Peter F Leadlay

University of Cambridge, Cambridge, England, United Kingdom

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Publications (236)1395 Total impact

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    ABSTRACT: Chemical probes capable of reacting with KS (ketosynthase)-bound biosynthetic intermediates were utilized for the investigation of the model type I iterative polyketide synthase 6-methylsalicylic acid synthase (6-MSAS) in vivo and in vitro. From the fermentation of fungal and bacterial 6-MSAS hosts in the presence of chain termination probes, a full range of biosynthetic intermediates was isolated and characterized for the first time. Meanwhile, in vitro studies of recombinant 6-MSA synthases with both nonhydrolyzable and hydrolyzable substrate mimics have provided additional insights into substrate recognition, providing the basis for further exploration of the enzyme catalytic activities.
    No preview · Article · Feb 2016 · Angewandte Chemie International Edition
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    ABSTRACT: Thiolactomycin (TLM) is a thiotetronate antibiotic that selectively targets bacterial fatty acid biosynthesis through inhibition of the b-ketoacyl-acyl carrier protein synthases (KASI/II) that catalyse chain elongation on the type II (dissociated) fatty acid synthase. It has proved effective in in vivo infection models of Mycobacterium tuberculosis and continues to attract interest as a template for drug discovery. We have used a comparative genomics approach to uncover the (hitherto elusive) biosynthetic pathway to TLM and related thiotetronates. Analysis of the whole-genome sequence of Streptomyces olivaceus T ¨ u 3010 producing the more ramified thiotetronate T ¨ u 3010 provided initial evidence that such thiotetronates are assembled by a novel iterative polyketide synthase-nonribosomal peptide synthetase, and revealed the identity of other pathway enzymes, encoded by adjacent genes. Subsequent genome sequencing of three other thiotetronate-producing actinomycetes, including the Lentzea sp. ATCC 31319 that produces TLM, confirmed that near-identical clusters were also present in these genomes. In-frame gene deletion within the cluster for T ¨ u 3010 from Streptomyces thiolactonus NRRL 15439, or within the TLM cluster, led to loss of production of the respective thiotetronate, confirming their identity. Each cluster houses at least one gene encoding a KASI/II enzyme, suggesting plausible mechanisms for self-resistance. A separate genetic locus encodes a cysteine desulfurase and a (thiouridylase-like) sulfur transferase to supply the sulfur atom for thiotetronate ring formation. Transfer of the main T ¨ u 3010 gene cluster (stu gene cluster) into Streptomyces avermitilis led to heterologous production of this thiotetronate, showing that an equivalent sulfur donor can be supplied by this host strain. Mutational analysis of the T ¨ u 3010 and TLM clusters has revealed the unexpected role of a cytochrome P450 enzyme in thiotetronate ring formation. These insights have allowed us to propose a mechanism for sulfur insertion, and have opened the way to engineering of the biosynthesis of TLM and other thiotetronates to produce novel analogues.
    Full-text · Article · Jan 2016 · Chemical Science
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    ABSTRACT: Desertomycin A is an aminopolyol polyketide containing a macrolactone ring. We have proposed that desertomycin A and similar compounds (marginolactones) are formed by polyketide synthases primed not with γ-aminobutanoyl-CoA but with 4-guanidinylbutanoyl-CoA, to avoid facile cyclization of the starter unit. This hypothesis requires that there be a final-stage de-amidination of the corresponding guanidino-substituted natural product, but no enzyme for such a process has been described. We have now identified candidate amidinohydrolase genes within the desertomycin and primycin clusters. Deletion of the putative desertomycin amidinohydrolase gene dstH in Streptomyces macronensis led to the accumulation of desertomycin B, the guanidino form of the antibiotic. Also, purified DstH efficiently catalyzed the in vitro conversion of desertomycin B into the A form. Hence this amidinohydrolase furnishes the missing link in this proposed naturally evolved example of protective-group chemistry.
