Structure and Substrate Sequestration in the Pyoluteorin Type II Peptidyl Carrier Protein PltL

Abstract

Type II non-ribosomal peptide synthetases (NRPS) generate exotic amino acid derivatives that, combined with additional pathways, form many bioactive natural products. One family of type II NRPSs produce pyrrole moieties, which commonly arise from proline oxidation while tethered to a conserved, type II peptidyl carrier protein (PCP), as exemplified by PltL in the biosynthesis of pyoluteorin. We sought to understand the structural role of pyrrole PCPs in substrate and protein interactions through the study of pyrrole analogs tethered to PltL. Solution-phase NMR structural analysis revealed key interactions in residues of helix II and III with a bound pyrrole moiety. Conservation of these residues among PCPs in other pyrrole containing pathways suggests a conserved mechanism for formation, modification, and incorporation of pyrrole moieties. Further NOE analysis provided a unique pyrrole binding motif, offering accurate substrate positioning within the cleft between helices II and III. The overall structure resembles other PCPs but contains a unique conformation for helix III. This provides evidence of sequestration by the PCP of aromatic pyrrole substrates, illustrating the importance of substrate protection and biosynthetic regulation in type II NRPSs.

Biosynthesis of coral settlement cue tetrabromopyrrole in marine bacteria by a uniquely adapted brominase–thioesterase enzyme pair

“…Sequestration of a pyrrole tethered to a type II peptidyl carrier protein (PCP) was recently demonstrated for a highly homologous PCP-PltL [57% amino acid similarity to Mm_Bmp1(ACP)] participating in the biosynthesis of the dichloropyrrole-containing natural product pyoluteorin (7,20). By analogy, in the biosynthesis of 1, it is possible that the terminal halogenation on the pyrrole partially liberates the sequestered substrate from the ACP, allowing access to the thioesterase.…”

New Structural Data Reveal the Motion of Carrier Proteins in Nonribosomal Peptide Synthesis

“…The secondary and tertiary structures of PCP domains are highly conserved, with only minor deviations from the prototypical four‐helix bundle being documented to date 19, 20, 21, 22, 23, 24, 25, 26, 27. At the level of primary structure, PCPs are more variable,21 which gives rise to variations in local shape and charge distributions of the exposed and buried surfaces.…”

“…This model was based on observations that a PCP domain from the tyrocidine synthetase exists in three different conformations (termed A, A/H, and H) depending on its loaded state 28. However, this has since been dismissed as an artifact of PCP domain excision from the larger synthetase since all other PCP structures elucidated to date adopt the A/H state 19, 20, 21, 22, 23, 24, 25, 26, 29, 30…”

Manipulating Protein–Protein Interactions in Nonribosomal Peptide Synthetase Type II Peptidyl Carrier Proteins

“…Holo -PigG and holo -PltL have similar structural features. Both possess a unique interruption in helix III, 2 and the N-terminal portion of helix II in both PCPs has slight positive potential that could form electrostatic interactions with an A domain, as this PCP region is proximal to the A domain in recent crystal structures PCP-A didomains (Figure 2C–D). 23–26 In contrast, loop 1 of these PCPs varies significantly between the two structures (Figure 2C–D).…”

“…Previously reported, NMR solution structures of PltL in holo - and pyrrolyl forms demonstrated that, like type II fatty acid and polyketide carrier proteins, 2, 27, 28 type II PCPs have the capacity to sequester their substrates. Holo -PigG and holo -PltL have similar structural features.…”