Modular Enzymatic Assembly Lines for Antibiotics

Summary

Principal Investigator: Christopher T Walsh
Abstract: DESCRIPTION (provided by applicant): Thiazolyl peptide antibiotics, including thiostrepton, the thiocillins and the GE2270 family, disrupt bacterial protein synthesis selectively with nanomolar affinity either for a site on the 50S ribosomal subunit or on elongation factor EF-TU. We and others have recently shown that the pyridine/dihydropyridine ring-containing macrocyclic scaffold necessary for the antibiotic activity of this natural product class arises from posttranslational modification of nascent ribosomally generated preproteins. Thus, the C-terminal 14 residues of the prethiocillin 52mer undergo a cascade of 14 posttranslational modifications, including 6 Cys to thiazoles, two threonines to dehydrobutyrines and two serines to the central pyridine heterocycle. In the thiocillin producer B, subtilis ATCC 14579 we have shown that preprotein gene replacement will allow in vivo analysis of requirements for the posttranslational cascade. This proposal will investigate the timing, order, and mechanisms of the posttranslational cascade modifications with the ultimate aim of reprogramming the machinery to understand target specificity (50S ribosomal subunit vs EF-Tu) and to optimize antibiotic activities. This will include study of the 26-atom macrocyclic thiocillin and also cloning, expression, and evaluation of the gene clusters encoding the 29 atom macrocyclic GE 37468 (which targets EF-Tu not the 50S ribosomal subunit) and the 35 atom macrocyclic berninamycin A, in which the 7 C-terminal serines are processed posttranslationally, five of them to dehydroAla, one to an oxazole, and the other condensed to form the embedded pyridine: we wish to understand how the distinct posttranslational fates of these serines during antibiotic scaffold maturation are controlled.
Funding Period: 1987-09-30 - 2013-06-30
more information: NIH RePORT

Top Publications

  1. ncbi Nonproteinogenic amino acid building blocks for nonribosomal peptide and hybrid polyketide scaffolds
    Christopher T Walsh
    Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, MA 02115, USA
    Angew Chem Int Ed Engl 52:7098-124. 2013
  2. pmc A head-to-head comparison of eneamide and epoxyamide inhibitors of glucosamine-6-phosphate synthase from the dapdiamide biosynthetic pathway
    Marie A Hollenhorst
    Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, Massachusetts 02115, United States
    Biochemistry 50:3859-61. 2011
  3. pmc The biosynthesis of cyanobacterial sunscreen scytonemin in intertidal microbial mat communities
    Emily P Balskus
    Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, MA 02115, USA
    FEMS Microbiol Ecol 77:322-32. 2011
  4. pmc Complexity generation in fungal peptidyl alkaloid biosynthesis: oxidation of fumiquinazoline A to the heptacyclic hemiaminal fumiquinazoline C by the flavoenzyme Af12070 from Aspergillus fumigatus
    Brian D Ames
    Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, Massachusetts 02115, United States
    Biochemistry 50:8756-69. 2011
  5. pmc Genetic interception and structural characterization of thiopeptide cyclization precursors from Bacillus cereus
    Albert A Bowers
    Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, Massachusetts 02115, USA
    J Am Chem Soc 132:12182-4. 2010
  6. pmc Manipulation of thiocillin variants by prepeptide gene replacement: structure, conformation, and activity of heterocycle substitution mutants
    Albert A Bowers
    Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, Massachusetts 02115, USA
    J Am Chem Soc 132:7519-27. 2010
  7. pmc The crystal structure of the novobiocin biosynthetic enzyme NovP: the first representative structure for the TylF O-methyltransferase superfamily
    Inmaculada Gómez García
    Department of Biological Chemistry, John Innes Centre, Norwich NR4 7UH, UK
    J Mol Biol 395:390-407. 2010
  8. pmc A three enzyme pathway for 2-amino-3-hydroxycyclopent-2-enone formation and incorporation in natural product biosynthesis
    Wenjun Zhang
    Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, Massachusetts 02115, USA
    J Am Chem Soc 132:6402-11. 2010
  9. pmc Thiazolyl peptide antibiotic biosynthesis: a cascade of post-translational modifications on ribosomal nascent proteins
    Christopher T Walsh
    Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, Massachusetts 02115, USA
    J Biol Chem 285:27525-31. 2010
  10. pmc Unraveling terminal C-domain-mediated condensation in fungal biosynthesis of imidazoindolone metabolites
    Stuart W Haynes
    Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, Massachusetts 02115, USA
    Biochemistry 50:5668-79. 2011