    No preview · Article · Dec 2015 · Angewandte Chemie International Edition
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    Full-text · Dataset · Oct 2015
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    ABSTRACT: Tetrahydropyran rings are a common feature of complex polyketide natural products, but much remains to be learned about the enzymology of their formation. The enzyme SalBIII from the salinomycin biosynthetic pathway resembles other polyether epoxide hydrolases/cyclases of the MonB family, but SalBIII plays no role in the conventional cascade of ring opening/closing. Mutation in the salBIII gene gave a metabolite in which ring A is not formed. Using this metabolite in vitro as a substrate analogue, SalBIII has been shown to form pyran ring A. We have determined the X-ray crystal structure of SalBIII, and structure-guided mutagenesis of putative active-site residues has identified Asp38 and Asp104 as an essential catalytic dyad. The demonstrated pyran synthase activity of SalBIII further extends the impressive catalytic versatility of a + b barrel fold proteins.
    Full-text · Article · Sep 2015 · Angewandte Chemie International Edition in English
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    ABSTRACT: Conglobatin is an unusual C2-symmetrical macrodiolide from the bacterium Streptomyces conglobatus with promising antitumor activity. Insights into the genes and enzymes that govern both the assembly-line production of the conglobatin polyketide and its dimerization are essential to allow rational alterations to be made to the conglobatin structure. We have used a rapid, direct in vitro cloning method to obtain the entire cluster on a 41-kbp fragment, encoding a modular polyketide synthase assembly line. The cloned cluster directs conglobatin biosynthesis in a heterologous host strain. Using a model substrate to mimic the conglobatin monomer, we also show that the conglobatin cyclase/thioesterase acts iteratively, ligating two monomers head-to-tail then re-binding the dimer product and cyclizing it. Incubation of two different monomers with the cyclase produces hybrid dimers and trimers, providing the first evidence that conglobatin analogs may in future become accessible through engineering of the polyketide synthase. Copyright © 2015 The Authors. Published by Elsevier Ltd.. All rights reserved.
    Preview · Article · Jun 2015 · Chemistry & biology
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    ABSTRACT: Elaiophylin is an unusual C2-symmetric antibiotic macrodiolide produced on a bacterial modular polyketide synthase assembly line. To probe the mechanism and selectivity of diolide formation, we sought to reconstitute ring formation in vitro by using a non-natural substrate. Incubation of recombinant elaiophylin thioesterase/cyclase with a synthetic pentaketide analogue of the presumed monomeric polyketide precursor of elaiophylin, specifically its N-acetylcysteamine thioester, produced a novel 16-membered C2-symmetric macrodiolide. A linear dimeric thioester is an intermediate in ring formation, which indicates iterative use of the thioesterase active site in ligation and subsequent cyclization. Furthermore, the elaiophylin thioesterase acts on a mixture of pentaketide and tetraketide thioesters to give both the symmetric decaketide diolide and the novel asymmetric hybrid nonaketide diolide. Such thioesterases have potential as tools for the in vitro construction of novel diolides.
    No preview · Article · Mar 2015 · Angewandte Chemie
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    ABSTRACT: Elaiophylin is an unusual C2 -symmetric antibiotic macrodiolide produced on a bacterial modular polyketide synthase assembly line. To probe the mechanism and selectivity of diolide formation, we sought to reconstitute ring formation in vitro by using a non-natural substrate. Incubation of recombinant elaiophylin thioesterase/cyclase with a synthetic pentaketide analogue of the presumed monomeric polyketide precursor of elaiophylin, specifically its N-acetylcysteamine thioester, produced a novel 16-membered C2 -symmetric macrodiolide. A linear dimeric thioester is an intermediate in ring formation, which indicates iterative use of the thioesterase active site in ligation and subsequent cyclization. Furthermore, the elaiophylin thioesterase acts on a mixture of pentaketide and tetraketide thioesters to give both the symmetric decaketide diolide and the novel asymmetric hybrid nonaketide diolide. Such thioesterases have potential as tools for the in vitro construction of novel diolides. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.
