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The biosynthetic pathway of actinorhodin in Streptomyces coelicolor A3(2) has been studied for decades as a model system of type II polyketide biosynthesis. The actinorhodin biosynthetic gene cluster includes a gene, actVI-orfA, that encodes a protein that belongs to the nuclear transport factor-2-like (NTF-2-like) superfamily. The function of this ActVI-ORFA protein has been a long-standing question in this field. Several hypothetical functions, including pyran ring cyclase, enzyme complex stability enhancer, and gene transcription regulator, have been proposed for ActVI-ORFA in previous studies. However, although the recent structural analysis of ActVI-ORFA revealed a solvent-accessible cavity, the protein displayed structural differences to the well-characterized cyclase SnoaL and did not possess a DNA-binding domain. The obtained crystal structure facilitates an inspection of the previous hypotheses regarding the function of ActVI-ORFA. In the present study, we investigated the effects of a series of actVI-orfA test plasmids with different mutations in an established vector/host system. Time-course analysis of dynamic metabolism profiles demonstrated that ActVI-ORFA prevented formation of shunt metabolites and may have a metabolic flux directing function, which shepherds the flux of unstable intermediates towards actinorhodin. The expression studies resulted in the isolation and structure elucidation of two new shunt metabolites from the actinorhodin pathway. Next, we utilized computational modeling to probe the active site of ActVI-ORFA and confirmed the importance of residues R76 and H78 in the flux directing functionality by expression studies. This is the first time such a function has been observed for a member of NTF-2-like superfamily in Streptomyces secondary metabolism.
Assuntos
Antraquinonas , Proteínas de Bactérias , Streptomyces coelicolor , Antraquinonas/metabolismo , Streptomyces coelicolor/metabolismo , Streptomyces coelicolor/genética , Proteínas de Bactérias/metabolismo , Proteínas de Bactérias/genética , Família Multigênica , Mutação , BenzoisocromanequinonasRESUMO
Streptomyces spp. are "nature's antibiotic factories" that produce valuable bioactive metabolites, such as the cytotoxic anthracycline polyketides. While the anthracyclines have hundreds of natural and chemically synthesized analogues, much of the chemical diversity stems from enzymatic modifications to the saccharide chains and, to a lesser extent, from alterations to the core scaffold. Previous work has resulted in the generation of a BioBricks synthetic biology toolbox in Streptomyces coelicolor M1152ΔmatAB that could produce aklavinone, 9-epi-aklavinone, auramycinone, and nogalamycinone. In this work, we extended the platform to generate oxidatively modified analogues via two crucial strategies. (i) We swapped the ketoreductase and first-ring cyclase enzymes for the aromatase cyclase from the mithramycin biosynthetic pathway in our polyketide synthase (PKS) cassettes to generate 2-hydroxylated analogues. (ii) Next, we engineered several multioxygenase cassettes to catalyze 11-hydroxylation, 1-hydroxylation, 10-hydroxylation, 10-decarboxylation, and 4-hydroxyl regioisomerization. We also developed improved plasmid vectors and S. coelicolor M1152ΔmatAB expression hosts to produce anthracyclinones. This work sets the stage for the combinatorial biosynthesis of bespoke anthracyclines using recombinant Streptomyces spp. hosts.
