Syntheses of Conjugated Pyrones for the Enzymatic Assay of Lovastatin Nonaketide Synthase, an Iterative Polyketide Synthase

Burr, Douglas A.; Chen, Xuan B; Vederas, John C.

doi:10.1021/ol062763i

Cited by 16 publications

(9 citation statements)

References 14 publications

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“…We also observed increase in the λ max of the α-pyrones, which is indicative of increased levels of conjugation. These observations, combined with inference from biosynthetic logic and comparison to the LovB system,2,11,24 led us to propose the structures of 11 and 12 as shown in Figure 3, of which the two compounds contain two and three α-methyl modifications, respectively. The failure of ApdA to synthesize tetramic acids containing unsaturated polyketide portions is evident of the substrate specificity of the NRPS module towards incorrectly tailored polyketide acyl substrates.…”

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confidence: 97%

Analysis of Intact and Dissected Fungal Polyketide Synthase-Nonribosomal Peptide Synthetase in Vitro and in Saccharomyces cerevisiae

et al. 2010

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The widely found fungal iterative PKS-NRPS hybrid megasynthetases are highly programmed biosynthetic machines involved in the synthesis of 3-acyltetramic acids and related natural products. In vitro analysis of iterative PKS-NRPS have been hampered by the difficulties associated with obtaining pure and functional forms of these large enzymes (>400 kDa). We successfully expressed Aspergillus nidulans aspyridone synthetase (ApdA) from an engineered Saccharomyces cerevisiae strain. The complete functions of ApdA and its enoylreductase partner ApdC are reconstituted in vitro and in S. cerevisiae with the production of preaspyridone 7. The programming rules of both the PKS and NRPS modules were then examined in vitro. The key interaction between the PKS and the NRPS was dissected and reconstituted in trans by using standalone modules. Analogs of 7 were synthesized through heterologous combinations of PKS and NRPS modules from different sources. Our results represent the largest, multidomain enzyme reconstituted to date; and offer new opportunities for engineered biosynthesis of fungal natural products.Filamentous fungi produce a diverse array of bioactive secondary metabolites. Among the small molecule natural products, polyketides and nonribosomal peptides are synthesized by multidomain enzymes such as polyketide synthases (PKSs) and nonribosomal peptide synthetases (NRPSs), respectively. The enzymology and biochemical properties of these mega-enzymes are highly complex and are different from the well-studied bacterial PKSs and NRPSs.1 In particular, the highly-reducing PKSs (HR-PKSs), in which individual domains are programmed to function in different permutations during the iterative process of chain elongation, are particularly enigmatic as exemplified by the lovastatin nonaketide synthase LovB.2 Notwithstanding the complexity of HR-PKSs, an even more impressive biosynthetic machinery that is found in nearly all filamentous fungi is the PKS-NRPS hybrid, in which a single module of NRPS is translationally fused to the C-terminus of a HR-PKS. A typical PKS-NRPS hybrid contains ~ 10 catalytic domains; exceeds 400 kDa as a standalone protein; and synthesizes acyltetramic acid (3-acyl-pyrrolidine-2,4-dione) as the product. The tetramic acids are further tailored into complex natural products, such as fusarin C,3 equisetin,4 aspyridone A 1 , 5 pseurotin A,6 cyclopiazonic acid 2 , 7 chaetoglobosin,8 tenellin 3 , 9 etc ( Figure 1A). The widespread presence of fungal PKS-NRPS machineries,10 along with their unique biosynthetic capabilities and highly evolved yitang@ucla.edu. Supporting Information Available: Experimental details and spectroscopic information are available free of charge via the internet at http://pubs.acs.org. ApdA is the PKS-NRPS that is involved in the synthesis of 1 ( Figure 1A). The role of ApdA was discovered via artificial transcription activation of the cryptic apd gene cluster in Aspergillus nidulans.5 The HR-PKS module of the enzyme, which consists of ketosynthase (KS), malonyl-CoA:ACP tr...

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Analysis of Intact and Dissected Fungal Polyketide Synthase-Nonribosomal Peptide Synthetase in Vitro and in Saccharomyces cerevisiae

et al. 2010

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“…After coupling of the first two fragments, we proceeded to treat phosphonate 19 with n -BuLi and directly added the ozonolysis product of 18 to the resulting stabilized phosphorus ylide (Scheme ). This one-pot protocol yielded the expected vinyl pyrone 20 in moderate amounts and with complete ( E )-selectivity.…”

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confidence: 99%

Biomimetic Approach Toward Enterocin and Deoxyenterocin

2018

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Enterocin (vulgamycin) is a structurally remarkable natural product with significant antibiotic activity. The synthesis of a linear polyketide resembling a biosynthetic precursor was achieved using an unusual acyloin reaction. A diazo group was introduced as a protecting group for an enolizable ketone. We were unable to bring about the envisioned biomimetic aldol addition cascade and gained insights into the feasibility of this process by DFT calculations. As an alternative approach to enterocin, we developed a Cu-catalyzed intramolecular cyclopropanation followed by a MgI2-induced fragmentation to install the 2-oxabicyclo[3.3.1]nonane core of the natural product.

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“…[52][53][54] Analysis of the detailed mechanisms of the expanding number of fungal HR PKS enzymes was initially very difficult because the amounts of enzyme produced are oen quite low as are the turnover rates. 46,55 This problem has now been solved in many cases by improved heterologous expression of the PKS proteins. In the case of lovastatin, our collaborator Yi Tang has used an engineered yeast strain developed by Nancy DaSilva to enable production of larger amounts of pure LovB and LovC proteins for mechanistic studies.…”

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confidence: 99%

Explorations of fungal biosynthesis of reduced polyketides – a personal viewpoint

Vederas

2014

Nat. Prod. Rep.

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This viewpoint on biosynthesis of reduced polyketides in fungi traces evolution of the research area over more than 4 decades. It is a companion to the related articles by two personal and scientific friends with whom there has been free exchange of ideas for over 30 years. Beginning with very rudimentary knowledge about assembly of such natural products, developments using stable isotope labelling and subsequently identification of biosynthetic genes, led to understanding of the processive nature of polyketide formation. Recent expression and isolation of fungal iterative polyketide synthase enzymes has enabled more detailed exploration of the mechanisms of these fascinating molecular machines.

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Syntheses of Conjugated Pyrones for the Enzymatic Assay of Lovastatin Nonaketide Synthase, an Iterative Polyketide Synthase

Cited by 16 publications

References 14 publications

Analysis of Intact and Dissected Fungal Polyketide Synthase-Nonribosomal Peptide Synthetase in Vitro and in Saccharomyces cerevisiae

Analysis of Intact and Dissected Fungal Polyketide Synthase-Nonribosomal Peptide Synthetase in Vitro and in Saccharomyces cerevisiae

Biomimetic Approach Toward Enterocin and Deoxyenterocin

Explorations of fungal biosynthesis of reduced polyketides – a personal viewpoint

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