    No preview · Article · Mar 2015 · Angewandte Chemie International Edition
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    ABSTRACT: Gentamicin C complex is a mixture of aminoglycoside antibiotics used worldwide to treat severe Gram-negative bacterial infections. Despite its clinical importance, the enzymology of its biosynthetic pathway has remained obscure. We report here insights into the four enzyme-catalyzed steps that lead from the first-formed pseudotrisaccharide gentamicin A2 to gentamicin X2, the last common intermediate for all components of the C complex. We have used both targeted mutations of individual genes and reconstitution of portions of the pathway in vitro to show that the secondary alcohol function at C-3″ of A2 is first converted to an amine, catalyzed by the tandem operation of oxidoreductase GenD2 and transaminase GenS2. The amine is then specifically methylated by the S-adenosyl-l-methionine (SAM)-dependent N-methyltransferase GenN to form gentamicin A. Finally, C-methylation at C-4″ to form gentamicin X2 is catalyzed by the radical SAM-dependent and cobalamin-dependent enzyme GenD1. Copyright © 2015 The Authors. Published by Elsevier Ltd.. All rights reserved.
    Full-text · Article · Jan 2015 · Chemistry & Biology
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    ABSTRACT: A library of functionalized chemical probes capable of reacting with ketosynthase-bound biosynthetic intermediates was prepared and utilized to explore in vivo polyketide diversification. Fermentation of ACP mutants of S. lasaliensis in the presence of the probes generated a range of unnatural polyketide derivatives, including novel putative lasalocid A derivatives characterized by variable aryl ketone moieties and linear polyketide chains (bearing alkyne/azide handles and fluorine) flanking the polyether scaffold. By providing direct information on microorganism tolerance and enzyme processing of unnatural malonyl-ACP analogues, as well as on the amenability of unnatural polyketides to further structural modifications, the chemical probes constitute invaluable tools for the development of novel mutasynthesis and synthetic biology.
    Preview · Article · Oct 2014 · Angewandte Chemie
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    ABSTRACT: The complex bis-spiroacetal polyether ionophore salinomycin has been identified as a uniquely selective agent against cancer stem cells and is also strikingly effective in an animal model of latent tuberculosis. The basis for these important activities is unknown. We show here that deletion of the salE gene abolishes salinomycin production and yields two new analogues, in both of which the C18C19 cis double bond is replaced by a hydroxy group stereospecifically located at C19, but which differ from each other in the configuration of the bis-spiroacetal. These results identify SalE as a novel dehydratase and demonstrate that biosynthetic engineering can be used to redirect the reaction cascade of oxidative cyclization to yield new salinomycin analogues for use in mechanism-of-action studies.
    Full-text · Article · Aug 2014 · ChemBioChem
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    ABSTRACT: Formation of Z-configured double bonds in reduced polyketides is uncommon and their origins have not been extensively studied. To investigate the origin of the Z-configured double bond in the macrolide borrelidin, the recombinant dehydratase domains BorDH2 and BorDH3 were assayed with a synthetic analogue of the predicted tetraketide substrate. The configuration of the dehydrated products was determined to be E in both cases by comparison to synthetic standards. Detailed NMR spectroscopic analysis of the biosynthetic intermediate pre-borrelidin confirmed the E,E-configuration of the full-length polyketide synthase product. In contrast to a previously-proposed hypothesis, our results show that in this case the Z-configured double bond is not formed via dehydration from a 3 L-configured precursor, but rather as the result of a later isomerization process.
    Full-text · Article · Jun 2014 · Chemical Science
  • Peter F Leadlay