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Antraciclinas , Policetídeo Sintases , Streptomyces coelicolor , Policetídeo Sintases/metabolismo , Policetídeo Sintases/genética , Antraciclinas/metabolismo , Streptomyces coelicolor/metabolismo , Streptomyces coelicolor/genética , Streptomyces/metabolismo , Streptomyces/genética , Vias Biossintéticas/genética , Hidroxilação , Antibacterianos/biossíntese , Antibacterianos/metabolismo , Antibacterianos/químicaRESUMO
Angucyclines are an important group of microbial natural products that display tremendous chemical diversity. Classical angucyclines are composed of a tetracyclic benz[a]anthracene scaffold with one ring attached at an angular orientation. However, in atypical angucyclines, the polyaromatic aglycone is cleaved at A-, B-, or C-rings, leading to structural rearrangements and enabling further chemical variety. Here, we have elucidated the branching points in angucycline biosynthesis leading toward cleavage of the C-ring in lugdunomycin and thioangucycline biosynthesis. We showed that 12-hydroxylation and 6-ketoreduction of UWM6 are shared steps in classical and C-ring-cleaved angucycline pathways, although the bifunctional 6-ketoreductase LugOIIred harbors additional unique 1-ketoreductase activity. We identified formation of the key intermediate 8-O-methyltetrangomycin by the LugN methyltransferase as the branching point toward C-ring-cleaved angucyclines. The final common step in lugdunomycin and thioangucycline biosynthesis is quinone reduction, catalyzed by the 7-ketoreductases LugG and TacO, respectively. In turn, the committing step toward thioangucyclines is 12-ketoreduction catalyzed by TacA, for which no orthologous protein exists on the lugdunomycin pathway. Our results confirm that quinone reductions are early tailoring steps and, therefore, may be mechanistically important for subsequent C-ring cleavage. Finally, many of the tailoring enzymes harbored broad substrate promiscuity, which we utilized in combinatorial enzymatic syntheses to generate the angucyclines SM 196 A and hydranthomycin. We propose that enzyme promiscuity and the competition of many of the enzymes for the same substrates lead to a branching biosynthetic network and formation of numerous shunt products typical for angucyclines rather than a canonical linear metabolic pathway.
Assuntos
Streptomyces , Streptomyces/metabolismo , Antraquinonas/metabolismo , Antraquinonas/química , Produtos Biológicos/metabolismo , Produtos Biológicos/química , Hidroxilação , Anguciclinas e AnguciclinonasRESUMO
Introduction: Daunorubicin and doxorubicin, two anthracycline polyketides produced by S. peucetius, are potent anticancer agents that are widely used in chemotherapy, despite severe side effects. Recent advances have highlighted the potential of producing improved derivatives with reduced side effects by incorporating l-rhodosamine, the N,N-dimethyl analogue of the native amino sugar moiety. Method: In this study, we aimed to produce N,N-dimethylated anthracyclines by engineering the doxorubicin biosynthetic pathway in the industrial Streptomyces peucetius strain G001. To achieve this, we introduced genes from the aclarubicin biosynthetic pathway encoding the sugar N-methyltransferases AclP and AknX2. Furthermore, the native gene for glycosyltransferase DnrS was replaced with genes encoding the aclarubicin glycosyltransferases AknS and AknT. Additionally, the gene for methylesterase RdmC from the rhodomycin biosynthetic pathway was introduced. Results: A new host was engineered successfully, whereby genes from the aclarubicin pathway were introduced and expressed. LC-MS/MS analysis of the engineered strains showed that dimethylated sugars were efficiently produced, and that these were incorporated ino the anthracycline biosynthetic pathway to produce the novel dimethylated anthracycline N,N-dimethyldaunorubicin. Further downstream tailoring steps catalysed by the cytochrome P450 monooxygenase DoxA exhibited limited efficacy with N,N-dimethylated substrates. This resulted in only low production levels of N,N-dimethyldaunorubicin and no N,N-dimethyldoxorubicin, most likely due to the low affinity of DoxA for dimethylated substrates. Discussion: S. peucetius G001 was engineered such as to produce N,N-dimethylated sugars, which were incorporated into the biosynthetic pathway. This allowed the successful production of N,N-dimethyldaunorubicin, an anticancer drug with reduced cytotoxicity. DoxA is the key enzyme that determines the efficiency of the biosynthesis of N,N-dimethylated anthracyclines, and engineering of this enzyme will be a major step forwards towards the efficient production of more N,N-dimethylated anthracyclines, including N,N-dimethyldoxorubicin. This study provides valuable insights into the biosynthesis of clinically relevant daunorubicin derivatives, highlighting the importance of combinatorial biosynthesis.
