetic origin remain to be determined. Herein we identify a new glucuronic acid decarboxylase from Micromonospora belonging to the superfamily of short-chain dehydrogenase/reductase enzymes, EvdS6. Biochemical characterization demonstrated that EvdS6 is an NAD+-dependent bifunctional enzyme that produces a mixture of two products, differing in the sugar C-4 oxidation state. This product distribution is atypical for glucuronic acid decarboxylating enzymes, most of which favor production of the reduced sugar and a minority of which favor release of the oxidized product. Spectroscopic and stereochemical analysis of reaction products revealed that the first product released is the oxidatively produced 4-keto-D-xylose and the second product is the reduced D-xylose. X-ray crystallographic analysis of EvdS6 at 1.51 Å resolution with bound co-factor and TDP demonstrated that the overall geometry of the EvdS6 active site is conserved with other SDR enzymes and enabled studies probing structural determinants for the reductive half of the net neutral catalytic cycle. Critical active site threonine and aspartate residues were unambiguously identified as essential in the reductive step of the reaction and resulted in enzyme variants producing almost exclusively the keto sugar. This work defines potential precursors for the G-ring L-lyxose and resolves likely origins of the H-ring ß-D-eurekanate sugar precursor.