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Chapter 9 The Enzymology of Polyether Biosynthesis
Abstract
Polyether ionophore antibiotics are a special class of polyketides widely used in veterinary medicine, and as food additives in animal husbandry. In this article, we review current knowledge about the mechanism of polyether biosynthesis, and the genetic and biochemical strategies used for its study. Several clear differences distinguish it from traditional type I modular polyketide biosynthesis: polyether backbones are assembled by modular polyketide synthases but are modified by two key enzymes, epoxidase and epoxide hydrolase, to generate the product. All double bonds involved in the oxidative cyclization in the polyketide backbone are of E geometry. Chain release in the polyether biosynthetic pathway requires a special type II thioesterase which specifically hydrolyzes the polyether thioester. All these discoveries should be very helpful for a deep understanding of the biosynthetic mechanism of this class of important natural compounds, and for the targeted engineering of polyether derivatives.
... A zipper mechanism leads to the formation of oxygen heterocycles in monensin biosynthesis [109][110][111]. ...
... During the biosynthesis of ionophoric terrestrial and marine polyethers, polyolefinic PKS products are first polyepoxidised and these epoxides are then opened in a so-called zipper mechanism that installs furan and/or pyran rings as well as cyclic acetals, if carbonyl groups are involved (shown for monensin in Scheme 17) [109][110][111]. While polyepoxidation is usually effected by only one epoxidase, one or more epoxide hydro-lases mediate regioselective epoxide opening and following controlled cyclisation. ...
... As the topic polyether biosynthesis is highly complex and a detailed discussion of further examples would go beyond the scope of this article, we would like to refer the reader to appropriate review literature [4,109]. ...
This review highlights the biosynthesis of heterocycles in polyketide natural products with a focus on oxygen and nitrogen-containing heterocycles with ring sizes between 3 and 6 atoms. Heterocycles are abundant structural elements of natural products from all classes and they often contribute significantly to their biological activity. Progress in recent years has led to a much better understanding of their biosynthesis. In this context, plenty of novel enzymology has been discovered, suggesting that these pathways are an attractive target for future studies.
... 183 In the biosynthesis of these compounds (as shown for monensin in Scheme 21), the flavin-dependent epoxidase (MonCI in the case of monensin) is believed to introduce multiple epoxide moieties into a polyunsaturated biosynthetic intermediate (141). A conserved epoxide hydrolase (encoded by each of the polyether biosynthetic gene clusters that have been sequenced, e.g., MonBI/MonBII in the case of monesin 184 ) is thought to generate the polycyclic ether ring system by initiating a cyclization cascade, 185 likely in a stepwise manner, 186 from the polyepoxy intermediate (142) using acid/base catalysis. [185][186][187] Support for this biosynthetic mechanism has been derived from studies of monensin biosynthesis, where disruption of the putative flavin epoxidase gene led to accumulation of a polyene precursor (141, Scheme 21). ...
... A conserved epoxide hydrolase (encoded by each of the polyether biosynthetic gene clusters that have been sequenced, e.g., MonBI/MonBII in the case of monesin 184 ) is thought to generate the polycyclic ether ring system by initiating a cyclization cascade, 185 likely in a stepwise manner, 186 from the polyepoxy intermediate (142) using acid/base catalysis. [185][186][187] Support for this biosynthetic mechanism has been derived from studies of monensin biosynthesis, where disruption of the putative flavin epoxidase gene led to accumulation of a polyene precursor (141, Scheme 21). 188 Recently, using a chemically synthesized substrate, the activity of the epoxide hydrolase, Lsa19, which catalyzes polyether formation by a cascade of epoxide opening reactions in the biosynthesis of lasalocid (137) was demonstrated in vitro (Scheme 21). ...
... 254 In a more recent study using a protein cell-free system of the producer, S. sahachiroi, the incorporation of valine, glycine and threonine into the tripeptidyl backbone was demonstrated. 255 Interestingly, ornithine (185) was also found to be an essential component for the in vitro reconstitution of azinomycin biosynthesis (Scheme 32). 255 These results provided valuable information regarding the possible building blocks used in the azinomycin pathway. ...
The established biosynthetic strategies for construction of cyclopropane, epoxide, and aziridine rings are illustrated. A strategy for the biosynthetic formation of cyclopropane moieties is by the displacement of a good leaving group by intramolecular attack of a carbanion using S N2-type chemistry. The biosynthetic gene cluster for hormaomycin, a nonribosomally synthesized peptide produced by Steptomyces griseoflavus that functions as a bacterial hormone and a narrow spectrum antibiotic, was also sequenced. In the case of PimD, recent structural studies provided an interesting twist to our understanding of the mechanism of P450 epoxidases. Recent bioinformatic studies suggest that flavin-dependent epoxidases are potentially widespread and are found in a number of natural product biosynthetic gene clusters. Several recent examples include the biosynthetic gene clusters for the enediyne compounds neocarzinostatin.
... They often contribute significantly to their structural and physical properties as well as to their biological activity [1][2][3]. Heterocycles can for example be involved in cation complexation as known for ionophoric polyethers or introduce conformational rigidity into a molecule, which is crucial for target binding [4]. ...
... As the topic polyether biosynthesis is highly complex and a detailed discussion of further examples would go beyond the scope of this article, we would like to refer the reader to appropriate review literature [4,109]. ...
This review highlights the biosynthesis of heterocycles in polyketide natural products with a focus on oxygen and nitrogen-containing heterocycles with ring sizes between 3 and 6 atoms. Heterocycles are abundant structural elements of natural products from all classes and they often contribute significantly to their biological activity. Progress in recent years has led to a much better understanding of their biosynthesis. In this context, plenty of novel enzymology has been discovered, suggesting that these pathways are an attractive target for future studies.
... Subsequent ring opening of the triene intermediate may be catalyzed by an epoxide hydrolase (MadI), and its homologs NanI, MonBI/BII [10], NigBI/BII, and Lsd19 [15,45,60] were found in other polyether biosynthetic pathways. These enzymatic functions have been summarized and inferred by Liu et al. [29]. ...
Maduramicin is the most efficient and possesses the largest market share of all anti-coccidiosis polyether antibiotics (ionophore); however, its biosynthetic gene cluster (BGC) has yet to been identified, and the associated strains have not been genetically engineered. Herein, we performed whole-genome sequencing of a maduramicin-producing industrial strain of Actinomadura sp. J1-007 and identified its BGC. Additionally, we analyzed the identified BGCs in silico to predict the biosynthetic pathway of maduramicin. We then developed a conjugation method for the non-spore-forming Actinomadura sp. J1-007, consisting of a site-specific integration method for gene overexpression. The maduramicin titer increased by 30% to 7.16 g/L in shake-flask fermentation following overexpression of type II thioesterase MadTE that is the highest titer at present. Our findings provide insights into the biosynthetic mechanism of polyethers and provide a platform for the metabolic engineering of maduramicin-producing microorganisms for overproduction and development of maduramicin analogs in the future.
... In the majority of bacterial and fungal polyketides, pendant methyl groups occur through the incorporation of propionate or the addition of the electrophilic methyl group of S-adenosyl methionine (SAM), catalyzed by the methyl transferase enzyme located in the polyketide biosynthetic gene cluster [61,62]. This reaction is described as α-alkylation because pendant alkylation occurs at a nucleophilic α-carbon in the polyketide chain ( Figure 5A). ...
Marine polyether toxins, mainly produced by marine dinoflagellates, are novel, complex, and diverse natural products with extensive toxicological and pharmacological effects. Owing to their harmful effects during outbreaks of marine red tides, as well as their potential value for the development of new drugs, marine polyether toxins have been extensively studied, in terms of toxicology, pharmacology, detection, and analysis, structural identification, as well as their biosynthetic mechanisms. Although the biosynthetic mechanisms of marine polyether toxins are still unclear, certain progress has been made. In this review, research progress and current knowledge on the biosynthetic mechanisms of polyether toxins are summarized, including the mechanisms of carbon skeleton deletion, pendant alkylation, and polyether ring formation, along with providing a summary of mined biosynthesis-related genes. Finally, future research directions and applications of marine polyether toxins are discussed.
... In most polyketide cases, these enzymes are unessential for the synthesis of the end product but appear to play an important role in maintaining the efficiency of the "enzymatic assembly line" leading to product formation. Particularly, they are involved in removing nonreactive acyl residues that block the PKS/NRPS megasynthase (4,5), maintaining the efficiency of the enzyme complex by editing misprimed precursors (6)(7)(8), removing aberrant intermediates (2), controlling starter units (3), providing key intermediates (9), or releasing final products (10)(11)(12)(13)(14). Consequently, it has been observed that deletion of TEII-encoding genes from gene clusters involved in polyketide biosynthesis usually leads to a decrease in the final product yield of the pathway concerned (6)(7)(8) rather than an absence thereof. ...