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Angucyclines are type II polyketide natural products, often characterized by unusual structural rearrangements through B- or C-ring cleavage of their tetracyclic backbone. While the enzymes involved in B-ring cleavage have been extensively studied, little is known of the enzymes leading to C-ring cleavage. Here, we unravel the function of the oxygenases involved in the biosynthesis of lugdunomycin, a highly rearranged C-ring cleaved angucycline derivative. Targeted deletion of the oxygenase genes, in combination with molecular networking and structural elucidation, showed that LugOI is essential for C12 oxidation and maintaining a keto group at C6 that is reduced by LugOII, resulting in a key intermediate towards C-ring cleavage. An epoxide group is then inserted by LugOIII, and stabilized by the novel enzyme LugOV for the subsequent cleavage. Thus, for the first time we describe the oxidative enzymatic steps that form the basis for a wide range of rearranged angucycline natural products.
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Streptomyces produce complex bioactive secondary metabolites with remarkable chemical diversity. Benzoisochromanequinone polyketides actinorhodin and naphthocyclinone are formed through dimerization of half-molecules via single or double carbon-carbon bonds, respectively. Here we sequenced the genome of S. arenae DSM40737 to identify the naphthocyclinone gene cluster and established heterologous production in S. albus J1074 by utilizing direct cluster capture techniques. Comparative sequence analysis uncovered ncnN and ncnM gene products as putative enzymes responsible for dimerization. Inactivation of ncnN that is homologous to atypical co-factor independent oxidases resulted in the accumulation of fogacin, which is likely a reduced shunt product of the true substrate for naphthocyclinone dimerization. In agreement, inactivation of the homologous actVA-3 in S. coelicolor M145 also led to significantly reduced production of actinorhodin. Previous work has identified the NAD(P)H-dependent reductase ActVA-4 as the key enzyme in actinorhodin dimerization, but surprisingly inactivation of the homologous ncnM did not abolish naphthocyclinone formation and the mutation may have been complemented by an endogenous gene product. Our data suggests that dimerization of benzoisochromanequinone polyketides require two-component reductase-oxidase systems.
Assuntos
Policetídeos , Streptomyces coelicolor , Oxirredutases/metabolismo , Antibacterianos/metabolismo , Dimerização , Antraquinonas/metabolismo , Carbono/metabolismo , Policetídeos/metabolismo , Streptomyces coelicolor/metabolismoRESUMO
Tetracenomycins and elloramycins are polyketide natural products produced by several actinomycetes that exhibit antibacterial and anticancer activities. They inhibit ribosomal translation by binding in the polypeptide exit channel of the large ribosomal subunit. The tetracenomycins and elloramycins are typified by a shared oxidatively modified linear decaketide core, yet they are distinguished by the extent of O-methylation and the presence of a 2',3',4'-tri-O-methyl-α-l-rhamnose appended at the 8-position of elloramycin. The transfer of the TDP-l-rhamnose donor to the 8-demethyl-tetracenomycin C aglycone acceptor is catalyzed by the promiscuous glycosyltransferase ElmGT. ElmGT exhibits remarkable flexibility toward transfer of many TDP-deoxysugar substrates to 8-demethyltetracenomycin C, including TDP-2,6-dideoxysugars, TDP-2,3,6-trideoxysugars, and methyl-branched deoxysugars in both d- and l-configurations. Previously, we developed an improved host, Streptomyces coelicolor M1146::cos16F4iE, which is a stable integrant harboring the required genes for 8-demethyltetracenomycin C biosynthesis and expression of ElmGT. In this work, we developed BioBricks gene cassettes for the metabolic engineering of deoxysugar biosynthesis in Streptomyces spp. As a proof of concept, we used the BioBricks expression platform to engineer biosynthesis for d-configured TDP-deoxysugars, including known compounds 8-O-d-glucosyl-tetracenomycin C, 8-O-d-olivosyl-tetracenomycin C, 8-O-d-mycarosyl-tetracenomycin C, and 8-O-d-digitoxosyl-tetracenomycin C. In addition, we generated four new tetracenomycins including one modified with a ketosugar, 8-O-4'-keto-d-digitoxosyl-tetracenomycin C, and three modified with 6-deoxysugars, including 8-O-d-fucosyl-tetracenomycin C, 8-O-d-allosyl-tetracenomycin C, and 8-O-d-quinovosyl-tetracenomycin C. Our work demonstrates the feasibility of BioBricks cloning, with the ability to recycle intermediate constructs, for the rapid assembly of diverse carbohydrate pathways and glycodiversification of a variety of natural products.