    No preview · Article · Jun 2014 · Nature
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    ABSTRACT: Covering: up to mid-2014Over the last several decades, the number of pharmacologically active natural products has significantly increased and several natural product families have taken shape. This review highlights the family of tetronate and spirotetronate compounds, which show a vast structural and functional diversity. The rapid growth of this group has created the need for a comprehensive overview and classification system, which we have devised based on structural characteristics. An updated overview is provided based on known tetronates, intended to spur further research in this field by identifying common structural features and general principles of their biosynthesis. We also compare a selection of chemical syntheses of representative compounds belonging to individual subtypes, both in terms of their efficiency as well as the extent to which they are biomimetic. This review also summarizes progress in unraveling some of the principles underlying the potent and varied bioactivities of natural tetronate antibiotics, and in identifying and better understanding their structure-activity relationships and modes of action.
    No preview · Article · Jun 2014 · Natural Product Reports
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    Full-text · Dataset · May 2014
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    ABSTRACT: Gentamicin C complex is a mixture of aminoglycoside antibiotics used to treat severe Gram-negative bacterial infections. We report here key features of the late-stage biosynthesis of gentamicins. We show that the intermediate gentamicin X2, a known substrate for C-methylation at C-6' to form G418 catalyzed by the radical SAM-dependent enzyme GenK, may instead undergo oxidation at C-6' to form an aldehyde, catalyzed by the flavin-linked dehydrogenase GenQ. Surprisingly, GenQ acts in both branches of the pathway, likewise oxidizing G418 to an analogous ketone. Amination of these intermediates, catalyzed mainly by aminotransferase GenB1, produces the known intermediates JI-20A and JI-20B, respectively. Other pyridoxal phosphate-dependent enzymes (GenB3 and GenB4) act in enigmatic dehydroxylation steps that convert JI-20A and JI-20B into the gentamicin C complex or (GenB2) catalyze the epimerization of gentamicin C2a into gentamicin C2.
    Full-text · Article · Apr 2014 · Chemistry & biology
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    Frank Hahn · Nadine Kandziora · Steffen Friedrich · Peter Francis Leadlay
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    ABSTRACT: Herein, we describe the syntheses of a complex biosynthesis-intermediate analogue of the potent antitumor polyketide borrelidin and of reference molecules to determine the stereoselectivity of the dehydratase of borrelidin polyketide synthase module 3. The target molecules were obtained from a common precursor aldehyde in the form of N-acetylcysteamine (SNAc) thioesters and methyl esters in 13 to 15 steps. Key steps for the assembly of the polyketide backbone of the dehydratase substrate analogue were a Yamamoto asymmetric carbocyclisation and a Sakurai allylation as well as an anti-selective aldol reaction. Reference compounds representing the E- and Z-configured double bond isomers as potential products of the dehydratase reaction were obtained from a common precursor aldehyde by Wittig olefination and Still-Gennari olefination. The final deprotection of TBS ethers and methyl esters was performed under mildly acidic conditions followed by pig liver esterase-mediated chemoselective hydrolysis. These conditions are compatible with the presence of a coenzyme A or a SNAc thioester, suggesting that they are generally applicable to the synthesis of complex polyketide-derived thioesters suited for biosynthesis studies.
    Preview · Article · Mar 2014 · Beilstein Journal of Organic Chemistry
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    Wolfgang Hüttel · Jonathan B Spencer · Peter F Leadlay
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    ABSTRACT: 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.
    Full-text · Article · Feb 2014 · Beilstein Journal of Organic Chemistry
  • Hui Hong · Taicia Fill · Peter F Leadlay
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    ABSTRACT: Keeping it basic: Arginine provides the exotic 4-guanidinobutanoate starter unit for two different types of zwitterionic polyketide (an example for one type is shown in the picture) produced by the same Streptomyces bacterium. The three-step precursor pathway is initiated by a remarkable decarboxylating monooxygenase with high specificity for arginine.
    No preview · Article · Dec 2013 · Angewandte Chemie International Edition
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    ABSTRACT: Mycolactones are polyketide-derived lipid virulence factors made by the slow-growing human pathogen, Mycobacterium ulcerans. Three unusually large and homologous plasmid-borne genes (mlsA1: 51 kb, mlsB: 42 kb and mlsA2: 7 kb) encode the mycolactone type I polyketide synthases (PKS). The extreme size and low sequence diversity of these genes has posed significant barriers for exploration of the genetic and biochemical basis of mycolactone synthesis. Here, we have developed a truncated, more tractable 3-module version of the 18-module mycolactone PKS and we show that this engineered PKS functions as expected in the natural host M. ulcerans to produce an additional polyketide; a triketide lactone (TKL). Cell fractionation experiments indicated that this 3-module PKS and the putative accessory enzymes encoded by mup045 and mup038 associated with the mycobacterial cell wall, a finding supported by confocal microscopy. We then assessed the capacity of the faster growing, Mycobacterium marinum to harbor and express the 3-module Mls PKS and accessory enzymes encoded by mup045 and mup038. RT-PCR, immunoblotting, and cell fractionation experiments confirmed that the truncated Mls PKS multienzymes were expressed and also partitioned with the cell wall material in M. marinum. However, this heterologous host failed to produce TKL. The systematic deconstruction of the mycolactone PKS presented here suggests that the Mls multienzymes are necessary but not sufficient for mycolactone synthesis and that synthesis is likely to occur (at least in part) within the mycobacterial cell wall. This research is also the first proof-of-principle demonstration of the potential of this enzyme complex to produce tailored small molecules through genetically engineered rearrangements of the Mls modules.
    Full-text · Article · Jul 2013 · PLoS ONE

Publication Stats

9k Citations
1,395.00 Total Impact Points

Institutions

  • 1981-2015
    • University of Cambridge
      • Department of Biochemistry
      Cambridge, England, United Kingdom
  • 2011
    • University of Freiburg
      Freiburg, Baden-Württemberg, Germany
  • 2008
    • Monash University (Australia)
      • Department of Microbiology
      Melbourne, Victoria, Australia
  • 2007
    • Newcastle University
      • School of Chemistry
      Newcastle-on-Tyne, England, United Kingdom
  • 2004
    • Center for Molecular Genetics
      Gif, Île-de-France, France