On the biosynthetic origins of the hydrogen atoms in the macrotetrolide antibiotics: and their mode of assembly catalysed by a nonactin polyketide synthase

Ashworth, Doreen M.; Clark, Carole A.

doi:10.1039/p19890001461

Cited by 27 publications

(28 citation statements)

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“…An equivalent role could be proposed for NonL, although the substrate for NonL is yet to be determined. The fact that the addition of ATP has no effect on the in vitro synthesis of macrotetrolides from NA and its homologs argues against the notion that the latter acids are activated into acyl CoAs that involve a CoA ligase like NonL before incorporation into macrotetrolides (2,36). NonD shows low but significant end-to-end similarity to several glutaryl 7-amino cephalosporanic acid (7-ACA) acylases, including the ones from Bacillus subtilis (28.4% identity and 40.8% similarity) (1) and from Zymomonas mobilis (19% identity and 27.3% similarity) (GenBank accession number AF124757), as well as to several proteins of unknown function, such as the hypothetical protein Rv2800 from Mycobacterium tuberculosis (31% identity and 40% similarity; GenBank accession number Z81331) and the hypothetical protein from a Synechocystis sp.…”

Section: Resultsmentioning

confidence: 96%

“…Sequential and stereospecific oligomerization of (ϩ)-and (Ϫ)-NA into NON was supported by the isolation of both enantiomers of NA and the dimeric NA (17), which could be viewed as derailed biosynthetic intermediates from the enzyme complex. In vitro synthesis of macrotetrolides from NA and its homologs was also demonstrated in the presence of an enzyme preparation that appeared to be a heteromeric complex with an apparent molecular weight of 350,000 (2,36).…”

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confidence: 99%

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Genetic Localization and Molecular Characterization of the nonS Gene Required for Macrotetrolide Biosynthesis in Streptomyces griseus DSM40695

Smith

Xiang

Shen

2000

Antimicrob Agents Chemother

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The macrotetrolides are a family of cyclic polyethers derived from tetramerization, in a stereospecific fashion, of the enantiomeric nonactic acid (NA) and its homologs. Isotope labeling experiments established that NA is of polyketide origin, and biochemical investigations demonstrated that 2-methyl-6,8-dihydroxynon-2E-enoic acid can be converted into NA by a cell-free preparation from Streptomyces lividans that expresses nonS. These results lead to the hypothesis that macrotetrolide biosynthesis involves a pair of enantiospecific polyketide pathways. In this work, a 55-kb contiguous DNA region was cloned from Streptomyces griseus DSM40695, a 6.3-kb fragment of which was sequenced to reveal five open reading frames, including the previously reported nonR and nonS genes. Inactivation of nonS in vivo completely abolished macrotetrolide production. Complementation of the nonS mutant by the expression of nonS in trans fully restored its macrotetrolide production ability, with a distribution of individual macrotetrolides similar to that for the wild-type producer. In contrast, fermentation of the nonS mutant in the presence of exogenous (؎)-NA resulted in the production of nonactin, monactin, and dinactin but not in the production of trinactin and tetranactin. These results prove the direct involvement of nonS in macrotetrolide biosynthesis. The difference in macrotetrolide production between in vivo complementation of the nonS mutant by the plasmid-borne nonS gene and fermentation of the nonS mutant in the presence of exogenously added (؎)-NA suggests that NonS catalyzes the formation of (؊)-NA and its homologs, supporting the existence of a pair of enantiospecific polyketide pathways for macrotetrolide biosynthesis in S. griseus. The latter should provide a model that can be used to study the mechanism by which polyketide synthase controls stereochemistry during polyketide biosynthesis.

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Section: Resultsmentioning

confidence: 96%

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confidence: 99%

Genetic Localization and Molecular Characterization of the nonS Gene Required for Macrotetrolide Biosynthesis in Streptomyces griseus DSM40695

Smith

Xiang

Shen

2000

Antimicrob Agents Chemother

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“…Methyl (±)-nonactate was obtained through methanolysis of nonactin as described by Ashworth et al .,13 adapted for a large scale as described by Dinges et al 26…”

Section: Methodsmentioning

confidence: 99%

Alternating Pattern of Stereochemistry in the Nonactin Macrocycle Is Required for Antibacterial Activity and Efficient Ion Binding

et al. 2009

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Nonactin is a polyketide antibiotic produced by Streptomyces griseus ETH A7796. It is active against a range of Gram positive organisms, acting as an ionophore that has a high selectivity for K + ions. Among the polyketides, nonactin has a unique structure. It is a 32-membered, cyclic tetraester generated from four monomers of nonactic acid, two of which are (+)-nonactic acid and two of which are (−)-nonactic acid. The monomers are arranged (+)-(−)-(+)-(−) in the macrocycle so that nonactin has S4 symmetry and is achiral. As the structure of nonactin is unique, so is the biosynthesis of the compound. There is much evidence that after an early achiral intermediate, there are two mirror image biosynthesis pathways, one for the synthesis of each enantiomer of nonactic acid. Emerging evidence suggests that the two pathways arose from gene duplication followed by divergent evolution. The central question, therefore, raised by both the structure and the biosynthesis of nonactin concerns the benefit, if any, to the producing organism in initially selecting for, and then maintaining, two independent pathways for nonactic acid synthesis and for generating only the achiral diastereoisomer of nonactin.To understand why achiral nonactin is the naturally generated diastereoisomer we prepared through synthesis two alternate diastereoisomers of nonactin, one prepared solely from (+)-nonactic acid and one prepared solely from (−)-nonactic acid, referred to here as 'all-(+)-nonactin' and 'all-(−)-nonactin' respectively. Both all-(+)-nonactin and all-(−)-nonactin were more than 500-fold less active against Gram positive organisms than nonactin confirming that the biosynthesis of both nonactic acid enantiomers, and their incorporation into nonactin, is necessary for biological activity. To understand the lack of antibacterial activity we used isothermal calorimetry to measure the association constant K a , ΔG, ΔH and ΔS of formation for the K + , Na + and NH 4 + complexes of nonactin and all-(−)-nonactin. The natural diastereoisomer had a high selectivity for K + to which it bound approximately 880-fold better than all-(−)-nonactin. We used a picrate partitioning assay to confirm that all-(−)-nonactin, unlike nonactin, could not partition K + ions into organic solvent. To complement the thermodynamic data we used a simple experimental model to mimic ion transport. Using two concentric glass tubes we arranged for two aqueous samples to be separated by a CHCl 3 layer. By following the co-transport of picrate anion from one aqueous layer to the other, through the organic phase, we were able to measure the rates of K + transport that were facilitated by the ionophores. Whereas nonactin allowed for efficient K + transport, all-(−)-nonactin was completely nigel.priestley@umontana.edu. Supporting Information Available: 1 H and 13 C NMR spectra and mass spectra for compounds 8 to 17. This material is available free of charge via the Internet at