Type II thioesterases typically function as editing enzymes removing acyl groups which have been mis-conjugated to acyl carrier proteins during polyketide secondary metabolite biosynthesis as a consequence of biosynthetic errors. Streptomyces chartreusis NRRL 3882 produces the pyrrole polyether ionophoric antibiotic and we have identified the presence of a putative type II thioesterase like sequence, calG, within the biosynthetic gene cluster involved in the antibiotic's synthesis. However targeted gene mutagenesis experiments in which calG was inactivated in the organism did not lead to a decrease in calcimycin production but rather reduced the strain's production of its main biosynthetic precursor, cezomycin. Results from in vitro activity assays of purified, recombinant CalG protein indicated that it was involved in the hydrolysis of cezomycin-CoA, as well as other acyl CoAs, but was not active toward 3-S-N-acetylcysteamine (SNAC - the mimic of the polyketide chain releasing precursor). Further investigation of the enzyme's activity showed that it possessed a cezomycin-CoA hydrolysis Km of 0.67 mM and a kcat of 17.77 min⁻¹ and was significantly inhibited by the presence of Mn²⁺, and Fe²⁺ divalent cations. Interestingly when S. chartreusis NRRL 3882 was cultured in the presence of inorganic nitrite, NaNO2, it was observed that the production of calcimycin rather than cezomycin was promoted. Also that the supplementation of S. chartreusis NRRL 3882 growth medium with the divalent cations Ca²⁺, Mg²⁺, Mn²⁺, and Fe²⁺ had a similar effect. Taken together these observations suggest that CalG is not responsible for mega-synthase polyketide precursor chain release during the synthesis of calcimycin nor for retaining the catalytic efficiency of the mega-synthase enzyme complex as is supposed to be the function for type II thioesterases. Rather our results suggest that CalG is a dedicated thioesterase that prevents the accumulation of cezomycin-CoA when intracellular nitrogen is limited, an apparently new and previously unreported function of Type II thioesterases.
Importance
Type II thioesterases (TEIIs) are generally regarded as being responsible for removing aberrant acyl groups that block polyketide production thereby maintaining the efficiency of the mega-synthase involved in this class of secondary metabolite's biosynthesis. Specifically this class of enzyme is believed to be involved in editing mis-primed precursors, controlling initial units, providing key intermediates and releasing final synthetic products in the biosynthesis of this class of secondary metabolite. Our results indicate that the putative TEII, CalG, present in the calcimycin (A23187) producing organism Streptomyces chartreusis NRRL 3882 is not important either for the retention of catalytic efficiency of, or the release of the product compound from, the mega-synthase involved in calcimycin biosynthesis. Rather the enzyme is involved in regulating/controlling the pool size of the calcimycin biosynthetic precursor, cezomycin, by hydrolysis of its CoA derivative. This novel function of CalG suggests a possible additional activity for enzymes belonging to the TEII protein family and promotes better understanding of the overall biosynthetic mechanisms involved in the production of this class of secondary metabolite.
... Polyether ionophores, represented by the widely used and intensively studied nanchangmycin (1, dianemycin), monensin (2), nigericin (3), and salinomycin (4) (Figure 1) are a large class of complex, branched chain polyketide carboxylic acids characterized by the presence of two or more tetrahydrofuran, tetrahydropyran, or acetal rings that serve as potent and selective ligands for monovalent or divalent metal cations. Polyethers disrupt physiological ion gradients by allowing diffusion of the complexed cations across cell membranes, thereby accounting for their antibacterial action and leading to their use as commercially important coccidiostats and veterinary growth promoters. ...
The polyketide synthases responsible for the biosynthesis of the polyether antibiotics nanchangmycin (1) and salinomycin (4) harbor a number of redox-inactive ketoreductase (KR⁰) domains that are implicated in the generation of C2-epimerized (2S)-2-methyl-3-ketoacyl-ACP intermediates. Evidence that the natural substrate for the polyether KR⁰ domains is, as predicted, a (2R)-2-methyl-3-ketoacyl-ACP intermediate, came from a newly developed coupled ketosynthase (KS)-ketoreductase (KR) assay that established that the decarboxylative condensation of methylmalonyl-CoA with S-propionyl-N-acetylcysteamine catalyzed by the Nan[KS1][AT1] didomain from module 1 of the nanchangmycin synthase generates exclusively the corresponding (2R)-2-methyl-3-ketopentanoyl-ACP (7a) product. In tandem equilibrium isotope exchange experiments, incubation of [2-²H]-(2R,3S)-2-methyl-3-hydroxypentanoyl-ACP (6a) with redox-active, epimerase-inactive EryKR6 from module 6 of the 6-deoxyerythronolide B synthase and catalytic quantities of NADP⁺ in the presence of redox-inactive, recombinant NanKR1⁰ or NanKR5⁰, from modules 1 and 5 of the nanchangmycin synthase, or recombinant SalKR7⁰ from module 7 of the salinomycin synthase, resulted in first-order, time-dependent washout of deuterium from 6a. Control experiments confirmed that this washout was due to KR⁰-catalyzed isotope exchange of the reversibly-generated, transiently-formed oxidation product [2-²H]-(2R)-2-methyl-3-ketopentanoyl-ACP (7a), con-sistent with the proposed epimerase activity of each of the KR0 domains. Although they belong to the superfamily of short chain dehydrogenase-reductases, the epimerase-active KR0 domains from polyether synthases lack one or both residues of the conserved Tyr-Ser dyad that has previously been implicated in KR-catalyzed epimerizations.
... Polyether ionophore antibiotics such as monensin, nanchangmycin, and nigericin are synthesized by modular PKSs which lack terminal TE domains (Liu et al. 2009). The polyunsaturated polyketide chain undergoes a cascade of oxidative cyclizations being still attached to the ACP, as opposed to classic macrolide polyketides which are released by a TEI domain and then are subject to post-polyketide modifications. ...
A large number of antibiotics and other industrially important microbial secondary metabolites are synthesized by polyketide synthases (PKSs) and nonribosomal peptide synthetases (NRPSs). These multienzymatic complexes provide an enormous flexibility in formation of diverse chemical structures from simple substrates, such as carboxylic acids and amino acids. Modular PKSs and NRPSs, often referred to as megasynthases, have brought about a special interest due to the colinearity between enzymatic domains in the proteins working as an "assembly line" and the chain elongation and modification steps. Extensive efforts toward modified compound biosynthesis by changing organization of PKS and NRPS domains in a combinatorial manner laid good grounds for rational design of new structures and their controllable biosynthesis as proposed by the synthetic biology approach. Despite undeniable progress made in this field, the yield of such "unnatural" natural products is often not satisfactory. Here, we focus on type II thioesterases (TEIIs)-discrete hydrolytic enzymes often encoded within PKS and NRPS gene clusters which can be used to enhance product yield. We review diverse roles of TEIIs (removal of aberrant residues blocking the megasynthase, participation in substrate selection, intermediate, and product release) and discuss their application in new biosynthetic systems utilizing PKS and NRPS parts.
... Monensin A (1) from Streptomyces cinnamonensis is one of the most prominent and best-studied of the polyether class of complex polyketides, an important and extensive group of natural products including a large number of antibiotic ionophores [1][2][3][4][5] as well as remarkable marine toxins such as the brevetoxins [6,7]. The antibiotic polyethers adopt a characteristic con-Scheme 1: The proposed pathway for monensin biosynthesis in Streptomyces cinnamonensis. ...
Polyether antibiotics such as monensin are biosynthesised via a cascade of directed ring expansions operating on a putative polyepoxide precursor. The resulting structures containing fused cyclic ethers and a lipophilic backbone can form strong ionophoric complexes with certain metal cations. In this work, we demonstrate for monensin biosynthesis that, as well as ether formation, a late-stage hydroxylation step is crucial for the correct formation of the sodium monensin complex. We have investigated the last two steps in monensin biosynthesis, namely hydroxylation catalysed by the P450 monooxygenase MonD and O-methylation catalysed by the methyl-transferase MonE. The corresponding genes were deleted in-frame in a monensin-overproducing strain of Streptomyces cinnamonensis. The mutants produced the expected monensin derivatives in excellent yields (ΔmonD: 1.13 g L(-1) dehydroxymonensin; ΔmonE: 0.50 g L(-1) demethylmonensin; and double mutant ΔmonDΔmonE: 0.34 g L(-1) dehydroxydemethylmonensin). Single crystals were obtained from purified fractions of dehydroxymonensin and demethylmonensin. X-ray structure analysis revealed that the conformation of sodium dimethylmonensin is very similar to that of sodium monensin. In contrast, the coordination of the sodium ion is significantly different in the sodium dehydroxymonensin complex. This shows that the final constitution of the sodium monensin complex requires this tailoring step as well as polyether formation.
... [30][31][32] The cyclization process in these compounds is probably associated to the formation of an epoxide intermediate presumably catalyzed by epoxidases and epoxide hydrolases. 33,34 The accompanying polyepoxide process proposed for marine polyether ladder toxins has clear precedents in several reports on epoxide and cycle formation during antibiotic polyether biosynthesis. 35 Although the process whereby 10 or more epoxides are formed and then coordinated to a polyepoxide cascade to yield polyether ladder toxins still remains unclear, an oxidase-catalyzed tandem epoxide formation and epoxide protection, followed by a hydrolase-catalyzed epoxide opening and cycle rearrangement, is likely involved. ...
Many marine invertebrates produce potent toxins, turning themselves poisonous as a defense strategy against predators. In contrast, other organisms can become poisonous by accumulating toxins from their own prey. Dinoflagellates are aquatic photosynthetic microbial eukaryotes, and some species produce highly toxic metabolites. These dinoflagellate toxins bioaccumulate up the food chain in various consumer organisms. Many filter-feeding organisms such as bivalves accumulate such toxins with no apparent adverse effects on them1 but causing intoxication when ingested by their predators, including fish and marine mammals, and ultimately also when humans consume contaminated seafood.
