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 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.

Fig. 1.

Schematic for the enterobactin synthetase and the condensation step of enterobactin biosynthesis.

Fig. 2.

Structural model of EntB-ArCP and regions of randomization. (A) Sequence alignment of TycC3-PCP and EntB-ArCP. Core residues in TycC3-PCP are shown in bold. The helical regions in the solution structure are indicated (25). The phosphopantetheinylated serine is indicated with an asterisk. The residues on EntB that were subjected to shotgun alanine scanning are indicated in red (the H2 library) and blue (L2H3 library). The residue numbering is for full-length EntB. (B) EntB-ArCP structural homology model based on optimized sequence alignment with TycC3-PCP. (C) The overlay of this structural homology model with TycC3-PCP (red), and FrnN (green) from frenolicin biosynthesis shows good structural conservation among carrier proteins and validates the EntB-ArCP homology model.

Fig. 3.

Conservation of residues from library selections. (A) Surface representation of the EntB-ArCP homology model with residues color-coded for the observed degree of conservation from the surviving pools after selection. Red indicates high WT/mut ratios (>50), orange indicates intermediate (6 <WT/mut <50), and green indicates low conservation (<6). The phosphopantetheinylated serine is shown in blue. The ratios shown here and numerically in Table 1 are the results from sequencing of >100 clones from each surviving pool. (B) Ribbon diagram representation of A with side chains for the hotspot near S245 shown as stick.

Fig. 4

Condensation assay. (A) Schematic of the Ser-S-NAC condensation assay. A soluble thioester substrate acts as a mimic for natural PCP-bound intermediate. Upon basic work-up, the DHB-Ser condensation product is obtained. (B) Kinetics of condensation catalyzed by EntF by using WT EntB-ArCP or mutants M249A, F264A, and A268Q. Full parameters are listed in Table 3.