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

Abstract

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 ...

Facile Detection of Acyl and Peptidyl Intermediates on Thiotemplate Carrier Domains via Phosphopantetheinyl Elimination Reactions during Tandem Mass Spectrometry

“…In Table 1, the elimination fragments ions are shown for pyrrolyl-S-PigG, pyrrolyl-β-ketoacyl-S-PigH-ACP 1 and pyrrolyl-β-ketoacyl-S-PigH-2ACP (12), the ACP and PCP domain of the 4 domain construct MycA loaded with acetoacetate, other β-ketoacids and the corresponding-β-aminobutyrates (16), pyrrolyl-S-PltL, mono-chloropyrrolyl-S-PltL, dichloropyrrolyl-S-PltL, di-bromopyrrolyl-S-PltL (9), seryl-S-PksN (25), phenylacetyl-S-PksJ (29), acetoacetyl-S-AcpK, acetyl-S-AcpK, acetoacetyl-S-PksL-2ACP, hydroxymethylglutaryl-S-PksL-2ACP, dehydromethylglutaryl-SPksL-2ACP, 2 Δ-isoprenyl-S-PksL-2ACP (19), and dihydroxybenzoyl-S-EntB(ArCP) (17,25). To determine if larger intact enzymes could be interrogated by the Top Down approach, the 74 kDa protein NikP1 on the nikkomycin biosynthetic pathway (13,25) and the undigested 127 kDa phenylalanyl-S-GrsA or D5-deutero phenylalanyl-S-GrsA were analyzed (13,14).…”

“…We provide support for the robustness and generality of this assay via 33 examples as this approach was used to characterize the loading of substrates, intermediates and products of proteins involved in the biosynthesis of the antibiotic nikkomycin (8), the antifungal agent pyoluteorin (9,10), the antibiotic prodigiosin (11,12), the antibacterial gramicidin (13,14), the antibacterial agent mycosubtilin (15,16), and the siderophore enterobactin (17,18). Further, intermediates were detected via this gas phase elimination on four proteins from the orphan orphan pksX cluster of B. subtilis that produces an unknown antibiotic (19)(20)(21).…”

Directed evolution can rapidly improve the activity of chimeric assembly-line enzymes

“…2a), an iron-scavenging small molecule excreted by E. coli in response to iron starvation (18). E. coli require enterobactin for growth on iron-deficient media, enabling us to develop a screen for enterobactin synthetase activity in which cell growth rate was highly correlated with the level of enterobactin production (19)(20)(21)(22).…”

Structure and Function of the RedJ Protein, a Thioesterase from the Prodiginine Biosynthetic Pathway in Streptomyces coelicolor

“…In contrast, RedJ has no selection against the foreign ACP of E. coli fatty acid biosynthesis, AcpP. Structural and mutagenesis studies of the enterobactin biosynthetic assembly line (Ent) have provided a coherent picture of the productive interaction of a monomeric TE with its cognate carrier domains in which the C terminus of helix 3 in the carrier domain contacts the surface of the TE near the entrance channel (22,42,43). Additional TE contacts occur with the C terminus of helix 1 in the carrier domains (22).…”