Biocatalytic Synthesis of Pikromycin, Methymycin, Neomethymycin, Novamethymycin, and Ketomethymycin

Hansen, Douglas A.; Rath, Christopher M.; Eisman, Eli B.; Narayan, Alison R. H.; Kittendorf, Jeffrey D.; Mortison, Jonathan D.; Yoon, Yeo Joon; Sherman, David H.

doi:10.1021/ja404134f

Cited by 52 publications

(73 citation statements)

References 58 publications

(94 reference statements)

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“…The reaction chamber also protects the module ACP from other reactants. For example, to generate the pentaketide-KS state of PikAIII (Figure 4b), the pentaketide intermediate was delivered as the thiophenol-activated mimic [53] of pentaketide-ACP 4 . The small-molecule mimic reacted with the PikAIII KS catalytic cysteine, but no adduct at the PikAIII ACP 5 phosphopantetheine thiol group was detected by mass spectrometry.…”

Section: Overall Architecturementioning

confidence: 99%

Architecture of the polyketide synthase module: surprises from electron cryo-microscopy

Smith

Skiniotis

Sherman

2015

Current Opinion in Structural Biology

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Modular polyketide synthases produce a vast array of bioactive molecules that are the basis of many highly valued pharmaceuticals. The biosynthesis of these compounds is based on ordered assembly lines of multi-domain modules, each extending and modifying a specific chain-elongation intermediate before transfer to the next module for further processing. The first 3D structures of a full polyketide synthase module in different functional states were obtained recently by electron cryo-microscopy. The unexpected module architecture revealed a striking evolutionary divergence of the polyketide synthase compared to its metazoan fatty acid synthase homolog, as well as remarkable conformational rearrangements dependent on its biochemical state during the full catalytic cycle. The design and dynamics of the module are highly optimized for both catalysis and fidelity in the construction of complex, biologically active natural products.

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Section: Overall Architecturementioning

confidence: 99%

Architecture of the polyketide synthase module: surprises from electron cryo-microscopy

Smith

Skiniotis

Sherman

2015

Current Opinion in Structural Biology

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“…The present results have therefore verified the success of this study for accessing novel chemical space. A number of methods based on PKS engineering have been developed and have produced diverse unnatural polyketide molecules [37][38][39][40][41][42] . These methods are undoubtedly powerful enough to generate analogues of structurally complex metabolites, although they do have some weakness regarding the simultaneous expansion of molecular frameworks.…”

Section: Discussionmentioning

confidence: 99%

Use of a biosynthetic intermediate to explore the chemical diversity of pseudo-natural fungal polyketides

et al. 2015

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The structural complexity and diversity of natural products make them attractive sources for potential drug discovery, with their characteristics being derived from the multi-step combination of enzymatic and non-enzymatic conversions of intermediates in each biosynthetic pathway. Intermediates that exhibit multipotent behaviour have great potential for use as starting points in diversity-oriented synthesis. Inspired by the biosynthetic pathways that form complex metabolites from simple intermediates, we developed a semi-synthetic process that combines heterologous biosynthesis and artificial diversification. The heterologous biosynthesis of fungal polyketide intermediates led to the isolation of novel oligomers and provided evidence for ortho-quinonemethide equivalency in their isochromene form. The intrinsic reactivity of the isochromene polyketide enabled us to access various new chemical entities by modifying and remodelling the polyketide core and through coupling with indole molecules. We thus succeeded in generating exceptionally diverse pseudo-natural polyketides through this process and demonstrated an advanced method of using biosynthetic intermediates.

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“…PikA4 docking domain was PCR amplified from pET24b-pikA4 33 using the primers Pik4dd_F and Pik4dd_R, and subsequently digested with the restriction enzyme Xba I. pET24a-Ery6TE was double digested with Xba I and Eco RV, and ligated with the digested PikA4 docking domain.…”

Section: Methodsmentioning

confidence: 99%

Inversion of Extender Unit Selectivity in the Erythromycin Polyketide Synthase by Acyltransferase Domain Engineering

et al. 2016

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Acyltransferase (AT) domains of polyketide synthases (PKSs) select extender units for incorporation into polyketides and dictate large portions of the structures of clinically relevant natural products. Accordingly, there is significant interest in engineering the substrate specificity of PKS ATs in order to site-selectively manipulate polyketide structure. However, previous attempts to engineer ATs have yielded mutant PKSs with relaxed extender unit specificity, rather than an inversion of selectivity from one substrate to another. Here, by directly screening the extender unit selectivity of mutants from active site saturation libraries of an AT from the prototypical PKS, 6-deoxyerythronolide B synthase, a set of single amino acid substitutions was discovered that dramatically impact the selectivity of the PKS with only modest reductions of product yields. One particular substitution (Tyr189Arg) inverted the selectivity of the wild-type PKS from its natural substrate towards a non-natural alkynyl-modified extender unit while maintaining more than twice the activity of the wild-type PKS with its natural substrate. The strategy and mutations described herein form a platform for combinatorial biosynthesis of site-selectively modified polyketide analogues that are modified with non-natural and non-native chemical functionality.

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Biocatalytic Synthesis of Pikromycin, Methymycin, Neomethymycin, Novamethymycin, and Ketomethymycin

Cited by 52 publications

References 58 publications

Architecture of the polyketide synthase module: surprises from electron cryo-microscopy

Architecture of the polyketide synthase module: surprises from electron cryo-microscopy

Use of a biosynthetic intermediate to explore the chemical diversity of pseudo-natural fungal polyketides

Inversion of Extender Unit Selectivity in the Erythromycin Polyketide Synthase by Acyltransferase Domain Engineering

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