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


Nonribosomal custom peptide synthesis 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 2/helix 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.

A number of medicinally useful natural products are biosynthesized in their cognate producer organisms by nonribosomal custom peptide synthesis (NRPS) and polyketide synthase (PKS) mutidomain enzymes Examples of NRPS- and PKS-derived compounds include the antibiotics erythromycin (PKS) and vancomycin (NRPS) the antitumor agents epothilone and bleomycin (both from hybrid PKS/NRPS systems)  and the immunosuppressant rapamycin (hybrid PKS/NRPS)  At various points during the biosynthesis of these molecules, substrates for catalytic operations are presented as thioesters tethered to carrier proteins through a 4′-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 . 

ACPs and PCPs are small (≈80–100 residues) and contain a central serine residue that is the site of attachment for the phosphopantetheine arm . Carrier proteins thus provide the protein scaffolding for display of the growing acyl chains in each module.

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