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Pseudouridimycin is a microbial C-nucleoside natural product that specifically inhibits bacterial RNA polymerases by binding to the active site and competing with uridine triphosphate for the nucleoside triphosphate (NTP) addition site. Pseudouridimycin consists of 5'-aminopseudouridine and formamidinylated, N-hydroxylated Gly-Gln dipeptide moieties to allow Watson-Crick base pairing and to mimic protein-ligand interactions of the triphosphates of NTP, respectively. The metabolic pathway of pseudouridimycin has been studied in Streptomyces species, but no biosynthetic steps have been characterized biochemically. Here, we show that the flavin-dependent oxidase SapB functions as a gate-keeper enzyme selecting pseudouridine (KM = 34 µM) over uridine (KM = 901 µM) in the formation of pseudouridine aldehyde. The pyridoxal phosphate (PLP)-dependent SapH catalyzes transamination, resulting in 5'-aminopseudouridine with a preference for arginine, methionine, or phenylalanine as cosubstrates as amino group donors. The binary structure of SapH in complex with pyridoxamine-5'-phosphate and site-directed mutagenesis identified Lys289 and Trp32 as key residues for catalysis and substrate binding, respectively. The related C-nucleoside oxazinomycin was accepted as a substrate by SapB with moderate affinity (KM = 181 µM) and was further converted by SapH, which opens possibilities for metabolic engineering to generate hybrid C-nucleoside pseudouridimycin analogues in Streptomyces.
Assuntos
Nucleosídeos , Pseudouridina , Vias Biossintéticas , RNA Polimerases Dirigidas por DNA/metabolismo , Nucleosídeos/metabolismo , Pseudouridina/biossíntese , Pseudouridina/metabolismo , Fosfato de Piridoxal/química , Streptomyces/química , Streptomyces/metabolismoRESUMO
Streptomyces soil bacteria produce hundreds of anthracycline anticancer agents with a relatively conserved set of genes. This diversity depends on the rapid evolution of biosynthetic enzymes to acquire novel functionalities. Previous work has identified S-adenosyl-l-methionine-dependent methyltransferase-like proteins that catalyze 4-O-methylation, 10-decarboxylation, or 10-hydroxylation, with additional differences in substrate specificities. Here we focused on four protein regions to generate chimeric enzymes using sequences from four distinct subfamilies to elucidate their influence in catalysis. Combined with structural studies we managed to depict factors that influence gain-of-hydroxylation, loss-of-methylation, and substrate selection. The engineering expanded the catalytic repertoire to include novel 9,10-elimination activity, and 4-O-methylation and 10-decarboxylation of unnatural substrates. The work provides an instructive account on how the rise of diversity of microbial natural products may occur through subtle changes in biosynthetic enzymes.
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Actinomycetes produce a variety of clinically indispensable molecules, such as antineoplastic anthracyclines. However, the actinomycetes are hindered in their further development as genetically engineered hosts for the synthesis of new anthracycline analogues due to their slow growth kinetics associated with their mycelial life cycle and the lack of a comprehensive genetic toolbox for combinatorial biosynthesis. In this report, we tackled both issues via the development of the BIOPOLYMER (BIOBricks POLYketide Metabolic EngineeRing) toolbox: a comprehensive synthetic biology toolbox consisting of engineered strains, promoters, vectors, and biosynthetic genes for the synthesis of anthracyclinones. An improved derivative of the production host Streptomyces coelicolor M1152 was created by deleting the matAB gene cluster that specifies extracellular poly-ß-1,6-N-acetylglucosamine (PNAG). This resulted in a loss of mycelial aggregation, with improved biomass accumulation and anthracyclinone production. We then leveraged BIOPOLYMER to engineer four distinct anthracyclinone pathways, identifying optimal combinations of promoters, genes, and vectors to produce aklavinone, 9-epi-aklavinone, auramycinone, and nogalamycinone at titers between 15-20 mg/L. Optimization of nogalamycinone production strains resulted in titers of 103 mg/L. We structurally characterized six anthracyclinone products from fermentations, including new compounds 9,10-seco-7-deoxy-nogalamycinone and 4-O-ß-d-glucosyl-nogalamycinone. Lastly, we tested the antiproliferative activity of the anthracyclinones in a mammalian cancer cell viability assay, in which nogalamycinone, auramycinone, and aklavinone exhibited moderate cytotoxicity against several cancer cell lines. We envision that BIOPOLYMER will serve as a foundational platform technology for the synthesis of designer anthracycline analogues.
