Hybrid Nonribosomal Peptide-Polyketide Interfaces in Epothilone Biosynthesis

Liu, Fei; Garneau, Sylvie; Walsh, Christopher T.

doi:10.1016/j.chembiol.2004.08.017

Cited by 36 publications

(32 citation statements)

References 43 publications

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“…In the three-protein synthase that produces the aglycone scaffold of erythromycin, 6-deoxyethronolide B, Khosla and coworkers (10) have demonstrated that appending these short linker regions onto noncognate donor carrier proteins allows for recognition by downstream elongation modules. In the hybrid PKS͞NRPS interface of the first two modules of epothilone biosynthesis, our laboratory has shown that removal of these linker regions disrupts the channeling of thiotemplated substrate intermediates to the downstream module (11,36). In addition, similar communication-mediating domains have been discovered in the NRPS that produces tyrocidine (12).…”

Section: Discussionmentioning

confidence: 87%

A protein interaction surface in nonribosomal peptide synthesis mapped by combinatorial mutagenesis and selection

Lai

Fischbach

Liu

et al. 2006

Proc. Natl. Acad. Sci. U.S.A.

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Nonribosomal peptide synthetases (NRPSs) and polyketide synthases are large, multidomain enzymes that biosynthesize a number of pharmaceutically important natural products. The recognition of biosynthetic intermediates, displayed via covalent attachment to carrier proteins, by catalytic domains is critical for NRPS and polyketide synthase function. We report the use of combinatorial mutagenesis coupled with in vivo selection for the production of the Escherichia coli NRPS product enterobactin to map the surface of the aryl carrier protein (ArCP) domain of EntB that interacts with the downstream elongation module EntF. Two libraries spanning the predicted helix 2 and loop 2͞helix 3 of EntB-ArCP were generated by shotgun alanine scanning and selected for their ability to support enterobactin production. From the surviving pools, we identified several hydrophobic residues (M249, F264, and A268) that were highly conserved. These residues cluster near the phosphopantetheinylated serine in a structural model, and two of these positions are in the predicted helix 3 region. Subsequent in vitro studies are consistent with the hypothesis that these residues form a surface on EntB required for interaction with EntF. These results suggest that helix 3 is a major recognition element in EntB-ArCP and demonstrate the utility of selection-based approaches for studying NRPS biosynthesis. nonribosomal peptide synthetase ͉ siderophore A number of medicinally useful natural products are biosynthesized in their cognate producer organisms by nonribosomal peptide synthetase (NRPS) and polyketide synthase (PKS) mutidomain enzymes (1). Examples of NRPS-and PKS-derived compounds include the antibiotics erythromycin (PKS) and vancomycin (NRPS) (2, 3), the antitumor agents epothilone and bleomycin (both from hybrid PKS͞NRPS systems) (4, 5), and the immunosuppressant rapamycin (hybrid PKS͞NRPS) (6). At various points during the biosynthesis of these molecules, substrates for catalytic operations are presented as thioesters tethered to carrier proteins through a 4Ј-phosphopantetheine cofactor. These carrier proteins, peptidyl carrier proteins (PCPs) in NRPSs and acyl carrier proteins (ACPs) in PKSs, are homologous to the ACPs that are involved in production of fatty acids from primary metabolism (7). ACPs and PCPs are small (Ϸ80-100 residues) and contain a central serine residue that is the site of attachment for the phosphopantetheine arm (1, 7). Carrier proteins thus provide the protein scaffolding for display of the growing acyl chains in each module.Critical to the function of NRPSs and PKSs are the interactions between domains (8). Because the biosynthetic intermediates are covalently tethered to carrier proteins, studying the determinants of carrier-protein recognition by other synthetase domains remains an important goal. In a typical cycle of NRPS elongation (that is, formation of an amide bond between two amino acids), a PCP must be recognized by the adenylation domain, which acylates the phosphopantetheine with an amino acyl group ...

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Section: Discussionmentioning

confidence: 87%

A protein interaction surface in nonribosomal peptide synthesis mapped by combinatorial mutagenesis and selection

Lai

Fischbach

Liu

et al. 2006

Proc. Natl. Acad. Sci. U.S.A.

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“…The N-terminal 55 residues of the EpoB protein make up the docking domain (EpoBdd), which recognizes the upstream EpoAdd to position the acetyl-S-EpoA-T domain for catalysis (24). EpoBdd adopts an αββαα fold, consisting of an initial α-helix, β-turn, and two final α-helices ( Fig.…”

Section: Resultsmentioning

confidence: 99%

Structural elements of an NRPS cyclization domain and its intermodule docking domain

Dowling

Kung

Croft

et al. 2016

Proc. Natl. Acad. Sci. U.S.A.

