FRET monitoring of a nonribosomal peptide synthetase

Abstract

Nonribosomal peptide synthetases (NRPSs) are multidomain enzyme templates for the synthesis of bioactive peptides. Large-scale conformational changes during peptide assembly are obvious from crystal structures, yet their dynamics and coupling to catalysis are poorly understood. We have designed an NRPS FRET sensor to monitor, in solution and in real time, the adoption of the productive transfer conformation between phenylalanine-binding adenylation (A) and peptidyl-carrier-protein domains of gramicidin synthetase I from Aneurinibacillus migulanus. The presence of ligands, substrates or intermediates induced a distinct fluorescence resonance energy transfer (FRET) readout, which was pinpointed to the population of specific conformations or, in two cases, mixtures of conformations. A pyrophosphate switch and lysine charge sensors control the domain alternation of the A domain. The phenylalanine-thioester and phenylalanine-AMP products constitute a mechanism of product inhibition and release that is involved in ordered assembly-line peptide biosynthesis. Our results represent insights from solution measurements into the conformational dynamics of the catalytic cycle of NRPSs.

The many faces and important roles of protein–protein interactions during non-ribosomal peptide synthesis

“…Adenylation domains have been shown to exist in at least two different catalytically relevant states, 40,46,73,74 with in solution studies conrming this general mechanism whilst also indicating the potential for different catalytic states to exist in mixed conformations as the reaction progresses. 75 One of the clearest examples of domain alternation in an A-domain has been demonstrated for LgrA (linear gramicidin synthetase A) as reported by Reimer et al in 2016. 46 Although structures of Adomains in different catalytic states were available before, Reimer et al provided snapshots of these catalytic states from the same machinery -LgrA ( Fig.…”

Repurposing Modular Polyketide Synthases and Non-ribosomal Peptide Synthetases for Novel Chemical Biosynthesis

“…However, the size limitation of this technique hindered to be utilized for the whole module structure. Other alternative techniques also reported were the integrated utilization of X-ray crystallography and electron microscopy (Tarry et al, 2017), and Small-angle X-ray scattering (SAXS) or spectroscopic methods for the study of time-resolved conformations (Edwards et al, 2014;Alfermann et al, 2017). Recently, cryo-EM technique was applied to obtain the high-resolution and dynamic structural information of module 5 of pikromycin PKS and pentaketidebound form of the module, but the homogeneous sample should be prepared carefully (Dutta et al, 2014;Whicher et al, 2014).…”

The Biotechnological Potential of Secondary Metabolites from Marine Bacteria

“…The significantly increased interest in obtaining new antibiotic agents derived from secondary metabolites of marine bacteria is associated with the advances in biotechnology that have been made in recent decades [12,13]. They are based on the revealed mechanism of synthesis of major microbial metabolite classes by means of polyketide synthase [14,15] and non-ribosomal peptide synthetase [16,17], which are biosynthetic pathways extensively utilized by marine microorganisms for producing antimicrobial substances [18]. Unlike primary metabolites, the biosynthetic pathways utilized to produce these molecules are numerous and have not been fully understood [8,10].…”

“…It catalyzes the ATP-dependent synthesis of important peptide products with antimicrobial activity from specific sequences of proteinogenic and non-encoded amino acid substrates. The process includes three key sequential stages: acetylation, thyolization, and condensation with the involvement of the same-name key domains and peptide carrier protein [18,59,62] ( Figure 2). Operation of this "assembly line" depends solely on the activity of peptide synthetase, which is a multi-domain modular enzyme complex.…”