Assuntos
Policetídeos , Streptomyces coelicolor , Streptomyces , Animais , Streptomyces coelicolor/genética , Streptomyces coelicolor/metabolismo , Engenharia Metabólica , Streptomyces/genética , Antraciclinas/metabolismo , Antibióticos Antineoplásicos/metabolismo , Policetídeos/metabolismo , Família Multigênica , Mamíferos/genéticaRESUMO
Transcription is the first and most highly regulated step in gene expression. Experimental techniques for monitoring transcription are, thus, important for studying gene expression and gene regulation as well as for translational research and drug development. Fluorescence methods are often superior to other techniques for real-time monitoring of biochemical processes. Green fluorescent proteins have long served as valuable tools for studying the process of translation. Here we present two methods that utilize fluorescent light-up RNA aptamers (FLAPs), the RNA mimics of green fluorescent proteins, to monitoring transcription and co-transcriptional RNA folding. FLAPs adopt defined three-dimensional folds that bind low molecular weight compounds called fluorogens with concomitant increase in fluorescence by many folds. FLAPs provide a strong fluorescence signal with low background that allows monitoring of transcription in real time in vitro and in vivo. However, it takes several seconds for RNA polymerase to synthesize FLAPs and the subsequent folding of the fluorogen-binding platform takes additional seconds or minutes. Here we show that Broccoli-FLAP is well suited for monitoring the rate of transcription initiation in a multi-round setup that mitigates the slow rate of the FLAP maturation. Furthermore, we demonstrate that a relatively slow and inefficient folding of iSpinach-FLAP can be taken advantage of for monitoring the action of RNA folding chaperones.
Assuntos
Aptâmeros de Nucleotídeos , Aptâmeros de Nucleotídeos/química , RNA Polimerases Dirigidas por DNA/genética , RNA Polimerases Dirigidas por DNA/metabolismo , Corantes Fluorescentes/química , Proteínas de Fluorescência Verde/genética , RNA , Dobramento de RNARESUMO
Actinomycetes are important producers of pharmaceuticals and industrial enzymes. However, wild type strains require laborious development prior to industrial usage. Here we present a generally applicable reporter-guided metabolic engineering tool based on random mutagenesis, selective pressure, and single-cell sorting. We developed fluorescence-activated cell sorting (FACS) methodology capable of reproducibly identifying high-performing individual cells from a mutant population directly from liquid cultures. Actinomycetes are an important source of catabolic enzymes, where product yields determine industrial viability. We demonstrate 5-fold yield improvement with an industrial cholesterol oxidase ChoD producer Streptomyces lavendulae to 20.4 U g-1 in three rounds. Strain development is traditionally followed by production medium optimization, which is a time-consuming multi-parameter problem that may require hard to source ingredients. Ultra-high throughput screening allowed us to circumvent medium optimization and we identified high ChoD yield production strains directly from mutant libraries grown under preset culture conditions. Genome-mining based drug discovery is a promising source of bioactive compounds, which is complicated by the observation that target metabolic pathways may be silent under laboratory conditions. We demonstrate our technology for drug discovery by activating a silent mutaxanthene metabolic pathway in Amycolatopsis. We apply the method for industrial strain development and increase mutaxanthene yields 9-fold to 99 mg l-1 in a second round of mutant selection. In summary, the ability to screen tens of millions of mutants in a single cell format offers broad applicability for metabolic engineering of actinomycetes for activation of silent metabolic pathways and to increase yields of proteins and natural products.