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“…To assess the incorporation of compound 11 into new macrotetrolide analogs, two fermentative cultures of S. griseus ETH A7796 were prepared from a single vegetative culture and grown for 48 hours under standard conditions 19. At 48 hours after inoculation of the fermentative culture (50 mL), 56 mg of 11 (56 mg in 0.5 mL of ethanol) was added to one culture; a blank sample (0.5 mL ethanol) was added to the equivalent control culture.…”

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confidence: 99%

“…At 48 hours after inoculation of the fermentative culture (50 mL), 56 mg of 11 (56 mg in 0.5 mL of ethanol) was added to one culture; a blank sample (0.5 mL ethanol) was added to the equivalent control culture. The cultures were allowed to grow for an additional 96 hours and the macrotetrolide mixture was isolated according to standard protocols 19. Analysis of the macrotetrolide mixtures by HPLC20 and LC-MS (TOF)21 showed an unexpected drastic reduction in macrotetrolide production in the fed culture compared to the control.…”

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confidence: 99%

Nonactin biosynthesis: Setting limits on what can be achieved with precursor-directed biosynthesis

Kusche

Phillips

Priestley

2009

Bioorganic & Medicinal Chemistry Letters

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Nonactin, produced by Streptomyces griseus ETH A7796, is a macrotetrolide assembled from nonactic acid. It is an effective inhibitor of drug efflux in multidrug resistant erythroleukemia K562 cells at sub-toxic concentrations and has been shown to possess both antibacterial and antitumor activity. As total synthesis is impractical for the generation of nonactin analogs we have studied precursor-directed biosynthesis as an alternative as it is known that nonactic acid can serve as a nonactin precursor in vivo. To determine the scope of the approach we prepared and evaluated a furan-based nonactic acid derivative, 11. Although no new nonactin analogs were detected when 11 was administered to S. griseus fermentative cultures, a significant inhibition of nonactin biosynthesis was noted (IC 50 ~ 100 μM). Cell mass, nonactic acid production and the generation of other secondary metabolites in the culture were unaffected by 11 demonstrating that 11 selectively inhibited the assembly of nonactin from nonactic acid. While we were unable to generate new nonactin analogs we have discovered, however, a useful inhibitor that we can use to probe the mechanism of nonactin assembly with the ultimate goal of developing more successful precursordirected biosynthesis transformations.Streptomyces griseus subsp. griseus ETH A7796 (DSM40695) makes a series of ionophore antibiotics known as the macrotetrolides (Figure 1). 1 Nonactin, the prototypical macrotetrolide (1) is assembled from two monomers of (-)-nonactic acid and two monomers of (+)-nonactic acid assembled (+)-(-)-(+)-(-) in a head-to-tail manner into a 32-membered macrocycle. Nonactin has both antibiotic and anticancer properties 2 and has been shown to be an inhibitor of drug efflux in multiple drug resistant cancers.3 The natural macrotetrolide homologues produced by S. griseus show a wide range potency with the minimum inhibitory concentration of 1 being an order of magnitude greater than that of dinactin (3) against Staphylococcus aureus and Mycobacterium bovis, a difference that is related to the stability constants of their respective Na + and K + complexes. 1,2,4 Nonactin is far too hydrophobic and insufficiently soluble to be an effective therapeutic. 5 The development of therapeutics based upon nonactin, therefore, will be dependent upon being able to make non-natural analogs in a direct and efficient manner. The total synthesis of nonactin has been achieved by a number of groups 6-9 as has the synthesis of nonactic acid. 10,11 The total synthesis of nonactin analogs is complicated as both enantiomers of nonactic acid, and its analogs, are required as is the sequential construction of a linear tetraester prior to a © 2008 Elsevier Ltd. All rights reserved. Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published ...

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On the biosynthetic origins of the hydrogen atoms in the macrotetrolide antibiotics: and their mode of assembly catalysed by a nonactin polyketide synthase

Cited by 27 publications

References 1 publication

Genetic Localization and Molecular Characterization of the nonS Gene Required for Macrotetrolide Biosynthesis in Streptomyces griseus DSM40695

Genetic Localization and Molecular Characterization of the nonS Gene Required for Macrotetrolide Biosynthesis in Streptomyces griseus DSM40695

Alternating Pattern of Stereochemistry in the Nonactin Macrocycle Is Required for Antibacterial Activity and Efficient Ion Binding

Nonactin biosynthesis: Setting limits on what can be achieved with precursor-directed biosynthesis

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