Dataset Information

FRET Monitoring of a Nonribosomal Peptide Synthetase Elongation Module Reveals Carrier Protein Shuttling between Catalytic Domains.

ABSTRACT: Nonribosomal peptide synthetases (NRPSs) employ multiple domains, specifically arranged in modules, for the assembly-line biosynthesis of a plethora of bioactive peptides. It is poorly understood how catalysis is correlated with the domain interplay and associated conformational changes. We developed FRET sensors of an elongation module to study in solution the intramodular interactions of the peptidyl carrier protein (PCP) with adenylation (A) and condensation (C) domains. Backed by HDX-MS analysis, we discovered dynamic mixtures of conformations that undergo distinct population changes in favor of the PCP-A and PCP-C interactions upon completion of the adenylation and thiolation reactions, respectively. To probe this model we blocked PCP binding to the C domain by photocaging and triggered peptide bond formation with light. Changing intramodular domain affinities of the PCP appear to result in conformational shifts according to the logic of the templated assembly process.

SUBMITTER: Ruschenbaum J

PROVIDER: S-EPMC9828546 | biostudies-literature | 2022 Nov

REPOSITORIES: biostudies-literature

ACCESS DATA

Publications

FRET Monitoring of a Nonribosomal Peptide Synthetase Elongation Module Reveals Carrier Protein Shuttling between Catalytic Domains.

Rüschenbaum Jennifer J Steinchen Wieland W Mayerthaler Florian F Feldberg Anna-Lena AL Mootz Henning D HD

Angewandte Chemie (International ed. in English) 20221026 48

Nonribosomal peptide synthetases (NRPSs) employ multiple domains, specifically arranged in modules, for the assembly-line biosynthesis of a plethora of bioactive peptides. It is poorly understood how catalysis is correlated with the domain interplay and associated conformational changes. We developed FRET sensors of an elongation module to study in solution the intramodular interactions of the peptidyl carrier protein (PCP) with adenylation (A) and condensation (C) domains. Backed by HDX-MS anal ...[more]

PMID: 36169151

Similar Datasets

Project description:Hybrid natural products are compounds that originate from diverse biosynthetic pathways and undergo a conjugation process, which enables them to expand their chemical diversity and biological functionality. Terpene-amino acid meroterpenoids have garnered increasing attention in recent years, driven by the discovery of noteworthy examples such as the anthelmintic CJ-12662, the insecticidal paeciloxazine, and aculene A (1). In the biosynthesis of terpene-amino acid natural products, single-module nonribosomal peptide synthetases (NRPSs) have been identified to be involved in the esterification step, catalyzing the fusion of modified terpene and amino acid components. Despite prior investigations into these NRPSs through gene deletion or in vivo experiments, the enzymatic basis and mechanistic insights underlying this family of single-module NRPSs remain unclear. In this study, we performed biochemical characterization of AneB by in vitro characterization, molecular docking, and site-directed mutagenesis. The enzyme reaction analyses, performed with L-proline and daucane/nordaucane sesquiterpene substrates, revealed that AneB specifically esterifies the C10-OH of aculenes with L-proline. Notably, in contrast to ThmA in CJ-12662 biosynthesis, which exclusively recognizes oxygenated amorpha-4,11-diene sesquiterpenes for L-tryptophan transfer, AneB demonstrates broad substrate selectivity, including oxygenated amorpha-4,11-diene and 2-phenylethanol, resulting in the production of diverse unnatural prolyl compounds. Furthermore, site-directed mutagenesis experiments indicated the involvement of H794 and D798 in the esterification catalyzed by AneB. Lastly, domain swapping between AneB and ThmA unveiled that the A‒T domains of ThmA can be effectively harnessed by the C domain of AneB for L-tryptophan transfer, thus highlighting the potential of the C domain of AneB for generating various terpene-amino acid meroterpenoid derivatives.One-sentence summaryThe enzymatic basis and mechanistic insights into AneB, a single-module NRPS, highlight its capacity to generate various terpene-amino acid meroterpenoid derivatives.

Dataset Information

FRET Monitoring of a Nonribosomal Peptide Synthetase Elongation Module Reveals Carrier Protein Shuttling between Catalytic Domains.

Publications

FRET Monitoring of a Nonribosomal Peptide Synthetase Elongation Module Reveals Carrier Protein Shuttling between Catalytic Domains.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets