RESUMEN
Bisindoles are biologically active natural products that arise from the oxidative dimerization of two molecules of l-tryptophan. In bacterial bisindole pathways, a core set of transformations is followed by the action of diverse tailoring enzymes that catalyze reactions that lead to diverse bisindole products. Among bisindoles, reductasporine is distinct due to its dimethylpyrrolinium structure. Its previously reported biosynthetic gene cluster encodes two unique tailoring enzymes, the imine reductase RedE and the dimethyltransferase RedM, which were shown to produce reductasporine from a common bisindole intermediate in recombinant E. coli. To gain more insight into the unique tailoring enzymes in reductasporine assembly, we reconstituted the biosynthetic pathway to reductasporine in vitro and then solved the 1.7 Å resolution structure of RedM. Our work reveals RedM adopts a variety of conformational changes with distinct open and closed conformations, and site-directed mutagenesis alongside sequence analysis identifies important active site residues. Finally, our work sets the stage for understanding how RedM evolved to react with a pyrrolinium scaffold and may enable the development of new dimethyltransferase catalysts.
Asunto(s)
Productos Biológicos , Metiltransferasas , Metiltransferasas/genética , Escherichia coli/genética , Escherichia coli/metabolismo , Oxidorreductasas/química , Mutagénesis Sitio-Dirigida , Productos Biológicos/metabolismo , Catálisis , Cristalografía por Rayos XRESUMEN
Imine reductases (IREDs) and reductive aminases have been used in the synthesis of chiral amine products for drug manufacturing; however, little is known about their biological contexts. Here we employ structural studies and site-directed mutagenesis to interrogate the mechanism of the IRED RedE from the biosynthetic pathway to the indolocarbazole natural product reductasporine. Cocrystal structures with the substrate-mimic arcyriaflavin A reveal an extended active site cleft capable of binding two indolocarbazole molecules. Site-directed mutagenesis of a conserved aspartate in the primary binding site reveals a new role for this residue in anchoring the substrate above the NADPH cofactor. Variants targeting the secondary binding site greatly reduce catalytic efficiency, while accumulating oxidized side-products. As indolocarbazole biosynthetic intermediates are susceptible to spontaneous oxidation, we propose the secondary site acts to protect against autooxidation, and the primary site drives catalysis through precise substrate orientation and desolvation effects. The structure of RedE with its extended active site can be the starting point as a new scaffold for engineering IREDs and reductive aminases to intercept large substrates relevant to industrial applications.
Asunto(s)
Iminas , Oxidorreductasas , Sitios de Unión , Catálisis , Cristalografía por Rayos X , Iminas/química , Iminas/metabolismo , Oxidación-Reducción , Oxidorreductasas/metabolismo , Estructura Terciaria de Proteína , Estructura Cuaternaria de Proteína , Modelos MolecularesRESUMEN
Entomopathogenic fungus Metarhizium majus contains the nine-gene PPZ cluster, with ppzA, encoding a peramine-producing nonribosomal peptide synthetase, as the central component. In this work, the roles of two α-ketoglutarate, iron-dependent oxygenases encoded by the PPZ genes ppzC and ppzD were elucidated. PpzD was found to produce both trans-4-hydroxy-l-proline and trans-3-hydroxy-l-proline in a 13.1:1 ratio, yielding a key precursor for peramine biosynthesis. PpzC was found to act directly on peramine, yielding the novel analogue 8-hydroxyperamine.
Asunto(s)
Compuestos Heterocíclicos con 2 Anillos , Hierro , Ácidos Cetoglutáricos , Metarhizium , Poliaminas , Familia de Multigenes , Compuestos FerrososRESUMEN
The mechanism by which molecular oxygen is activated by the organic cofactor pyridoxal phosphate (PLP) for oxidation reactions remains poorly understood. Recent work has identified arginine oxidases that catalyze desaturation or hydroxylation reactions. Here, we investigate a desaturase from the Pseudoalteromonas luteoviolacea indolmycin pathway. Our work, combining X-ray crystallographic, biochemical, spectroscopic, and computational studies, supports a shared mechanism with arginine hydroxylases, involving two rounds of single-electron transfer to oxygen and superoxide rebound at the 4' carbon of the PLP cofactor. The precise positioning of a water molecule in the active site is proposed to control the final reaction outcome. This proposed mechanism provides a unified framework to understand how oxygen can be activated by PLP-dependent enzymes for oxidation of arginine and elucidates a shared mechanistic pathway and intertwined evolutionary history for arginine desaturases and hydroxylases.
Asunto(s)
Aminoácido Oxidorreductasas/metabolismo , Fosfato de Piridoxal/metabolismo , Aminoácido Oxidorreductasas/química , Dominio Catalítico , Cristalografía por Rayos X , Evolución Química , Oxigenasas de Función Mixta/metabolismo , Conformación ProteicaRESUMEN
Pyridoxal phosphate-dependent enzymes able to use oxygen as a co-substrate have emerged in multiple protein families. Here, we use crystallography to solve the 2.40 Å resolution crystal structure of Cap15, a nucleoside biosynthetic enzyme that catalyzes the oxidative decarboxylation of glycyl uridine. Our structural study captures the internal aldimine, pinpointing the active site lysine as K230 and showing the site of phosphate binding. Our docking studies reveal how Cap15 is able to catalyze a stereoselective deprotonation reaction, and bioinformatic analysis reveals active site residues that distinguish Cap15 from the structurally related d-glucosaminate-6-phosphate ammonia lyase and l-seryl-tRNA(Sec) selenium transferase (SelA). Our work provides the structural basis for further mechanistic investigation of a unique biosynthetic enzyme and provides a blueprint for understanding how oxygen reactivity emerged in the SelA-like protein family.
Asunto(s)
Aminoglicósidos , Fosfato de Piridoxal , Fosfato de Piridoxal/metabolismo , Fosfatos , Proteínas Recombinantes , Cristalografía por Rayos XRESUMEN
l-Alanosine is a diazeniumdiolate (N-nitrosohydroxylamine) antibiotic that inhibits MTAP-deficient tumor cells by blocking de novo adenine biosynthesis. Previous work revealed the early steps in the biosynthesis of l-alanosine. In the present study, we used genome mining to discover two new l-alanosine-producing strains that lack the aspartate-nitrosuccinate pathway genes found in the original l-alanosine producer. Instead, nitrate is reduced with a unique set of nitrate-nitrite reductases. These enzymes are typically used as part of the nitrogen cycle for denitrification or assimilation, and our report here shows how enzymes from the nitrogen cycle can be repurposed for the biosynthesis of specialized metabolites. The widespread distribution of nitric-oxide-producing reductases also indicates a potential for the discovery of new nitric-oxide-derived natural products.
Asunto(s)
Nitratos , Óxido Nítrico , Oxidorreductasas/genética , Nitrito Reductasas , Nitrato ReductasasRESUMEN
The nitrogen-nitrogen bond is a core feature of diverse functional groups like hydrazines, nitrosamines, diazos, and pyrazoles. Such functional groups are found in >300 known natural products. Such N-N bond-containing functional groups are also found in significant percentage of clinical drugs. Therefore, there is wide interest in synthetic and enzymatic methods to form nitrogen-nitrogen bonds. In this review, we summarize synthetic and biosynthetic approaches to diverse nitrogen-nitrogen-bond-containing functional groups, with a focus on biosynthetic pathways and enzymes.
Asunto(s)
Productos Biológicos , Nitrógeno , Productos Biológicos/química , Vías Biosintéticas , Hidrazinas/química , Hidrazinas/metabolismo , Nitrógeno/químicaRESUMEN
Piperazic acid (Piz) is a nonproteinogenic amino acid possessing a rare nitrogen-nitrogen bond. However, little is known about how Piz is incorporated into nonribosomal peptides, including whether adenylation domains specific to Piz exist. In this study, we show that free piperazic acid is directly adenylated and then incorporated into the incarnatapeptin nonribosomal peptides through isotopic incorporation studies. We also use in vitro reconstitution to demonstrate adenylation of free piperazic acid with a three-domain nonribosomal peptide synthetase from the incarnatapeptin gene cluster. We furthermore use bioinformatics and site-directed mutagenesis to outline consensus sequences for the adenylation of piperazic acid, which can now be used for the prediction of gene clusters linked to piperazic-acid-containing peptides. Finally, we discover a fusion protein of a piperazate synthase and an adenylation domain, highlighting the close biosynthetic relationship of piperazic acid formation and its adenylation. Altogether, our work demonstrates the evolution of biosynthetic systems for the activation of free piperazic acid through adenylation, a pathway we suggest is likely to be employed in the majority of pathways to piperazic-acid-containing peptides.
Asunto(s)
Péptido Sintasas , Piridazinas , Nitrógeno , Péptido Sintasas/metabolismo , Péptidos/química , Piridazinas/química , Especificidad por SustratoRESUMEN
Natural nonproteinogenic amino acids vastly outnumber the well-known 22 proteinogenic amino acids. Such amino acids are generated in specialized metabolic pathways. In these pathways, diverse biosynthetic transformations, ranging from isomerizations to the stereospecific functionalization of C-H bonds, are employed to generate structural diversity. The resulting nonproteinogenic amino acids can be integrated into more complex natural products. Here we review recently discovered biosynthetic routes to freestanding nonproteinogenic α-amino acids, with an emphasis on work reported between 2013 and mid-2019.
Asunto(s)
Aminoácidos/biosíntesis , Aminoácidos/química , Humanos , IsomerismoRESUMEN
Laboratory cultures of two 'biosynthetically talented' bacterial strains harvested from tropical and temperate Pacific Ocean sediment habitats were examined for the production of new natural products. Cultures of the tropical Salinispora arenicola strain RJA3005, harvested from a PNG marine sediment, produced salinorcinol (3) and salinacetamide (4), which had previously been reported as products of engineered and mutated strains of Amycolatopsis mediterranei, but had not been found before as natural products. An S. arenicola strain RJA4486, harvested from marine sediment collected in the temperate ocean waters off British Columbia, produced the new aminoquinone polyketide salinisporamine (5). Natural products 3, 4, and 5 are putative shunt products of the widely distributed rifamycin biosynthetic pathway.
Asunto(s)
Actinomycetales , Productos Biológicos , Micromonosporaceae , Productos Biológicos/metabolismo , Sedimentos Geológicos/microbiología , Micromonosporaceae/genéticaRESUMEN
Microbes produce specialized metabolites to thrive in their natural habitats. However, it is rare that a given specialized metabolite is biosynthesized via pathways with distinct intermediates and enzymes. Here, we show that the core assembly mechanism of the antibiotic indolmycin in marine gram-negative Pseudoalteromonas luteoviolacea is distinct from its counterpart in terrestrial gram-positive Streptomyces species, with a molecule that is a shunt product in the Streptomyces pathway employed as a biosynthetic substrate for a novel metal-independent N-demethylindolmycin synthase in the P. luteoviolacea pathway. To provide insight into this reaction, we solved the 1.5 Å resolution structure in complex with product and identified the active site residues. Guided by our biosynthetic insights, we then engineered the Streptomyces indolmycin producer for titer improvement. This study provides a paradigm for understanding how two unique routes to a microbial specialized metabolite can emerge from convergent biosynthetic transformations.
Asunto(s)
Bacterias/metabolismo , Vías Biosintéticas , Bacterias/genética , Biocatálisis , Familia de MultigenesRESUMEN
Bifidobacterium longum subsp. infantis ATCC 15697 has emerged as a model for infant gut-associated bifidobacterial strains. Here we present a genetic system for B. longum subsp. infantis ATCC 15697 using its own DNA restriction-modification systems and create a fucose permease deletion mutant lacking the ability to use free fucose as a carbon source.
Asunto(s)
Proteínas Bacterianas/genética , Bifidobacterium longum subspecies infantis/enzimología , Fucosa/metabolismo , Proteínas de Transporte de Membrana/genética , Proteínas Bacterianas/metabolismo , Bifidobacterium longum subspecies infantis/genética , Bifidobacterium longum subspecies infantis/metabolismo , Eliminación de Gen , Proteínas de Transporte de Membrana/metabolismoRESUMEN
Pyrazomycin is a rare C-nucleoside antibiotic containing a naturally occurring pyrazole ring, the biosynthetic origin of which has remained obscure for decades. In this study we report the identification of the gene cluster responsible for pyrazomycin biosynthesis in Streptomyces candidus NRRLâ 3601, revealing that the StrR-family regulator PyrR is the cluster-situated transcriptional activator governing pyrazomycin biosynthesis. Furthermore, our results from in vivo reconstitution and stable-isotope feeding experiments provide support for the hypothesis that PyrN is a new nitrogen-nitrogen bond-forming enzyme that catalyzes the linkage of the ϵ-NH2 nitrogen atom of l-N6 -OH-lysine and the α-NH2 nitrogen atom of l-glutamic acid. This study lays the foundation for further genetic and biochemical characterization of pyrazomycin pathway enzymes involved in constructing the characteristic pyrazole ring.
Asunto(s)
Antibacterianos/biosíntesis , Ribosa/biosíntesis , Streptomyces/metabolismo , Amidas , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Familia de Multigenes , Pentosiltransferasa/genética , Pentosiltransferasa/metabolismo , Pirazoles , Proteínas Represoras/genética , Proteínas Represoras/metabolismo , Streptomyces/genéticaRESUMEN
The formation of a N-N bond is a unique biochemical transformation, and nature employs diverse biosynthetic strategies to activate nitrogen for bond formation. Among molecules that contain a N-N bond, biosynthetic routes to diazeniumdiolates remain enigmatic. We here report the biosynthetic pathway for the diazeniumdiolate-containing amino acid l-alanosine. Our work reveals that the two nitrogen atoms in the diazeniumdiolate of l-alanosine arise from glutamic acid and aspartic acid, and we clarify the early steps of the biosynthetic pathway by using both inâ vitro and inâ vivo approaches. Our work demonstrates a peptidyl-carrier-protein-based mechanism for activation of the precursor l-diaminopropionate, and we also show that nitric oxide can participate in non-enzymatic diazeniumdiolate formation. Furthermore, we demonstrate that the gene alnA, which encodes a fusion protein with an N-terminal cupin domain and a C-terminal AraC-like DNA-binding domain, is required for alanosine biosynthesis.
Asunto(s)
Alanina/análogos & derivados , Alanina/biosíntesis , Alanina/química , Alanina/genética , Ácido Aspártico/química , Ácido Aspártico/metabolismo , Ácido Glutámico/química , Ácido Glutámico/metabolismo , Estructura Molecular , Familia de Multigenes , Streptomyces/genética , Streptomyces/metabolismoRESUMEN
Nitric oxide (NO) has wide-ranging roles in biology, but less is known about its role in building chemical diversity. Here we report a new route to NO from the biosynthetic pathway to the N-nitroso compound streptozocin. We show that the N-nitroso group of streptozocin comes from the biosynthetic reassembly of l-arginine, with the guanidino nitrogens forming a nitrogen-nitrogen bond. To understand this biosynthetic process, we identify the biosynthetic gene cluster of streptozocin and demonstrate that free l-arginine is N-methylated by StzE to give Nω-monomethyl-l-arginine. We show that this product is then oxidized by StzF, a nonheme iron-dependent enzyme unrelated to known nitric oxide synthases, generating a urea compound and NO. Our work implies that formation and capture of NO is the likely route to N-nitroso formation in vivo. Altogether, our work unveils a new enzyme pair for the production of NO from l-arginine and sets the stage for understanding biosynthetic routes to N-nitroso natural products.
Asunto(s)
Arginina/metabolismo , Óxido Nítrico Sintasa/metabolismo , Óxido Nítrico/metabolismo , Compuestos Nitrosos/metabolismo , Proteínas de Hierro no Heme/metabolismo , Arginina/química , Estructura Molecular , Óxido Nítrico/química , Compuestos Nitrosos/químicaRESUMEN
Acyclic imines are unstable in aqueous conditions. For this reason, known imine reductases, which enable the synthesis of chiral amines, mainly intercept stable cyclic imines. Here we report the detailed biochemical and structural characterization of Bsp5, an imino acid reductase from the d-2-hydroxyacid dehydrogenase family that reduces acyclic imino acids produced in situ by a partner oxidase. We determine a 1.6 Å resolution structure of Bsp5 in complex with d-arginine and coenzyme NADPH. Combined with mutagenesis work, our study reveals the minimal structural constraints for its biosynthetic activity. Furthermore, we demonstrate that Bsp5 can intercept more complex products from an alternate oxidase partner, suggesting that this oxidase-imino acid reductase pair could be evolved for biocatalytic conversion of l-amino acids to d-amino acids.
Asunto(s)
Iminoácidos/química , Iminoácidos/metabolismo , Oxidorreductasas/química , Oxidorreductasas/metabolismo , Biocatálisis , Modelos Moleculares , Dominios ProteicosRESUMEN
Covering: up to mid-2018 Pyridoxal 5'-phosphate (PLP) is a versatile organic cofactor used to catalyze diverse reactions on amino acid, oxoacid, and amine substrates. Here we review the reactions catalyzed by PLP-dependent enzymes, highlighting enzymes reported in the natural product biosynthetic literature. We describe enzymes that catalyze transaminations, Claisen-like condensations, and ß- and γ-eliminations and substitutions, along with epimerizations, decarboxylations, and transaldolations. Finally, we describe a newly reported group of O2-, PLP-dependent enzymes. Altogether, natural product biosynthesis showcases the incredible versatility of PLP-dependent transformations for building chemical complexity.
Asunto(s)
Productos Biológicos/metabolismo , Fosfato de Piridoxal/fisiología , Imidazoles , Lactamas/metabolismo , Lipoproteínas/biosíntesis , Macrólidos/metabolismo , Oligopéptidos/biosíntesis , Pactamicina/biosíntesis , Saxitoxina/biosíntesis , Tiazoles/metabolismo , Tionas/metabolismoRESUMEN
Covering: up to the end of 2018 Piperazic acid is a cyclic hydrazine and a non-proteinogenic amino acid found in diverse non-ribosomal peptide (NRP) and hybrid NRP-polyketide (PK) structures. Piperazic acid was first identified as a residue in the monamycins in 1959. Since then, the piperazic acid residue has been found in >30 families of natural products, representing >140 compounds. Many of these compounds have potent biological activity, ranging from anti-malarial to anti-apoptotic to anti-bacterial activity, although high toxicity often accompanies this potent biological activity. Recently, we identified a piperazate synthase, responsible for N-N bond formation to give piperazic acid. Here, we review piperazic acid-containing natural products discovered from 1959 to 2018, with an emphasis on the biosynthetic routes to these natural products.
Asunto(s)
Productos Biológicos/química , Productos Biológicos/metabolismo , Piridazinas/química , Productos Biológicos/farmacología , Estructura Molecular , Familia de Multigenes , Péptidos/química , Péptidos/metabolismo , Péptidos Cíclicos/biosíntesis , Péptidos Cíclicos/química , Piridazinas/metabolismoRESUMEN
Molecules containing a nitrogen-nitrogen (N-N) linkage have a variety of structures and biological activities; however, no enzyme has yet been demonstrated to catalyze N-N bond formation in an organic molecule. Here we report that the heme-dependent enzyme KtzT from Kutzneria sp. 744 catalyzes N-N bond formation in the biosynthesis of piperazate, a building block for nonribosomal peptides.
Asunto(s)
Oxigenasas de Función Mixta/metabolismo , Nitrógeno/metabolismo , Piridazinas/metabolismo , Actinomycetales/enzimología , Oxigenasas de Función Mixta/química , Conformación Molecular , Nitrógeno/química , Piridazinas/químicaRESUMEN
Nitroimidazoles are one of the most effective ways to treat anaerobic bacterial infections. Synthetic nitroimidazoles are inspired by the structure of azomycin, isolated from Streptomyces eurocidicus in 1953. Despite its foundational role, no biosynthetic gene cluster for azomycin has been found. Guided by bioinformatics, we identified a cryptic biosynthetic gene cluster in Streptomyces cattleya and then carried out inâ vitro reconstitution to deduce the enzymatic steps in the pathway linking l-arginine to azomycin. The gene cluster we discovered is widely distributed among soil-dwelling actinobacteria and proteobacteria, suggesting that azomycin and related nitroimidazoles may play important ecological roles. Our work sets the stage for development of biocatalytic approaches to generate azomycin and related nitroimidazoles.