Four major groups of dinoflagellate toxins have been described, namely, saxitoxins, ladder-shaped polyether compounds, long-chain polyketides, and macrolides.2 The dinoflagellate species Gambierdiscus toxicus produces several potent polyether toxins, some of which were initially identified in connection with a common type of food poisoning called ciguatera, caused by consumption of certain contaminated tropical and subtropical fish. Ciguatera involves a combination of gastrointestinal, neurological, and cardiovascular disorders. The two most common toxin classes associated with ciguatera are ciguatoxin (CTx) and maitotoxin (MTx), and they are among the most lethal natural substances known to man.
Most of the neurological symptoms of ciguatera are caused by CTx, which exert their effects due primarily to the activation of voltage-gated sodium channels, causing cell membrane depolarization.
MTx displays diverse pharmacological activities, which seem to be derived from its ability to activate Ca2+-uptake processes in a variety of cell types. MTx is the largest and most toxic known nonbiopolymeric toxin, with a molecular weight of 3422 Da. MTx is a very interesting compound given its extremely potent biological activity, and it has been used as a powerful pharmacological tool for the elucidation ofCa2+-dependent cellular processes.
Covering: up to the end of 2023Type I modular polyketide synthases construct polyketide natural products in an assembly line-like fashion, where the growing polyketide chain attached to an acyl carrier protein is passed from catalytic domain to catalytic domain. These enzymes have immense potential in drug development since they can be engineered to produce non-natural polyketides by strategically adding, exchanging, and deleting individual catalytic domains. In practice, however, this approach frequently results in complete failures or dramatically reduced product yields. A comprehensive understanding of modular polyketide synthase architecture is expected to resolve these issues. We summarize the three-dimensional structures and the proposed mechanisms of three full-length modular polyketide synthases, Lsd14, DEBS module 1, and PikAIII. We also describe the advantages and limitations of using X-ray crystallography, cryo-electron microscopy, and AlphaFold2 to study intact type I polyketide synthases.
A new linear type 1 polyketide, ionostatin (1) has been fully defined using a combined genomic and bioinformatics approach coupled with confirmatory chemical analyses. The 41 carbon-containing polyether is the product of the 101 kbp ion biosynthetic cluster containing seven modular type 1 polyketide synthases. Ionostatin is composed of 15 chiral centers that were proposed using the stereospecificities installed by the different classes of ketoreductases and enoylreductases and confirmed by rigorous NMR analysis. Incorporated into the structure are two tetrahydrofuran rings that appear to be the product of stereospecific epoxidation followed by stereospecific ring opening and cyclization. These transformations are proposed to be catalyzed by conserved enzymes analogous to those found in other bacterial-derived polyether biosynthetic clusters. Ionostatin shows moderate cancer cell cytotoxicity against U87 glioblastoma and SKOV3 ovarian carcinoma at 7.4 μg/mL.
Natural product biosynthetic pathways are composed of enzymes that use powerful chemistry to assemble complex molecules. Small molecule neurotoxins are examples of natural products with intricate scaffolds which often have high affinities for their biological targets. The focus of this Minireview is small molecule neurotoxins targeting voltage‐gated sodium channels (VGSCs) and the state of knowledge on their associated biosynthetic pathways. There are three small molecule neurotoxin receptor sites on VGSCs associated with three different classes of molecules: guanidinium toxins, alkaloid toxins, and ladder polyethers. Each of these types of toxins have unique structural features which are assembled by biosynthetic enzymes and the extent of information known about these enzymes varies among each class. The biosynthetic enzymes involved in the formation of these toxins have the potential to become useful tools in the efficient synthesis of VGSC probes.
The potential of the polyether salinomycin as an inhibitory agent against cancer stem cells has attracted interest in this family of compounds. In this study, we found that the aglycone polyether nanchangmycin and its homologues show promising activities against breast cancer stem cells as well as 38 other different types of cancer cells in in vitro assays. We found that aglycone polyethers caused elevations in calcium levels, an accumulation of reactive oxygen species and mitochondrial inner membrane permeability to H+ and K+, resulting in the release of cytochrome c and apoptosis-inducing factor and the triggering of caspase-dependent apoptosis. Our analyses also indicate that aglycone polyethers are potent Wnt/β-catenin signaling inhibitors, blocking the Wnt pathway and resulting in reduced cell survival. Notably, the key autophagy-related proteins LC3A/B were also activated by aglycone polyether treatment. Furthermore, nanchangmycin showed inhibitory effects towards somatic tumors developed from MCF-7 paclitaxel-resistant breast cancer cells injected into Balb/c mice. Our study not only provides promising candidates for therapy against cancer stem cells but also provides the groundwork for identifying stronger therapeutic agents among the natural polyether compounds.
Cascade reactions in organic synthesis, especially in natural product campaigns with biomimetic strategies, have previously been reviewed. Here we take up the complementary topic of enzyme‐mediated cascade reactions and note their widespread occurrence. To facilitate comparison with the mechanistic categorizations of cascade reactions by synthetic chemists and delineate the common underlying chemistry, we discuss four types of enzymatic cascade reactions: those mediated by nucleophilic, electrophilic, pericyclic, and radical‐based chemistries. Two subtypes of enzyme generating radical cascades exist at opposite ends of the oxygen abundance spectrum. Iron‐based enzymes use O2 to generate high valent iron‐oxo species to homolyze unactivated C‐H bonds in substrates that set off skeletal rearrangements. At the anaerobic end, enzymes reversibly cleave S‐adenosylmethionine (SAM) to generate the 5'‐deoxyadenosyl radical as a powerful oxidant to initiate C‐H bond homolysis in bound substrates. The latter enzymes are termed radical SAM enzymes. We categorize the former as "thwarted oxygenases".
Enzyme‐mediated cascade reactions are widespread in biosynthesis. To facilitate comparison with the mechanistic categorizations of cascade reactions by synthetic chemists and delineate the common underlying chemistry, we discuss four types of enzymatic cascade reactions: those involving nucleophilic, electrophilic, pericyclic, and radical reactions. Two subtypes of enzymes that generate radical cascades exist at opposite ends of the oxygen abundance spectrum. Iron‐based enzymes use O2 to generate high valent iron‐oxo species to homolyze unactivated C−H bonds in substrates to initiate skeletal rearrangements. At anaerobic end, enzymes reversibly cleave S‐adenosylmethionine (SAM) to generate the 5′‐deoxyadenosyl radical as a powerful oxidant to initiate C−H bond homolysis in bound substrates. The latter enzymes are termed radical SAM enzymes. We categorize the former as “thwarted oxygenases”.
The enzymology of 135 assembly lines containing primarily cis-acyltransferase modules is comprehensively analyzed, with greater attention paid to less common phenomena. Diverse online transformations, in which the substrate and/or product of the reaction is an acyl chain bound to an acyl carrier protein, are classified so that unusual reactions can be compared and underlying assembly-line logic can emerge. As a complement to the chemistry surrounding the loading, extension, and offloading of assembly lines that construct primarily polyketide products, structural aspects of the assembly-line machinery itself are considered. This review of assembly-line phenomena, covering the literature up to 2017, should thus be informative to the modular polyketide synthase novice and expert alike.
An iterative strategy for the stereodivergent synthesis of unbranched 1,5,9,n-polyenes (and -polyynes) was investigated. Starting from a terminal alkyne the iteration cycle consists of a C3 extension (allylation), a chemoselective hydroboration, an alkyne reduction, and an oxidation of the associated alcohol with subsequent C1 homologation. Double bond geometry is controlled using stereoselective alkyne reductions, employing either the Lindlar hydrogenation protocol or an aluminum hydride reduction. In a model sequence it was demonstrated that the strategy is applicable to the synthesis of 1,5,9,n-polyenes with any possible double bond configuration accessible in equally high efficiency and selectivity. It is worth noting that our approach does not require any protecting group chemistry. Furthermore, using the same strategy, the first total synthesis of chatenaytrienin-4, the proposed unsaturated biosynthetic precursor of the bis-THF acetogenin membranacin, was examined. Thus, the all-cis 1,5,9-triene natural product was prepared in 15 steps from commercially available starting materials in 6% overall yield.
Marine natural products (MNPs) produced by marine microorganisms are extensively explored in recent years as potential therapeutic agents for medical treatments. MNPs isolated from marine bacteria, marine fungi, and marine cyanobacteria have shown a wide range of useful medical activities including antimicrobial, anticancer, antiviral, and anti-inflammatory among others. On the other hand, a vast array of novel natural products produced by marine microorganisms, for example, polyketides, peptides, alkaloids, and terpenes are being explored as lead compounds or drug candidates to increase the healthy life span of human being. High-throughput genome sequencing technology enables the identification of tremendous biosynthetic genes for MNPs biosynthesis, and the combination of omics technology, genetic and mass spectrometry studies with classical chemical studies is promoting the rapid discovery of more MNPs. In this chapter, the most recently reported novel chemical structures and activity studies of marine microorganisms are presented, and progress on genome-based studies for MNPs discovery including gene-based screening, metagenomic studies, as well as genome-mining approaches is discussed. Understanding the biosynthesis machinery of MNPs provides a basis for creating novel drug molecules of enhanced medical properties. Harnessing the chemical and genetic diversities of marine microorganisms opens the door for a complete exploration of MNPs in drug discovery for medical treatments.
The first total synthesis of muricadienin, the unsaturated putative precursor in the biosynthesis of trans- and cis-solamin is described. Key steps in the synthesis are a chemoselective hydroboration, a Z-selective Wittig reaction, and a Fries rearrangement for introducing the terminal α-substituted butenolide. Thus, muricadienin can be synthesized in 11 steps from commercially available starting materials in 42% overall yield.