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Epothilones are thiazole-containing natural products with anticancer activity that are biosynthesized by polyketide synthase (PKS)-nonribosomal peptide synthetase (NRPS) enzymes EpoA-F. A cyclization domain of EpoB (Cy) assembles the thiazole functionality from an acetyl group and L-cysteine via condensation, cyclization, and dehydration. The PKS carrier protein of EpoA contributes the acetyl moiety, guided by a docking domain, whereas an NRPS EpoB carrier protein contributes L-cysteine. To visualize the structure of a cyclization domain with an accompanying docking domain, we solved a 2.03-Å resolution structure of this bidomain EpoB unit, comprising residues M1-Q497 (62 kDa) of the 160-kDa EpoB protein. We find that the N-terminal docking domain is connected to the V-shaped Cy domain by a 20-residue linker but otherwise makes no contacts to Cy. Molecular dynamic simulations and additional crystal structures reveal a high degree of flexibility for this docking domain, emphasizing the modular nature of the components of PKS-NRPS hybrid systems. These structures further reveal two 20-Å-long channels that run from distant sites on the Cy domain to the active site at the core of the enzyme, allowing two carrier proteins to dock with Cy and deliver their substrates simultaneously. Through mutagenesis and activity assays, catalytic residues N335 and D449 have been identified. Surprisingly, these residues do not map to the location of the conserved HHxxxDG motif in the structurally homologous NRPS condensation (C) domain. Thus, although both C and Cy domains have the same basic fold, their active sites appear distinct.crystal structure | molecular dynamics | epothilone | natural product

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“…The boundary between the 57 kDa amino-terminal Cy domain and the subsequent internal A domain was assigned to a loop identified by secondary structure prediction, as undertaken for the EntF C domain (8). Since the initial 56 amino acids of the N-terminus of EpoB have previously been shown to be required for communication with the upstream subunit EpoA(ACP), this amino-terminal region was retained in its entirety (20). Two possible sites for the EpoB(Cy) C-terminus were designated Glu492 and Gln497.…”

Section: Construction and Purification Of Epob(cy)mentioning

confidence: 99%

Excision of the Epothilone Synthetase B Cyclization Domain and Demonstration of in Trans Condensation/Cyclodehydration Activity

2005

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The epothilones are potent anticancer natural products produced by a polyketide synthase (PKS)-nonribosomal peptide synthetase (NRPS) hybrid involving proteins EpoA-F. The single NRPS module of the epothilone assembly line, EpoB, is a distinct subunit of approximately 160 kDa and consists of four successive domains: cyclization, adenylation, oxidation, and peptidyl carrier protein (Cy-AOx-PCP). The cyclization domain is responsible for introduction of the thiazoline heterocycle into the growing polyketide/nonribosomal peptide chain from the precursors malonyl-CoA and cysteine through the multiple steps of condensation, cyclization, and dehydration. This enzyme-bound thiazoline intermediate is subsequently oxidized to a thiazole by the EpoB Ox domain. The EpoB module was dissected to provide 57 kDa EpoB(Cy) and 102 kDa EpoB(A-Ox-PCP) as subunit fragments to evaluate Cy as a freestanding domain. EpoB was reconstituted by these fragments in trans to generate the methylthiazole product. Using this system, apparent kinetic constants for the upstream acyl donor EpoA(ACP) and EpoB(Cy) were determined, providing a measure of affinity for the naturally occurring interface of the amino terminus of EpoB and the EpoA carboxy terminus. Site-directed mutants in excised EpoB(Cy) were prepared and used to examine residues involved in condensation and heterocycle formation. This work demonstrates the ability to define a functional Cy domain by excision from its native NRPS module, and examine both its protein-protein interactions and mechanism of activity.

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Hybrid Nonribosomal Peptide-Polyketide Interfaces in Epothilone Biosynthesis

Cited by 36 publications

References 43 publications

A protein interaction surface in nonribosomal peptide synthesis mapped by combinatorial mutagenesis and selection

A protein interaction surface in nonribosomal peptide synthesis mapped by combinatorial mutagenesis and selection

Structural elements of an NRPS cyclization domain and its intermodule docking domain

Excision of the Epothilone Synthetase B Cyclization Domain and Demonstration of in Trans Condensation/Cyclodehydration Activity

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