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Actinobacteria , Engenharia Metabólica , Actinobacteria/genética , Actinomyces , Engenharia Metabólica/métodos , Redes e Vias Metabólicas , MutagêneseRESUMO
Showdomycin produced by Streptomyces showdoensis ATCC 15227 is a C-nucleoside microbial natural product with antimicrobial and cytotoxic properties. The unique feature of showdomycin in comparison to other nucleosides is its maleimide base moiety, which has the distinct ability to alkylate nucleophilic thiol groups by a Michael addition reaction. In order to understand structure-activity relationships of showdomycin, we synthesized a series of derivatives with modifications in the maleimide ring at the site of alkylation to moderate its reactivity. The showdomycin congeners were designed to retain the planarity of the base ring system to allow Watson-Crick base pairing and preserve the nucleosidic character of the compounds. Consequently, we synthesized triphosphates of showdomycin derivatives and tested their activity against RNA polymerases. Bromo, methylthio, and ethylthio derivatives of showdomycin were incorporated into RNA by bacterial and mitochondrial RNA polymerases and somewhat less efficiently by the eukaryotic RNA polymerase II. Showdomycin derivatives acted as uridine mimics and delayed further extension of the RNA chain by multi-subunit, but not mitochondrial RNA polymerases. Bioactivity profiling indicated that the mechanism of action of ethylthioshowdomycin was altered, with approximately 4-fold reduction in both cytotoxicity against human embryonic kidney cells and antibacterial activity against Escherichia coli. In addition, the ethylthio derivative was not inactivated by medium components or influenced by addition of uridine in contrast to showdomycin. The results explain how both the maleimide ring and the nucleoside nature contribute to the bioactivity of showdomycin and demonstrates for the first time that the two activities can be separated.
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Nucleosídeos , Showdomicina , Antibacterianos/farmacologia , Humanos , Maleimidas/farmacologia , RNA , Showdomicina/farmacologia , Relação Estrutura-Atividade , UridinaRESUMO
Covering: January 1995 to June 2021Anthracyclines are glycosylated microbial natural products that harbour potent antiproliferative activities. Doxorubicin has been widely used as an anticancer agent in the clinic for several decades, but its use is restricted due to severe side-effects such as cardiotoxicity. Recent studies into the mode-of-action of anthracyclines have revealed that effective cardiotoxicity-free anthracyclines can be generated by focusing on histone eviction activity, instead of canonical topoisomerase II poisoning leading to double strand breaks in DNA. These developments have coincided with an increased understanding of the biosynthesis of anthracyclines, which has allowed generation of novel compound libraries by metabolic engineering and combinatorial biosynthesis. Coupled to the continued discovery of new congeners from rare Actinobacteria, a better understanding of the biology of Streptomyces and improved production methodologies, the stage is set for the development of novel anthracyclines that can finally surpass doxorubicin at the forefront of cancer chemotherapy.