Covering: up to 2013Dinoflagellates produce unique polyketides characterized by their size and complexity. The biosynthesis of a limited number of such metabolites has been reported, with studies largely hampered by the low yield of compounds and the severe scrambling of label in the isotopically-labeled precursors. Nonetheless, of the successful biosynthetic experiments that have been reported, many surprising and unique processes have been discovered. This knowledge has been accessed through a series of biochemical labeling studies, and while limited molecular genetic data has been amassed, it is still in the early stages of development. In an attempt to meet this challenge, this review has compared some of the biosynthetic processes with similar ones identified in other microbes such as bacteria and myxobacteria, with the idea that similar genes and enzymes are employed by dinoflagellates.
Mangrove-derived actinomycetes are promising sources of bioactive natural products. In this study, using homologous screening of the biosynthetic genes and anti-microorganism/tumor assaying, 163 strains of actinomycetes isolated from mangrove sediments were investigated for their potential to produce halogenated metabolites. The FADH2-dependent halogenase genes, identified in PCR-screening, were clustered in distinct clades in the phylogenetic analysis. The coexistence of either polyketide synthase (PKS) or nonribosomal peptide synthetase (NRPS) as the backbone synthetases in the strains harboring the halogenase indicated that these strains had the potential to produce structurally diversified antibiotics. As a validation, a new enduracidin producer, Streptomyces atrovirens MGR140, was identified and confirmed by gene disruption and HPLC analysis. Moreover, a putative ansamycin biosynthesis gene cluster was detected in Streptomyces albogriseolus MGR072. Our results highlight that combined genome mining is an efficient technique to tap promising sources of halogenated natural products synthesized by mangrove-derived actinomycetes.
Enantioselective epoxidation followed by regioselective epoxide opening reaction are the key processes in construction of the polyether skeleton. Recent genetic analysis of ionophore polyether biosynthetic gene clusters suggested that flavin-containing monooxygenases (FMOs) could be involved in the oxidation steps. In vivo and in vitro analyses of Lsd18, an FMO involved in the biosynthesis of polyether lasalocid, using simple olefin or truncated diene of a putative substrate as substrate mimics demonstrated that enantioselective epoxidation affords natural type mono- or bis-epoxide in a stepwise manner. These findings allow us to figure out enzymatic polyether construction in lasalocid biosynthesis.
Incubation of recombinant module 2 of the polyether nanchangmycin synthase (NANS), carrying an appended thioesterase domain, with the ACP-bound substrate (2RS)-2-methyl-3-ketobutyryl-NANS_ACP1 (2-ACP1) and methylmalonyl-CoA in the presence of NADPH gave diastereomerically pure (2S,4R)-2,4-dimethyl-5-ketohexanoic acid (4a). These results contrast with the previously reported weak discrimination by NANS module 2+TE between the enantiomers of the corresponding N-acetylcysteamine-conjugated substrate analogue (±)-2-methyl-3-ketobutyryl-SNAC (2-SNAC), which resulted in formation of a 5:3 mixture of 4a and its (2S,4S)-diastereomer 4b. Incubation of NANS module 2+TE with 2-ACP1 in the absence of NADPH gave unreduced 3,5,6-trimethyl-4-hydroxypyrone (3) with a k(cat) of 4.4 ± 0.9 min⁻¹ and a k(cat)/K(m) of 67 min⁻¹ mM⁻¹, corresponding to a ∼2300-fold increase compared to the k(cat)/K(m) for the diffusive substrate 2-SNAC. Covalent tethering of the 2-methyl-3-ketobutyryl thioester substrate to the NANS ACP1 domain derived from the natural upstream PKS module of the nanchangmycin synthase significantly enhanced both the stereospecificity and the kinetic efficiency of the sequential polyketide chain translocation and condensation reactions catalyzed by the ketosynthase domain of NANS module 2.
The naturally occurring antibiotic lasalocid A contains a chemical
structure that is not expected to form readily. The enzyme that
catalyses the formation of this structure has been identified, and its
activity is a revelation. See Letter p.355
Caught in the act: Intermediates in the biosynthesis of lasalocid A are captured in vivo by malonyl carba(dethia)-N-acetyl cysteamine probes. These species constitute novel snapshots of the timing of ether and aromatic ring formation, thus providing valuable insights for the reconstruction and the engineering of polyether biosynthetic pathways.
Salinomycin is widely used in animal husbandry as a food additive due to its antibacterial and anticoccidial activities. However,
its biosynthesis had only been studied by feeding experiments with isotope-labeled precursors. A strategy with degenerate
primers based on the polyether-specific epoxidase sequences was successfully developed to clone the salinomycin gene cluster.
Using this strategy, a putative epoxidase gene, slnC, was cloned from the salinomycin producer Streptomyces albus XM211. The targeted replacement of slnC and subsequent trans-complementation proved its involvement in salinomycin biosynthesis. A 127-kb DNA region containing slnC was sequenced, including genes for polyketide assembly and release, oxidative cyclization, modification, export, and regulation.
In order to gain insight into the salinomycin biosynthesis mechanism, 13 gene replacements and deletions were conducted. Including
slnC, 7 genes were identified as essential for salinomycin biosynthesis and putatively responsible for polyketide chain release,
oxidative cyclization, modification, and regulation. Moreover, 6 genes were found to be relevant to salinomycin biosynthesis
and possibly involved in precursor supply, removal of aberrant extender units, and regulation. Sequence analysis and a series
of gene replacements suggest a proposed pathway for the biosynthesis of salinomycin. The information presented here expands
the understanding of polyether biosynthesis mechanisms and paves the way for targeted engineering of salinomycin activity
and productivity.
Say “when”: Analysis of the biosynthetic gene cluster for the polyether antibiotic and anticancer agent salinomycin (1) shows that its core structure is synthesised by a nine-multienzyme modular polyketide synthase. Deletion of the salC gene, which is required for oxidative cyclisation, has led to the detection of a novel metabolite whose structure reveals the timing of a key dehydration step.
Recombinant nanchangmycin synthase module 2 (NANS module 2), with the thioesterase domain from the 6-deoxyerythronolide B synthase (DEBS TE) appended to the C-terminus, was cloned and expressed in Escherichia coli. Incubation of NANS module 2+TE with (±)-2-methyl-3-keto-butyryl-N-acetylcysteamine thioester (1), the SNAC analog of the natural ACP-bound substrate, with methylmalonyl-CoA (MM-CoA) in the absence of NADPH gave 3,5,6-trimethyl-4-hydroxypyrone (2), identified by direct comparison with synthetic 2 by radio-TLC-phosphorimaging and LC-ESI(+)-MS-MS. The reaction showed k(cat) 0.5 ± 0.1 min(-1) and K(m)(1) 19 ± 5 mM at 0.5 mM MM-CoA and k(cat)(app) 0.26 ± 0.02 min(-1) and K(m)(MM-CoA) 0.11 ± 0.02 mM at 8 mM 1. Incubation in the presence of NADPH generated the fully saturated triketide chain elongation product as a 5:3 mixture of (2S,4R)-2,4-dimethyl-5-ketohexanoic acid (3a) and the diastereomeric (2S,4S)-3b. The structure and stereochemistry of each product was established by comparison with synthetic 3a and 3b by a combination of radio-TLC-phosphorimaging and LC-ESI(-)-MS-MS, as well as chiral capillary GC-MS analysis of the corresponding methyl esters 3a-Me and 3b-Me. The recombinant dehydratase domain from NANS module 2, NANS DH2, was shown to catalyze the formation of an (E)-double bond by syn-dehydration of the ACP-bound substrate anti-(2R,3R,4S,5R)-2,4-dimethyl-3,5-dihydroxyheptanoyl-ACP6 (4), generated in situ by incubation of (2S,3R)-2-methyl-3-hydroxypentanoyl-SNAC (5), methylmalonyl-CoA, and NADPH with the recombinant [KS6][AT6] didomain and ACP6 from DEBS module 6 along with the ketoreductase from the tylactone synthase module 1 (TYLS KR1). These results also indirectly establish the stereochemistry of the reactions catalyzed by the KR and enoylreductase (ER) domains of NANS module 2.
The discovery of brevisin, the first example of an "interrupted" polycyclic ether, obtained from the dinoflagellate Karenia brevis, posed some important questions regarding the mechanism of the cyclization process. Consequently, we have established absolute configurations of brevisin and its related metabolite brevisamide using a modified Mosher's esterification method. For brevisin, analysis was carried out on both the 31-monokis- and the 10,31-bis-MTPA esters. The results suggest that both metabolites, like other polyethers from K. brevis, result from polyepoxide precursors with uniform (S, S) configurations for all epoxides and provide further support for a universal stereochemical model for dinoflagellate polyether formation.
This review covers the recent literature on the release mechanisms for polyketides and nonribosomal peptides produced by microorganisms. The emphasis is on the novel enzymology and mechanistic insights revealed by the biosynthetic studies of new natural products.