Research Grants

Detail Information

Publications43

  1. ncbi Nonproteinogenic amino acid building blocks for nonribosomal peptide and hybrid polyketide scaffolds
    Christopher T Walsh
    Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, MA 02115, USA
    Angew Chem Int Ed Engl 52:7098-124. 2013
    ....
  2. pmc A head-to-head comparison of eneamide and epoxyamide inhibitors of glucosamine-6-phosphate synthase from the dapdiamide biosynthetic pathway
    Marie A Hollenhorst
    Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, Massachusetts 02115, United States
    Biochemistry 50:3859-61. 2011
    ....
  3. pmc The biosynthesis of cyanobacterial sunscreen scytonemin in intertidal microbial mat communities
    Emily P Balskus
    Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, MA 02115, USA
    FEMS Microbiol Ecol 77:322-32. 2011
    ..aestuarii, is likely responsible for sunscreen production. This study is the first to utilize an understanding of the molecular basis of scytonemin assembly to explore its synthesis and function within natural microbial communities...
  4. pmc Complexity generation in fungal peptidyl alkaloid biosynthesis: oxidation of fumiquinazoline A to the heptacyclic hemiaminal fumiquinazoline C by the flavoenzyme Af12070 from Aspergillus fumigatus
    Brian D Ames
    Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, Massachusetts 02115, United States
    Biochemistry 50:8756-69. 2011
    ..2'-epi-FQA is processed by Af12070 to epi-FQD, not epi-FQC, illustrating that the delicate balance in product cyclization regiochemistry can be perturbed by a remote stereochemical center...
  5. pmc Genetic interception and structural characterization of thiopeptide cyclization precursors from Bacillus cereus
    Albert A Bowers
    Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, Massachusetts 02115, USA
    J Am Chem Soc 132:12182-4. 2010
    ..These results identify TclM and its homologues in other thiazolyl peptide producing strains as the enzymes responsible for the trans-annular heteroannulation at core of this class of molecules...
  6. pmc Manipulation of thiocillin variants by prepeptide gene replacement: structure, conformation, and activity of heterocycle substitution mutants
    Albert A Bowers
    Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, Massachusetts 02115, USA
    J Am Chem Soc 132:7519-27. 2010
    ..These structures demonstrate that the tight conformational rigidity of the natural product is severely disrupted by loss of even a single heterocycle, perhaps accounting for the attendant loss of activity in such mutants...
  7. pmc The crystal structure of the novobiocin biosynthetic enzyme NovP: the first representative structure for the TylF O-methyltransferase superfamily
    Inmaculada Gómez García
    Department of Biological Chemistry, John Innes Centre, Norwich NR4 7UH, UK
    J Mol Biol 395:390-407. 2010
    ..These observations could inform future attempts to utilize NovP for methylating a range of glycosylated compounds...
  8. pmc A three enzyme pathway for 2-amino-3-hydroxycyclopent-2-enone formation and incorporation in natural product biosynthesis
    Wenjun Zhang
    Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, Massachusetts 02115, USA
    J Am Chem Soc 132:6402-11. 2010
    ..This three enzyme pathway is likely the general route to the C(5)N ring system in other natural products, including the antibiotic moenomycin...
  9. pmc Thiazolyl peptide antibiotic biosynthesis: a cascade of post-translational modifications on ribosomal nascent proteins
    Christopher T Walsh
    Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, Massachusetts 02115, USA
    J Biol Chem 285:27525-31. 2010
    ..The trithiazolyl pyridine framework thus arises from post-translational modification of the peptide backbone of three Cys and two Ser residues of the prepeptide...
  10. pmc Unraveling terminal C-domain-mediated condensation in fungal biosynthesis of imidazoindolone metabolites
    Stuart W Haynes
    Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, Massachusetts 02115, USA
    Biochemistry 50:5668-79. 2011
    ..The Af12050 and TqaB condensation domains are thus a paired set of diastereospecific annulation catalysts that act on the fumiquinazoline F scaffold...
  11. pmc Codon randomization for rapid exploration of chemical space in thiopeptide antibiotic variants
    Travis S Young
    Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, 240 Longwood Avenue, Armenise 608, Boston, MA 02115, USA