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Antineoplásicos , Policetídeos , Antraciclinas/metabolismo , Antraciclinas/farmacologia , Antineoplásicos/farmacologia , DNA Topoisomerases Tipo II/metabolismo , Doxorrubicina/metabolismo , Doxorrubicina/farmacologiaRESUMO
Serine proteases regulate many physiological processes and play a key role in a variety of cancers. Aeruginosins are a family of natural products produced by cyanobacteria that exhibit pronounced structural diversity and potent serine protease inhibition. Here, we sequenced the complete genome of Nodularia sphaerocarpa UHCC 0038 and identified the 43.7 kb suomilide biosynthetic gene cluster. Bioinformatic analysis demonstrated that suomilide belongs to the aeruginosin family of natural products. We identified 103 complete aeruginosin biosynthetic gene clusters from 12 cyanobacterial genera and showed that they encode an unexpected chemical diversity. Surprisingly, purified suomilide inhibited human trypsin-2 and -3, with IC50 values of 4.7 and 11.5 nM, respectively, while trypsin-1 was inhibited with an IC50 of 104 nM. Molecular dynamics simulations suggested that suomilide has a long residence time when bound to trypsins. This was confirmed experimentally for trypsin-1 and -3 (residence times of 1.5 and 57 min, respectively). Suomilide also inhibited the invasion of aggressive and metastatic PC-3M prostate cancer cells without affecting cell proliferation. The potent inhibition of trypsin-3, together with a long residence time and the ability to inhibit prostate cancer cell invasion, makes suomilide an attractive drug lead for targeting cancers that overexpress trypsin-3. These results substantially broaden the genetic and chemical diversity of the aeruginosin family and suggest that aeruginosins may be a source of selective inhibitors of human serine proteases.
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Compostos Azabicíclicos/farmacologia , Produtos Biológicos/farmacologia , Inibidores da Tripsina/farmacologia , Produtos Biológicos/química , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Genes Bacterianos , Humanos , Nodularia/genética , Inibidores da Tripsina/isolamento & purificaçãoRESUMO
RNA polymerases (RNAPs) synthesize RNA from NTPs, whereas DNA polymerases synthesize DNA from 2'dNTPs. DNA polymerases select against NTPs by using steric gates to exclude the 2'OH, but RNAPs have to employ alternative selection strategies. In single-subunit RNAPs, a conserved Tyr residue discriminates against 2'dNTPs, whereas selectivity mechanisms of multi-subunit RNAPs remain hitherto unknown. Here, we show that a conserved Arg residue uses a two-pronged strategy to select against 2'dNTPs in multi-subunit RNAPs. The conserved Arg interacts with the 2'OH group to promote NTP binding, but selectively inhibits incorporation of 2'dNTPs by interacting with their 3'OH group to favor the catalytically-inert 2'-endo conformation of the deoxyribose moiety. This deformative action is an elegant example of an active selection against a substrate that is a substructure of the correct substrate. Our findings provide important insights into the evolutionary origins of biopolymers and the design of selective inhibitors of viral RNAPs.
Assuntos
Proteínas de Bactérias/metabolismo , RNA Polimerases Dirigidas por DNA/metabolismo , Desoxirribonucleotídeos/metabolismo , Desoxirribose/metabolismo , Arginina/metabolismo , Proteínas de Bactérias/genética , Proteínas de Bactérias/isolamento & purificação , Proteínas de Bactérias/ultraestrutura , Cristalografia por Raios X , RNA Polimerases Dirigidas por DNA/genética , RNA Polimerases Dirigidas por DNA/isolamento & purificação , RNA Polimerases Dirigidas por DNA/ultraestrutura , Escherichia coli/enzimologia , Escherichia coli/genética , Cinética , Simulação de Acoplamento Molecular , Regiões Promotoras Genéticas , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/ultraestrutura , Especificidade por Substrato , Thermus thermophilus/enzimologia , Thermus thermophilus/genéticaRESUMO
Synthetic biology-based approaches have been employed to generate advanced natural product (NP) pathway intermediates to overcome obstacles in NP drug discovery and production. Type II polyketides (PK-IIs) comprise a major subclass of NPs that provide attractive structures for antimicrobial and anticancer drug development. Herein, we have assembled five biosynthetic pathways using a generalized operon design strategy in Streptomyces coelicolor M1152 to allow comparative analysis of metabolite production in an improved heterologous host. The work resulted in production of four distinct PK-II core structures, namely benzoisochromanequinone, angucycline, tetracenomycin, and pentangular compounds, which serve as precursors to diverse pharmaceutically important NPs. Our bottom-up design strategy provided evidence that the biosynthetic pathway of BE-7585A proceeds via an angucycline core structure, instead of rearrangement of an anthracycline aglycone, and led to the discovery of a novel 26-carbon pentangular polyketide. The synthetic biology platform presented here provides an opportunity for further controlled production of diverse PK-IIs in a heterologous host.