Several independent gene clusters containing varying lengths of type I polyketide synthase genes were isolated from 'Streptomyces nanchangensis' NS3226, a producer of nanchangmycin and meilingmycin. The former is a polyether compound similar to dianemycin and the latter is a macrolide compound similar to milbemycin, which shares the same macrolide ring as avermectin but has different side groups. Clusters A-H spanned about 133, 132, 104, 174, 122, 54, 37 and 59 kb, respectively. Two systems were developed for functional analysis of the gene clusters by gene disruption or replacement. (1) Streptomyces phage phiC31 and its derived vectors can infect and lysogenize this strain. (2) pSET152, an Escherichia coli plasmid with phiC31 attP site, and pHZ1358, a Streptomyces-Escherichia coli shuttle cosmid vector, both carrying oriT from RP4, can be mobilized from E. coli into NS3226 by conjugation. pHZ1358 was shown to be generally useful for generating mutant strains by gene disruption and replacement in NS3226 as well as in several other Streptomyces strains. A region in cluster A (approximately 133 kb) seemed to be involved in nanchangmycin production because replacement of several DNA fragments in this region by an apramycin resistance gene [aac3(IV)] gave rise to nanchangmycin non-producing mutants.
Macrocyclic polyketides exhibit an impressive range of medically useful activities, and there is great interest in manipulating the genes that govern their synthesis. The 6-deoxyerythronolide B synthase (DEBS) of Saccharopolyspora erythraea, which synthesizes the aglycone core of the antibiotic erythromycin A, has been modified by repositioning of a chain-terminating cyclase domain to the carboxyl-terminus of DEBS1, the multienzyme that catalyzes the first two rounds of polyketide chain extension. The resulting mutant markedly accelerates formation of the predicted triketide lactone, compared to a control in which the repositioned domain is inactive. Repositioning of the cyclase should be generally useful for redirecting polyketide synthesis to obtain polyketides of specified chain lengths.
Large-scale in vitro screening of different types of ionophores previously pinpointed nine compounds that were very active and selective in vitro against Plasmodium falciparum; their in vitro and in vivo antimalarial effects were further studied. Addition of the ionophores to synchronized P. falciparum suspensions revealed that all P. falciparum stages were sensitive to the drugs. However, the schizont stages were three- to ninefold more sensitive, and 12 h was required for complete parasite clearance. Pretreatment of healthy erythrocytes with toxic doses of ionophores for 24 to 48 h showed that the activity was not due to an irreversible effect on the host erythrocyte. No preferential ionophore adsorption in infected or uninfected erythrocytes occurred. On the other hand, ionophore molecules strongly bound to serum proteins since increasing the serum concentration from 2 to 50% led to almost a 25-fold parallel increase in the ionophore 50% inhibitory concentration. Mice infected with the malaria parasites Plasmodium vinckei petteri or Plasmodium chabaudi were successfully treated with eight ionophores in a 4-day suppressive test. The 50% effective dose after intraperitoneal administration ranged from 0.4 to 4.1 mg/kg of body weight, and the therapeutic indices were about 5 for all ionophores except monensin A methyl ether, 5-bromo lasalocid A, and gramicidin D, whose therapeutic indices were 12, 18, and 344, respectively. These three compounds were found to be curative, with no recrudescence. Gramicidin D, which presented impressive antimalarial activity, requires parenteral administration, while 5-bromo lasalocid A has the major advantage of being active after oral administration. Overall, the acceptable levels of toxicity and the good in vivo therapeutic indices in the rodent model highlight the interesting potential of these ionophores for the treatment of malaria in higher animals.
The BLAST programs are widely used tools for searching protein and DNA databases for sequence similarities. For protein comparisons,
a variety of definitional, algorithmic and statistical refinements described here permits the execution time of the BLAST
programs to be decreased substantially while enhancing their sensitivity to weak similarities. A new criterion for triggering
the extension of word hits, combined with a new heuristic for generating gapped alignments, yields a gapped BLAST program
that runs at approximately three times the speed of the original. In addition, a method is introduced for automatically combining
statistically significant alignments produced by BLAST into a position-specific score matrix, and searching the database using
this matrix. The resulting Position-Specific Iterated BLAST (PSIBLAST) program runs at approximately the same speed per iteration
as gapped BLAST, but in many cases is much more sensitive to weak but biologically relevant sequence similarities. PSI-BLAST
is used to uncover several new and interesting members of the BRCT superfamily.
The sigE gene of Streptomyces coelicolor A3(2) encodes an RNA polymerase sigma factor belonging to the extracytoplasmic function (ECF) subfamily. Constructed sigE deletion and disruption mutants were more sensitive than the parent to muramidases such as hen egg white lysozyme and to the CwlA amidase from Bacillus subtilis. This correlated with an altered muropeptide profile, as determined by reverse-phase high-performance liquid chromatography analysis of lytic digests of purified peptidoglycan. The sigE mutants required high levels of magnesium for normal growth and sporulation, overproducing the antibiotic actinorhodin and forming crenellated colonies in its absence. Together, these data suggest that sigE is required for normal cell wall structure. The role of sigmaE was further investigated by analyzing the expression of hrdD, which is partially sigE dependent. The hrdD gene, which encodes the sigmaHrdD subunit of RNA polymerase, is transcribed from two promoters, hrdDp1 and hrdDp2, both similar to promoters recognized by other ECF sigma factors. The activities of hrdDp1 and hrdDp2 were reduced 20- and 3-fold, respectively, in sigE mutants, although only hrdDp1 was recognized by EsigmaE in vitro. Growth on media deficient in magnesium caused the induction of both hrdD promoters in a sigE-dependent manner.
Streptomyces coelicolor is a representative of the group of soil-dwelling, filamentous bacteria responsible for producing most natural antibiotics used in human and veterinary medicine. Here we report the 8,667,507 base pair linear chromosome of this organism, containing the largest number of genes so far discovered in a bacterium. The 7,825 predicted genes include more than 20 clusters coding for known or predicted secondary metabolites. The genome contains an unprecedented proportion of regulatory genes, predominantly those likely to be involved in responses to external stimuli and stresses, and many duplicated gene sets that may represent 'tissue-specific' isoforms operating in different phases of colonial development, a unique situation for a bacterium. An ancient synteny was revealed between the central 'core' of the chromosome and the whole chromosome of pathogens Mycobacterium tuberculosis and Corynebacterium diphtheriae. The genome sequence will greatly increase our understanding of microbial life in the soil as well as aiding the generation of new drug candidates by genetic engineering.
Modular polyketide synthases (PKSs) synthesize the polyketide cores of pharmacologically important natural products such as erythromycin and picromycin. Understanding PKSs at high resolution could present new opportunities for chemoenzymatic synthesis of complex molecules. The crystal structures of macrocycle-forming thioesterase (TE) domains from the picromycin synthase (PICS) and 6-deoxyerythronolide B synthase (DEBS) were determined to 1.8-3.0 A with an R(crys) of 19.2-24.4%, including three structures of PICS TE (crystallized at pH 7.6, 8.0, and 8.4) and a second crystal form of DEBS TE. As predicted by the previous work on DEBS TE [Tsai, S. C., et al. (2001) Proc. Natl. Acad. Sci. U.S.A. 98, 14808-14813], PICS TE contains an open substrate channel and a hydrophobic dimer interface. Notwithstanding their similarity, the dimer interfaces and substrate channels of DEBS TE and PICS TE reveal key differences. The structural basis for the divergent substrate specificities of DEBS TE and PICS TE is analyzed. The size of the substrate channel increases with increasing pH, presumably due to electrostatic repulsion in the channel at elevated pH. Together, these structures support previous predictions that macrocycle-forming thioesterases from PKSs share the same protein fold, an open substrate channel, a similar catalytic mechanism, and a hydrophobic dimer interface. They also provide a basis for the design of enzymes capable of catalyzing regioselective macrocyclization of natural or synthetic substrates. A series of high-resolution snapshots of a protein channel at different pHs is presented alongside analysis of channel residues, which could help in the redesign of the protein channel architecture.
Species of the genus Streptomyces are of major pharmaceutical interest because they synthesize a variety of bioactive secondary metabolites. We have determined the complete nucleotide sequence of the linear chromosome of Streptomyces avermitilis. S. avermitilis produces avermectins, a group of antiparasitic agents used in human and veterinary medicine. The genome contains 9,025,608 bases (average GC content, 70.7%) and encodes at least 7,574 potential open reading frames (ORFs). Thirty-five percent of the ORFs (2,664) constitute 721 paralogous families. Thirty gene clusters related to secondary metabolite biosynthesis were identified, corresponding to 6.6% of the genome. Comparison with Streptomyces coelicolor A3(2) revealed that an internal 6.5-Mb region in the S. avermitilis genome was highly conserved with respect to gene order and content, and contained all known essential genes but showed perfectly asymmetric structure at the oriC center. In contrast, the terminal regions were not conserved and preferentially contained nonessential genes.
The PKS genes for biosynthesis of the polyether nanchangmycin are organized to encode two sets of proteins (six and seven ORFs, respectively), but are separated by independent ORFs that encode an epimerase, epoxidase, and epoxide hydrolase, and, notably, an independent ACP. One of the PKS modules lacks a corresponding ACP. We propose that the process of oxidative cyclization to form the polyether structure occurs when the polyketide chain is still anchored on the independent ACP before release. 4-O-methyl-L-rhodinose biosynthesis and its transglycosylation involve four putative genes, and regulation of nanchangmycin biosynthesis seems to involve activation as well as repression. In-frame deletion of a KR6 domain generated the nanchangmycin aglycone with loss of 4-O-methyl-L-rhodinose and antibacterial activity, in agreement with the assignments of the PKS domains catalyzing specific biosynthetic steps.