    Chem Biol 19:1600-10. 2012
    ..Second, the activity of producing mutants was detected in an antibiotic overlay assay. In total, 29 of the 133 variants produced mature compound, 12 of which retained antibiotic activity and 1 that had improved activity...
  12. pmc Assembly of asperlicin peptidyl alkaloids from anthranilate and tryptophan: a two-enzyme pathway generates heptacyclic scaffold complexity in asperlicin E
    Stuart W Haynes
    Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, 240 Longwood Avenue, Boston, Massachusetts 02115, USA
    J Am Chem Soc 134:17444-7. 2012
    ..This report provides the first genetic characterization of a NRPS assembly line that efficiently activates two anthranilate building blocks and illustrates the remarkably efficient biosynthesis of the complex heptacyclic asperlicin E...
  13. pmc An iterative, bimodular nonribosomal peptide synthetase that converts anthranilate and tryptophan into tetracyclic asperlicins
    Xue Gao
    Department of Chemical and Biomolecular Engineering, University of California, Los Angeles, 420 Westwood Plaza, Los Angeles, CA 90095, USA
    Chem Biol 20:870-8. 2013
    ....
  14. pmc Short pathways to complexity generation: fungal peptidyl alkaloid multicyclic scaffolds from anthranilate building blocks
    Christopher T Walsh
    Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, 25 Shattuck Street, Boston, Massachusetts
    ACS Chem Biol 8:1366-82. 2013
    ..Use of the nonproteinogenic amino acid anthranilate as chain-initiating building block and chain-terminating intramolecular nucleophile leads efficiently to peptidyl alkaloid scaffolds with two to seven fused rings. ..
  15. pmc The posttranslational modification cascade to the thiopeptide berninamycin generates linear forms and altered macrocyclic scaffolds
    Steven J Malcolmson
    Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, MA 02115, USA
    Proc Natl Acad Sci U S A 110:8483-8. 2013
    ..Expression of the gene cluster in S. venezuelae generates a variant of the 35-atom skeleton of berninamycin, containing a methyloxazoline in the place of a methyloxazole within the macrocyclic framework...
  16. pmc Complexity generation in fungal peptidyl alkaloid biosynthesis: a two-enzyme pathway to the hexacyclic MDR export pump inhibitor ardeemin
    Stuart W Haynes
    Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, 240 Longwood Avenue, Boston, MA 02115, USA
    ACS Chem Biol 8:741-8. 2013
    ..The two-enzyme ArdAB pathway reveals remarkable efficiency in construction of the hexacyclic peptidyl alkaloid scaffold...
  17. pmc Action and timing of BacC and BacD in the late stages of biosynthesis of the dipeptide antibiotic bacilysin
    Jared B Parker
    Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, 240 Longwood Avenue, Boston, MA 02115, United States
    Biochemistry 52:889-901. 2013
    ....
  18. pmc Flavoenzymes: versatile catalysts in biosynthetic pathways
    Christopher T Walsh
    Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, 240 Longwood Ave, Boston, MA 02115, USA
    Nat Prod Rep 30:175-200. 2013
    ....
  19. pmc Cyclization of fungal nonribosomal peptides by a terminal condensation-like domain
    Xue Gao
    Department of Chemical and Biomolecular Engineering, University of California Los Angeles, Los Angeles, CA, USA
    Nat Chem Biol 8:823-30. 2012
    ..Our work provides evidence of a likely universal macrocyclization strategy used by fungal NRPSs...
  20. pmc Generation of thiocillin ring size variants by prepeptide gene replacement and in vivo processing by Bacillus cereus
    Albert A Bowers
    Department of Medicinal Chemistry and Molecular Pharmacology, Purdue University, West Lafayette, Indiana 47907, USA
    J Am Chem Soc 134:10313-6. 2012
    ..Cyclization proceeds in many cases to provide altered ring sizes, giving access to several variant rings sizes that have not previously been observed in nature...
  21. pmc Natural products version 2.0: connecting genes to molecules
    Christopher T Walsh
    Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, 240 Longwood Avenue, Boston, Massachusetts 02115, USA
    J Am Chem Soc 132:2469-93. 2010
    ..We conclude by considering two questions: What would it take to find all natural product scaffolds? What kind of scientists will be studying natural products in the future?..
  22. pmc A flavin-dependent halogenase catalyzes the chlorination step in the biosynthesis of Dictyostelium differentiation-inducing factor 1
    Christopher S Neumann
    Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, 240 Longwood Avenue, Boston, MA 02115, USA
    Proc Natl Acad Sci U S A 107:5798-803. 2010
    ....
  23. pmc Cascade reactions during coronafacic acid biosynthesis: elongation, cyclization, and functionalization during Cfa7-catalyzed condensation
    Eric R Strieter
    Harvard Medical School, Department of Biological Chemistry and Molecular Pharmacology, 240 Longwood Avenue, Boston, Massachusetts 02115, USA
    J Am Chem Soc 131:2113-5. 2009
    ..This reactivity translates into an intramolecular cyclization, which can proceed in a facile manner as observed by the enzyme-independent cyclization of a linear beta-ketothioester intermediate...
  24. pmc beta-Hydroxylation of the aspartyl residue in the phytotoxin syringomycin E: characterization of two candidate hydroxylases AspH and SyrP in Pseudomonas syringae
    Gitanjali M Singh
    Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, Massachusetts 02115, USA
    Biochemistry 47:11310-20. 2008
    ..The hydroxylation gives the anticipated L- threo-3-OH-Asp diastereomer found in syringomycin. The knockout of syrP abolishes the production of the mature syringomycin E, while knockout of aspH has no effect on syringomycin production...
  25. pmc Tandem action of the O2- and FADH2-dependent halogenases KtzQ and KtzR produce 6,7-dichlorotryptophan for kutzneride assembly
    John R Heemstra
    Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, Massachusetts 02115, USA
    J Am Chem Soc 130:14024-5. 2008
    ..These findings provide further insights into the enzymatic logic of carbon-chloride bond formation during the biosynthesis of halogenated secondary metabolites...
  26. pmc An eight residue fragment of an acyl carrier protein suffices for post-translational introduction of fluorescent pantetheinyl arms in protein modification in vitro and in vivo
    Zhe Zhou
    Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, Massachusetts 02115, USA
    J Am Chem Soc 130:9925-30. 2008
    ....
  27. pmc Investigating the initial steps in the biosynthesis of cyanobacterial sunscreen scytonemin
    Emily P Balskus
    Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, Massachusetts 02115, USA
    J Am Chem Soc 130:15260-1. 2008
    ..A regioselective acyloin reaction between indole-3-pyruvic acid (4) and p-hydroxyphenylpyruvic acid (5) is a key step in assembling the carbon framework of a proposed monomeric scytonemin precursor (2)...
  28. pmc Structural basis for the selectivity of the external thioesterase of the surfactin synthetase
    Alexander Koglin
    Institute of Biophysical Chemistry and Center for Biomolecular Magnetic Resonance and Cluster of Excellence Macromolecular Complexes CEF, J W Goethe University, 60438 Frankfurt am Main, Germany
    Nature 454:907-11. 2008
    ..Furthermore, we show that the TEII enzyme exists in several conformations of which only one is selected on interaction with its native substrate, a modified holo-T domain...
  29. pmc What's new in enzymatic halogenations
    Danica Galonic Fujimori
    Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, 240 Longwood Avenue, Boston, MA 02115, USA
    Curr Opin Chem Biol 11:553-60. 2007
    ..The mechanism of halogen incorporation is tailored to the electronic demands of specific substrates and involves enzymes with distinct redox coenzyme requirements...
  30. pmc BluB cannibalizes flavin to form the lower ligand of vitamin B12
    Michiko E Taga
    Department of Biology, Massachusetts Institute of Technology, 77 Massachusetts Avenue, Cambridge, Massachusetts 02139, USA
    Nature 446:449-53. 2007
    ..This investigation of the biosynthesis of DMB provides clarification of an aspect of vitamin B12 that was otherwise incomplete, and may contribute to a better understanding of vitamin B12-related disease...
  31. pmc CD and MCD of CytC3 and taurine dioxygenase: role of the facial triad in alpha-KG-dependent oxygenases
    Michael L Neidig
    Department of Chemistry, Stanford University, Stanford, California 94305, USA
    J Am Chem Soc 129:14224-31. 2007
    ..These studies provide initial insight into the active-site features that favor chlorination by CytC3 over the hydroxylation reactions occurring in related enzymes...
  32. pmc Biosynthesis of (-)-(1S,2R)-allocoronamic acyl thioester by an Fe(II)-dependent halogenase and a cyclopropane-forming flavoprotein
    Christopher S Neumann
    Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, Massachusetts 02115, USA
    J Am Chem Soc 130:14022-3. 2008