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Produtos Biológicos/metabolismo , Descoberta de Drogas/métodos , Policetídeos/metabolismo , Streptomyces coelicolor/genética , Streptomyces coelicolor/metabolismo , Genes Bacterianos , Engenharia Metabólica/métodos , Naftacenos/metabolismo , Plasmídeos/genética , Tioaçúcares/metabolismoRESUMO
The anthracyclines are structurally diverse anticancer natural products that bind to DNA and poison the topoisomerase II-DNA complex in cancer cells. Rational modifications in the deoxysugar functionality are especially advantageous for synthesizing drugs with improved potency. Combinatorial biosynthesis of glycosyltransferases and deoxysugar synthesis enzymes is indispensable for the generation of glycodiversified anthracyclines. This Synopsis considers recent advances in glycosyltransferase structural biology and site-directed mutagenesis, pathway engineering, and deoxysugar combinatorial biosynthesis with a focus on the generation of "new-to-nature" anthracycline analogues.
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Produtos Biológicos , Policetídeos , Antraciclinas , Glicosilação , Glicosiltransferases/metabolismoRESUMO
Proteins belonging to the NTF2-like superfamily are present in the biosynthetic pathways of numerous polyketide natural products, such as anthracyclins and benzoisochromanequinones. Some have been found to be bona fide polyketide cyclases, but many of them have roles that are currently unknown. Here, the X-ray crystal structures of three NTF2-like proteins of unknown function are reported: those of ActVI-ORFA from Streptomyces coelicolor A3(2) and its homologs Caci_6494, a protein from an uncharacterized biosynthetic cluster in Catenulispora acidiphila, and Aln2 from Streptomyces sp. CM020, a protein in the biosynthetic pathway of alnumycin. The presence of a solvent-accessible cavity and the conservation of the His/Asp dyad that is characteristic of many polyketide cyclases suggest a potential enzymatic role for these enzymes in polyketide biosynthesis.
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Actinobacteria/química , Proteínas de Bactérias/química , Policetídeos/química , Streptomyces coelicolor/química , Streptomyces/química , Actinobacteria/enzimologia , Sequência de Aminoácidos , Antraquinonas/química , Antraquinonas/metabolismo , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Sítios de Ligação , Clonagem Molecular , Cristalografia por Raios X , Escherichia coli/genética , Escherichia coli/metabolismo , Expressão Gênica , Vetores Genéticos/química , Vetores Genéticos/metabolismo , Modelos Moleculares , Família Multigênica , Naftoquinonas/química , Naftoquinonas/metabolismo , Policetídeos/metabolismo , Ligação Proteica , Conformação Proteica em alfa-Hélice , Conformação Proteica em Folha beta , Domínios e Motivos de Interação entre Proteínas , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Streptomyces/enzimologia , Streptomyces coelicolor/enzimologia , Especificidade por SubstratoRESUMO
Angucyclines are a structurally diverse class of actinobacterial natural products defined by their varied polycyclic ring systems, which display a wide range of biological activities. We recently discovered lugdunomycin (1), a highly rearranged polyketide antibiotic derived from the angucycline backbone that is synthesized via several yet unexplained enzymatic reactions. Here, we show via in vivo, in vitro, and structural analysis that the promiscuous reductase LugOII catalyzes both a C6 and an unprecedented C1 ketoreduction. This then sets the stage for the subsequent C-ring cleavage that is key to the rearranged scaffolds of 1. The 1.1 Å structures of LugOII in complex with either ligand 8-O-Methylrabelomycin (4) or 8-O-Methyltetrangomycin (5) and of apoenzyme were resolved, which revealed a canonical Rossman fold and a remarkable conformational change during substrate capture and release. Mutational analysis uncovered key residues for substrate access, position, and catalysis as well as specific determinants that control its dual functionality. The insights obtained in this work hold promise for the discovery and engineering of other promiscuous reductases that may be harnessed for the generation of novel biocatalysts for chemoenzymatic applications.