Epoxide hydrolases are essential for the processing of epoxide-containing compounds in detoxification or metabolism. The classic epoxide hydrolases have an alpha/beta hydrolase fold and act via a two-step reaction mechanism including an enzyme-substrate intermediate. We report here the structure of the limonene-1,2-epoxide hydrolase from Rhodococcus erythropolis, solved using single-wavelength anomalous dispersion from a selenomethionine-substituted protein and refined at 1.2 A resolution. This enzyme represents a completely different structure and a novel one-step mechanism. The fold features a highly curved six-stranded mixed beta-sheet, with four alpha-helices packed onto it to create a deep pocket. Although most residues lining this pocket are hydrophobic, a cluster of polar groups, including an Asp-Arg-Asp triad, interact at its deepest point. Site-directed mutagenesis supports the conclusion that this is the active site. Further, a 1.7 A resolution structure shows the inhibitor valpromide bound at this position, with its polar atoms interacting directly with the residues of the triad. We suggest that several bacterial proteins of currently unknown function will share this structure and, in some cases, catalytic properties.
Aclacinomycin methylesterase (RdmC) is one of the tailoring enzymes that modify the aklavinone skeleton in the biosynthesis
of anthracyclines in Streptomyces species. The crystal structures of this enzyme from Streptomyces purpurascens in complex with the product analogues 10-decarboxymethylaclacinomycin T and 10-decarboxymethylaclacinomycin A were determined
to nominal resolutions of 1.45 and 1.95 Å, respectively. RdmC is built up of two domains. The larger α/β domain shows the
common α/β hydrolase fold, whereas the smaller domain is α-helical. The active site and substrate binding pocket are located
at the interface between the two domains. Decarboxymethylaclacinomycin T and decarboxymethylaclacinomycin A bind close to
the catalytic triad (Ser102-His276-Asp248) in a hydrophobic pocket, with the sugar moieties located at the surface of the enzyme. The binding of the ligands is dominated
by hydrophobic interactions, and specificity appears to be controlled mainly by the shape of the binding pocket rather than
through specific hydrogen bonds. Mechanistic key features consistent with the structure of complexes of RdmC with product
analogues are Ser102 acting as nucleophile and transition state stabilization by an oxyanion hole formed by the backbone amides of residues Gly32 and Met103.
Streptomycetes are high G+C Gram-positive, antibiotic-producing, mycelial soil bacteria. The 8.7-Mb Streptomyces coelicolor genome was previously sequenced by using an ordered library of Supercos-1 clones. Here, we describe an efficient procedure for creating precise gene replacements in the cosmid clones by using PCR targeting and lambda-Red-mediated recombination. The cloned Streptomyces genes are replaced with a cassette containing a selectable antibiotic resistance and oriT(RK2) for efficient transfer to Streptomyces by RP4-mediated intergeneric conjugation. Supercos-1 does not replicate in Streptomyces, but the clones readily undergo double-crossover recombination, thus creating gene replacements. The antibiotic resistance cassettes are flanked by yeast FLP recombinase target sequences for removal of the antibiotic resistance and oriT(RK2) to generate unmarked, nonpolar mutations. The technique has been used successfully by >20 researchers to mutate around 100 Streptomyces genes. As an example, we describe its application to the discovery of a gene involved in the production of geosmin, the ubiquitous odor of soil. The gene, Sco6073 (cyc2), codes for a protein with two sesquiterpene synthase domains, only one of which is required for geosmin biosynthesis, probably via a germacra-1 (10) E,5E-dien-11-ol intermediate generated by the sesquiterpene synthase from farnesyl pyrophosphate.
MODBASE (http://salilab.org/modbase) is a database of annotated comparative protein structure models for all available protein sequences that can be matched
to at least one known protein structure. The models are calculated by MODPIPE, an automated modeling pipeline that relies
on MODELLER for fold assignment, sequence–structure alignment, model building and model assessment (http:/salilab.org/modeller). MODBASE is updated regularly to reflect the growth in protein sequence and structure databases, and improvements in the
software for calculating the models. MODBASE currently contains 3 094 524 reliable models for domains in 1 094 750 out of
1 817 889 unique protein sequences in the UniProt database (July 5, 2005); only models based on statistically significant
alignments and models assessed to have the correct fold despite insignificant alignments are included. MODBASE also allows
users to generate comparative models for proteins of interest with the automated modeling server MODWEB (http://salilab.org/modweb). Our other resources integrated with MODBASE include comprehensive databases of multiple protein structure alignments (DBAli,
http://salilab.org/dbali), structurally defined ligand binding sites and structurally defined binary domain interfaces (PIBASE, http://salilab.org/pibase) as well as predictions of ligand binding sites, interactions between yeast proteins, and functional consequences of human
nsSNPs (LS-SNP, http://salilab.org/LS-SNP).
The polyketide synthase (PKS) for the biosynthesis of the polyether nanchangmycin lacks an apparent thioesterase comparable to the type I thioesterase domains of the modular PKSs responsible for macrolide biosynthesis. Three candidate polyether chain-releasing factors were examined. Both the putative CR domain and the NanE protein appeared to be genetically relevant. Among the three heterologously expressed soluble proteins (recombinant CR domain, the ACP-CR didomain, and NanE) tested, only NanE hydrolyzed the polyether-SNAC. By contrast, recombinant DEBS TE from the erythromycin pathway, and the recombinant MonAX, a type II TE associated with the polyether monensin biosynthesis for which a homolog has not been detected in the nanchangmycin cluster, hydrolyzed a diketide-SNAC but not the polyether-SNAC. We could thus conclude that NanE is a dedicated thioesterase mediating the specific release of the polyether chain during nanchangmycin biosynthesis.
Saccharopolyspora erythraea is used for the industrial-scale production of the antibiotic erythromycin A, derivatives of which play a vital role in medicine. The sequenced chromosome of this soil bacterium comprises 8,212,805 base pairs, predicted to encode 7,264 genes. It is circular, like those of the pathogenic actinomycetes Mycobacterium tuberculosis and Corynebacterium diphtheriae, but unlike the linear chromosomes of the model actinomycete Streptomyces coelicolor A3(2) and the closely related Streptomyces avermitilis. The S. erythraea genome contains at least 25 gene clusters for production of known or predicted secondary metabolites, at least 72 genes predicted to confer resistance to a range of common antibiotic classes and many sets of duplicated genes to support its saprophytic lifestyle. The availability of the genome sequence of S. erythraea will improve insight into its biology and facilitate rational development of strains to generate high-titer producers of clinically important antibiotics.
The total synthesis is reported, from a pool of optically pure starting materials, of a tritium labelled form of a putative intermediate in monensin biosynthesis, in which the terminal carboxyl group is activated as a caprylcysteamine thiol ester.
The incorporation of [2-2H2]- and (S)-[2-2H1]-propionate into monensin-A in cultures of Streptomyces cinnamonensis occurs with retention of label only at C(4) and C(6) in the antibiotic, whereas during the incorporation of (R)-[2-2H1]propionate the deuterium label is lost to the medium.
The biosynthesis of monensin-A in cultures of Streptomyces cinnamonensis is shown to occur with the incorporation of four oxygen atoms from molecular oxygen.
Syntheses of [2-2H,3-13C]- and [2-2H,3′-2H2]proclavaminic acid, (22) and (29) respectively, are described. These labeled substrates were incubated with clavaminate synthase, a key enzyme acting in the biosynthesis of the lactamase inhibitor clavulanic acid, in the presence of ferrous ion, α-ketoglutaric acid and molecular oxygen. The apparent oxidative cyclization/desaturation chemistry evident in the conversion of proclavaminic acid (8) to clavaminic acid (9) takes place without loss or exchange of label at C-2 and C-3′ in the substrate. These observations point to notable similarities to sulfur insertion reactions in natural product biosynthesis and lead to the proposition of a generalized mechanism for oxidative cyclization invoking substrate heteroatom participation. By extension of this mechanistic hypothesis, a new biogenetic speculation is advanced to account for polyether formation, for example, in monensin (52) and brevetoxin A (55).
Feeding of [1-13C] acetate to cultures of Streptomyces cinnamonensis gave monensin A labeled at carbons 7, 9, 13, 19, and 25, as established by 13C NMR analysis. Similarly, incorporation of [1-13C]propionate resulted in enrichment of carbons 1, 3, 5, 11, 17, 21, and 23. Further incorporations of [1,2-13C2]acetate, [1,2-13C2]propionate, [2-13C]propionate, and [2,3-13C2]succinate and analysis by 13C NMR, including extensive homonuclear 13C{13C} decoupling, established the biosynthetic origins of all the carbon atoms of monensin, while allowing a complete assignment of the 13C NMR spectrum. When [1-13C,1-18O2]propionate was fed, isotopically shifted peaks indicating the presence of oxygen-18 at C-1, C-3, and C-5 were observed, whereas feeding of [1-13C,1-18O2]acetate gave rise to excess oxygen-18 at C-7, C-9, and C-25. Three of the remaining ether oxygens, O(7), O(8), and O(9), were shown to be derived from molecular oxygen by growth of S. cinnamonensis in an atmosphere of 18O2 and 13C NMR analysis of the resulting labeled monensin A. These results are consistent with initial formation of the all-E-triene 7, which can be converted to monensin by cyclization of the triepoxide 8.