    ..This work introduces a new monomer for potential incorporation into nonribosomal peptides and validates the unique strategy for its biosynthesis...
  33. pmc Gatekeeping versus promiscuity in the early stages of the andrimid biosynthetic assembly line
    Nathan A Magarvey
    Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, Massachusetts 02115, USA
    ACS Chem Biol 3:542-54. 2008
    ....
  34. pmc Inhibitors of sterol biosynthesis as Staphylococcus aureus antibiotics
    Christopher T Walsh
    Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, 240 Longwood Avenue, Boston, MA 02115, USA
    Angew Chem Int Ed Engl 47:5700-2. 2008
  35. pmc Substrate-triggered formation and remarkable stability of the C-H bond-cleaving chloroferryl intermediate in the aliphatic halogenase, SyrB2
    Megan L Matthews
    Department of Chemistry, The Pennsylvania State University, University Park, Pennsylvania 16802, USA
    Biochemistry 48:4331-43. 2009
    ....
  36. pmc Generation of thiocillin variants by prepeptide gene replacement and in vivo processing by Bacillus cereus
    Michael G Acker
    Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, Massachusetts 02115, USA
    J Am Chem Soc 131:17563-5. 2009
    ....
  37. pmc An enzymatic cyclopentyl[b]indole formation involved in scytonemin biosynthesis
    Emily P Balskus
    Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, Massachusetts 02115, USA
    J Am Chem Soc 131:14648-9. 2009
    ..Additional mechanistic experiments have revealed that cyclization likely precedes decarboxylation and that the latter event may provide a driving force for cyclopentane formation...
  38. pmc Characterization of cyclo-acetoacetyl-L-tryptophan dimethylallyltransferase in cyclopiazonic acid biosynthesis: substrate promiscuity and site directed mutagenesis studies
    Xinyu Liu
    Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, 240 Longwood Avenue, Boston, Massachusetts 02115, USA
    Biochemistry 48:11032-44. 2009
    ..Subsequent site-directed mutagenesis studies identified five polar/charged residues and five tyrosine residues within these motifs that are critical for CpaD activity...
  39. pmc How nature morphs peptide scaffolds into antibiotics
    Elizabeth M Nolan
    Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, MA 02115, USA
    Chembiochem 10:34-53. 2009
    ..Others might provide new platforms for the design of novel therapeutics to combat emerging antibiotic-resistant bacterial pathogens...
  40. pmc Antibiotics for emerging pathogens
    Michael A Fischbach
    Department of Molecular Biology and Center for Computational and Integrative Biology, Massachusetts General Hospital, Boston, MA 02114, USA
    Science 325:1089-93. 2009
    ....
  41. pmc Cyclopiazonic acid biosynthesis in Aspergillus sp.: characterization of a reductase-like R* domain in cyclopiazonate synthetase that forms and releases cyclo-acetoacetyl-L-tryptophan
    Xinyu Liu
    Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, 240 Longwood Avenue, Boston, Massachusetts 02115, USA
    Biochemistry 48:8746-57. 2009
    ..It is likely that cyclization of beta-ketoacylaminoacyl-S-pantetheinyl intermediates to released tetramates represents a default cyclization/release route for redox-incompetent R* domains embedded in NRPS assembly lines...
  42. pmc Structural insights into nonribosomal peptide enzymatic assembly lines
    Alexander Koglin
    Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, MA 02115, USA
    Nat Prod Rep 26:987-1000. 2009
    ..This Highlight reviews recent breakthrough achievements in both X-ray and NMR spectroscopic studies that illuminate the architecture of NRPS PCP domains, PCP-containing didomain-fragments and of a full termination module (C-A-PCP-TE)...
  43. pmc Biosynthetic tailoring of microcin E492m: post-translational modification affords an antibacterial siderophore-peptide conjugate
    Elizabeth M Nolan
    Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, Massachusetts 02115, USA
    J Am Chem Soc 129:14336-47. 2007
    ..Nonenzymatic and base-catalyzed migration of the peptide to the C6' position affords the C6' glycosyl ester linkage observed in the mature toxin, MccE492m, isolated from bacterial cultures...

Research Grants30

  1. Biosynthesis of the thiopeptide antibiotic thiostrepton
    Wendy L Kelly; Fiscal Year: 2013
    ..We are proposing to study how these compounds are naturally constructed and apply the knowledge gained toward the production the first therapeutic agents based upon the thiopeptide framework. ..