A unified stereochemical model is proposed which correlates the structure and stereochemistry of a large number of polyether antibiotics and which suggests the biosynthetic basis for this perceived structural regularity. Two stereochemical prototypes, illustrated in Figure 3, parts A and B, summarize the stereochemical patterns of more than 30 different polyether antibiotics of the APPA and PAPA structural families.
As a direct test of the Cane-Westley hypothesis concerning the mode of assembly of ether rings in the polyether class of ionophore antibiotics, we describe experiments culminating in the synthesis of three putative intermediates on the monensin biosynthetic pathway and incorporation experiments with these materials and the monensin-producing organism Streptomyces cinnamonensis. The putative intermediates synthesised include the trienes [21-3H]-7 and [13-3H]-10, and the diene [9-3H]-11. The results of the incorporation experiments conducted with whole cell cultures suggest that [13-3H]-10 and [21-3H]-7 are unable to cross the intact cell membrane of S. cinnamonensis, whereas diene [9-3H]-11 can gain entry to the cellular interior, but is then degraded efficiently, most likely by a pathway closely related to β-oxidation, without being specifically incorporated into the antibiotic.
FramePlot is a web-based tool for predicting protein-coding regions in bacterial DNA with a high G+C content, such as Streptomyces. The graphical output provides for easy distinction of protein-coding regions from non-coding regions. The plot is a clickable map. Clicking on an ORF provides not only the nucleotide sequence but also its deduced amino acid sequence. These sequences can then be compared to the NCBI sequence database over the Internet. The program is freely available for academic purposes at http://www.nih.go.jp/~jun/cgi-bin/frameplot.pl.
FUGUE, a program for recognizing distant homologues by sequence-structure comparison (http://www-cryst.bioc.cam.ac.uk/fugue/), has three key features. (1) Improved environment-specific substitution tables. Substitutions of an amino acid in a protein structure are constrained by its local structural environment, which can be defined in terms of secondary structure, solvent accessibility, and hydrogen bonding status. The environment-specific substitution tables have been derived from structural alignments in the HOMSTRAD database (http://www-cryst.bioc.cam.ac.uk/homstrad/). (2) Automatic selection of alignment algorithm with detailed structure-dependent gap penalties. FUGUE uses the global-local algorithm to align a sequence-structure pair when they greatly differ in length and uses the global algorithm in other cases. The gap penalty at each position of the structure is determined according to its solvent accessibility, its position relative to the secondary structure elements (SSEs) and the conservation of the SSEs. (3) Combined information from both multiple sequences and multiple structures. FUGUE is designed to align multiple sequences against multiple structures to enrich the conservation/variation information. We demonstrate that the combination of these three key features implemented in FUGUE improves both homology recognition performance and alignment accuracy.
Polyether metabolites are an important class of natural products. Although their biosynthesis, especially construction of polyether skeletons, attracted organic chemists for many years, no experimental data on the enzymatic polyether formation has been obtained. In this study, a putative epoxide hydrolase gene lsd19 found on the biosynthetic gene cluster of an ionophore polyether lasalocid was cloned and successfully overexpressed in Escherichia coli. Using the purified Lsd19, a proposed substrate, bisepoxyprelasalocid, and its synthesized analogue were successfully converted into lasalocid A and its derivative via a 6-endo-tet cyclization mode. On the other hand, treatment of the bisepoxide with trichloroacetic acid gave isolasalocid A via a 5-exo-tet cyclization mode. Therefore, the enzymatic conversion observed in this study unambiguously showed that the bisepoxyprelasalocid is an intermediate of the lasalocid biosynthesis and that Lsd19 catalyzes the sequential cyclic ether formations involving an energetically disfavored 6-endo-tet cyclization. This is the first example of the enzymatic epoxide-opening reactions leading to a polyether natural product.
We examined the inhibitory activities of 10 polyether antibiotics on human immunodeficiency virus (HIV) type 1. These compounds
caused concentration-dependent inhibition of HIV replication in primary infected cultures of human T-lymphoblastoid H9 cells.
The ratio of 50% effective concentrations for cellular cytotoxicity (MTT assay) to antiviral activity (reverse transcriptase
assay) was over 5. Anti-HIV activity was also observed in cultures of monocytic lineage U937 cells chronically infected with
HIV.
In Saccharopolyspora erythraea, the genes that govern synthesis of the polyketide portion of the macrolide antibiotic erythromycin
are organized in six repeated units that encode fatty acid synthase (FAS)-like activities. Each repeated unit is designated
a module, and two modules are contained in a single open reading frame. A model for the synthesis of this complex polyketide
is proposed, where each module encodes a functional synthase unit and each synthase unit participates specifically in one
of the six FAS-like elongation steps required for formation of the polyketide. In addition, genetic organization and biochemical
order of events appear to be colinear. Evidence for the model is provided by construction of a selected mutant and by isolation
of a polyketide of predicted structure.
Erythromycin A, a clinically important polyketide antibiotic, is produced by the Gram-positive bacterium Saccharopolyspora erythraea. In an arrangement that seems to be generally true of antibiotic biosynthetic genes in Streptomyces and related bacteria like S. erythraea, the ery genes encoding the biosynthetic pathway to erythromycin are clustered around the gene (ermE) that confers self-resistance on S. erythraea. The aglycone core of erythromycin A is derived from one propionyl-CoA and six methylmalonyl-CoA units, which are incorporated head-to-tail into the growing polyketide chain, in a process similar to that of fatty-acid biosynthesis, to generate a macrolide intermediate, 6-deoxyerythronolide B. 6-Deoxyerythronolide B is converted into erythromycin A through the action of specific hydroxylases, glycosyltransferases and a methyltransferase. We report here the analysis of about 10 kilobases of DNA from S. erythraea, cloned by chromosome 'walking' outwards from the erythromycin-resistance determinant ermE, and previously shown to be essential for erythromycin biosynthesis. Partial sequencing of this region indicates that it encodes the synthase. Our results confirm this, and reveal a novel organization of the erythromycin-producing polyketide synthase, which provides further insight into the mechanism of chain assembly.
The biosynthesis of monensin by Streptomyces cinnamonensis was studied by using 14C-labeled glucose, acetate, propionate, butyrate, and methionine. The results indicated that the antibiotic is synthesized
from five acetate, seven propionate, and one butyrate molecules. The o-methyl group of monensin is derived from methionine, whereas the terminal hydroxymethyl group is incorporated from acetate.
Complexes of monovalent metal cations with the antibiotics monensins, nigericin, and dianemycin have been shown by X-ray crystallography to be functionally very similar. Differences in cation specificities can be attributed to differences in size and flexibility of the antibiotic ligands. Strong similarities between the three antibiotics suggest a common biosynthetic mechanism.
Three antibiotic resistance gene cassettes, derived from the omega interposon (Prentki and Krisch (1984) Gene 29, 303-313) were constructed. These cassettes carry different antibiotic resistance genes, conferring resistance to geneticin, hygromycin or viomycin, flanked by short inverted repeats containing transcription and translation termination signals and synthetic polylinkers. These cassettes were designated omega aac, omega hyg and omega vph. Resistance phenotypes conferred by these constructions are selectable in E. coli and Streptomyces. These cassettes can be used for insertional mutagenesis or for vector construction.
Polyketides are important compounds with antibiotic and anticancer activities. Several modular polyketide synthases (PKSs) contain a terminal thioesterase (TE) domain probably responsible for the release and concomitant cyclization of the fully processed polyketide chain. Because the TE domain influences qualitative aspects of product formation by engineered PKSs, its mechanism and specificity are of considerable interest.
The TE domain of the 6-deoxyerythronolide B synthase was overexpressed in Escherichia coli. When tested against a set of N-acetyl cysteamine thioesters the TE domain did not act as a cyclase, but showed significant hydrolytic specificity towards substrates that mimic important features of its natural substrate. Also the overall rate of polyketide chain release was strongly enhanced by a covalent connection between the TE domain and the terminal PKS module (by as much as 100-fold compared with separate TE and PKS 'domains').
The inability of the TE domain alone to catalyze cyclization suggests that macrocycle formation results from the combined action of the TE domain and a PKS module. The chain-length and stereochemical preferences of the TE domain might be relevant in the design and engineered biosynthesis of certain novel polyketides. Our results also suggest that the TE domain might loop back to catalyze the release of polyketide chains from both terminal and pre-terminal modules, which may explain the ability of certain naturally occurring PKSs, such as the picromycin synthase, to generate both 12-membered and 14-membered macrolide antibiotics.
The structural basis for the striking stereochemical discrimination among triketide analogs has been investigated by incubating a series of N-acetyl cysteamine (-SNAC) esters of unsaturated triketides with DEBS module 2+TE. The triketide analogs were first screened under a standard set of short-term incubation conditions in the presence of the extender substrate methylmalonyl-CoA and NADPH. For those triketide analogs that served as substrates for module 2+TE, the relative specificity, represented by the k(cat)/K(M) values, was quantitated. Triketide diastereomers that were converted in precursor-directed biosynthesis experiments to unsaturated 16-membered ring macrolides by DEBS(KS1(0)) were good to excellent substrates for DEBS module 2+TE, whereas analogs that were converted to the 14-membered ring analogs of 10,11-dehydro-6-deoxyerythronolide B by DEBS(KS1(0)) were not turned over at all by module 2+TE.
The thioesterase (TE) domain of the methymycin/picromycin synthase (PICS) was functionally expressed in Escherichia coli, and the optimal N-terminal boundary of the recombinant TE was determined. A series of diketide-N-acetylcysteamine (SNAC) thioesters were tested as substrates. PICS TE showed a strong preference for the 2-methyl-3-ketopentanoyl-SNAC substrate 5 over the stereoisomers of the reduced diketides 1-4, with an approximately 1.6:1 preference for the (2R,3S)-2-methyl-3-hydroxy diastereomer 2 over the (2S,3R)-diketide 1. The closely related DEBS TE, the thioesterase from the 6-deoxyerythronolide B synthase, showed a more marked 4.4:1 preference for 2 over 1, with only a slightly greater preference for the 3-ketoacyl-SNAC substrate 5. The roles of several active site residues in PICS TE were examined by site-directed mutagenesis. Serine 148, which is part of the apparent catalytic triad consisting of S148, H268, and D176, was found to be essential for thioesterase activity, while replacement of D176 with asparagine (D176N) gave a mutant thioesterase that retained substantial, albeit reduced, hydrolytic activity toward diketide-SNAC substrates. Mutation of E187 and R191, each of which is thought to play a role in substrate binding, had only minor effects on the relative specificity for diketide substrates 1, 2, and 5. Finally, when PICS TE was fused to the C-terminus of DEBS module 3, the resultant chimeric protein converted diketide 1 with methylmalonyl-CoA to triketide ketolactone 6 with improved catalytic efficiency compared to that of the previously developed DEBS module 3-(DEBS)TE construct.
Cloning of polyether polyketide synthase (PKS) genes for salinomycin biosynthesis was attempted from Streptomyces albus. Seven beta-ketoacyl synthase (KS) core regions were obtained by PCR amplification using primers designed based on the conserved KS domains of type I PKSs. Using the KS fragment as a probe, screening of an S. albus genomic DNA library was carried out by colony hybridization. From the positive cosmid clone isolated, a 4.5-kbBamHI fragment was subcloned and sequenced. It showed high homology with bacterial type I PKSs and was deduced to code for KS, malonyl transferase, and ketoreductase motifs. By gene disruption with this 4.5-kb BamHI fragment, the cloned gene was shown to be a part of the salinomycin biosynthetic gene cluster of S. albus.
The analysis of a candidate biosynthetic gene cluster (97 kbp) for the polyether ionophore monensin from Streptomyces cinnamonensis has revealed a modular polyketide synthase composed of eight separate multienzyme subunits housing a total of 12 extension modules, and flanked by numerous other genes for which a plausible function in monensin biosynthesis can be ascribed. Deletion of essentially all these clustered genes specifically abolished monensin production, while overexpression in S. cinnamonensis of the putative pathway-specific regulatory gene monR led to a fivefold increase in monensin production. Experimental support is presented for a recently-proposed mechanism, for oxidative cyclization of a linear polyketide intermediate, involving four enzymes, the products of monBI, monBII, monCI and monCII. In frame deletion of either of the individual genes monCII (encoding a putative cyclase) or monBII (encoding a putative novel isomerase) specifically abolished monensin production. Also, heterologous expression of monCI, encoding a flavin-linked epoxidase, in S. coelicolor was shown to significantly increase the ability of S. coelicolor to epoxidize linalool, a model substrate for the presumed linear polyketide intermediate in monensin biosynthesis.
Details zu einer Biosynthese: Die E-Formen eines Tetra- und eines Pentaketids sind Zwischenstufen in der Biosynthese des antibiotischen Ionophors Monensin A (siehe Struktur) – das konnte durch Einbringen von Hybrid-Polyketid-Synthasen in Streptomyces cinnamonensis nachgewiesen werden. Die trans-konfigurierte Doppelbindung in den Zwischenstufen ist eine Zielstruktur der Epoxidase, die die oxidative Cyclisierung induziert.
A type I PKS gene probe obtained from RAPB of the rapamycin producer Streptomyces hygroscopicus, strongly hybridised to 92 out of 1120 cosmids from a genomic library of the elaiophylin-producing strain Streptomyces sp. DSM4137. Partial cosmid sequencing suggested the presence of 10 separate sequences encoding type I PKS genes. One entire DNA sequence was obtained and found exactly to match the gene organisation expected for the biosynthesis of the unusual macrodiolide polyketide elaiophylin. The putative elaiophylin gene cluster contains five large open-reading frames encoding typical modular polyketide synthases, which together catalyse the synthesis of the octaketide monomer of elaiophylin. Other genes were identified that would be required for provision of the ethylmalonate extender unit, for the synthesis and attachment of 2-deoxy-L-fucose and in regulation, or in export of the product. Immediately adjacent to the putative elaiophylin biosynthetic gene cluster is a 30-kbp region containing the gene for adenosylcobalamin-dependent methylmalonyl CoA mutase and also genes involved in the biosynthesis of the cobalamin cofactor. Analysis of the latter gene set confirms the view that cbiD of the anaerobic pathway and cobF in the aerobic pathway catalyse the same methylation of precorrin-5. The proximity of these genes to the putative elaiophylin gene cluster can best be rationalised if in this organism succinyl-CoA is a significant source of the methylmalonate units for complex polyketide biosynthesis.
(Chemical Equation Presented) Evidence for the intermediate in the polyether biosynthesis of the ionophore antibiotic monensin A has been obtained. A tridecaketide E,E,E-triene (see formula) has been isolated by using mutant strains of Streptomyces cinnamonensis. Characterization of this intermediate allows the likely biosynthetic route to monensin to be discriminated.
The recombinant thioesterase (TE) domain of the picromycin/methymycin synthase (PICS) catalyzes the macrolactonization of 3, the N-acetylcysteamine thioester of seco-10-deoxymethynolide to generate 10-deoxymethynolide (1) with high efficiency. By contrast, 4, the 7-dihydro derivative of seco-thioester 3, undergoes exclusive hydrolysis by PICS TE to seco-acid 5. The recombinant TE domain of 6-deoxyerythronolide B synthase (DEBS TE) shows the same reaction specificity as PICS TE, but with significantly lower activity.
The alpha-amino acid ester hydrolase (AEH) from Acetobacter turbidans is a bacterial enzyme catalyzing the hydrolysis and synthesis of beta-lactam antibiotics. The crystal structures of the native enzyme, both unliganded and in complex with the hydrolysis product D-phenylglycine are reported, as well as the structures of an inactive mutant (S205A) complexed with the substrate ampicillin, and an active site mutant (Y206A) with an increased tendency to catalyze antibiotic production rather than hydrolysis. The structure of the native enzyme shows an acyl binding pocket, in which D-phenylglycine binds, and an additional space that is large enough to accommodate the beta-lactam moiety of an antibiotic. In the S205A mutant, ampicillin binds in this pocket in a non-productive manner, making extensive contacts with the side chain of Tyr(112), which also participates in oxyanion hole formation. In the Y206A mutant, the Tyr(112) side chain has moved with its hydroxyl group toward the catalytic serine. Because this changes the properties of the beta-lactam binding site, this could explain the increased beta-lactam transferase activity of this mutant.
Ionophoric polyethers are produced by the exquisitely stereoselective oxidative cyclization of a linear polyketide, probably via a triepoxide intermediate. We report here that deletion of either or both of the monBI and monBII genes from the monensin biosynthetic gene cluster gave strains that produced, in place of monensins A and B, a mixture of C-3-demethylmonensins and a number of minor components, including C-9-epi-monensin A. All the minor components were efficiently converted into monensins by subsequent acid treatment. These data strongly suggest that epoxide ring opening and concomitant polyether ring formation are catalyzed by the MonB enzymes, rather than by the enzyme MonCII as previously thought. Consistent with this, homology modeling shows that the structure of MonB-type enzymes closely resembles the recently determined structure of limonene-1,2-epoxide hydrolase from Rhodococcus erythropolis.
A 45 kb DNA sequencing analysis from Streptomyces hygroscopicus 5008 involved in validamycin A (VAL-A) biosynthesis revealed 16 structural genes, 2 regulatory genes, 5 genes related transport, transposition/integration or tellurium resistance; another 4 genes had no obvious identity. The VAL-A biosynthetic pathway was proposed, with assignment of the required genetic functions confined to the sequenced region. A cluster of eight reassembled genes was found to support VAL-A synthesis in a heterologous host, S. lividans 1326. In vivo inactivation of the putative glycosyltransferase gene (valG) abolished the final attachment of glucose for VAL production and resulted in accumulation of the VAL-A precursor, validoxylamine, while the normal production of VAL-A could be restored by complementation with valG. The role of valG in the glycosylation of validoxylamine to VAL-A was demonstrated in vitro by enzymatic assay.
Polyether ionophores, such as monensin A, are known to be biosynthesised, like many other antibiotic polyketides, on giant modular polyketide synthases (PKSs), but the intermediates and enzymes involved in the subsequent steps of oxidative cyclisation remain undefined. In particular there has been no agreement on the mechanism and timing of the final polyketide chain release. We now report evidence that MonCII from the monensin biosynthetic gene cluster in Streptomyces cinnamonensis, which was previously thought to be an epoxide hydrolase, is a novel thioesterase that belongs to the alpha/beta-hydrolase structural family and might catalyse this step. Purified recombinant MonCII was found to hydrolyse several thioester substrates, including an N-acetylcysteamine thioester derivative of monensin A. Further, incubation with a hallmark inhibitor of such enzymes, phenylmethanesulfonyl fluoride, led to inhibition of the thioesterase activity and to the accumulation of an acylated form of MonCII. These findings require a reassessment of the role of other enzymes implicated in the late stages of polyether ionophore biosynthesis.