Issue 3, 2021

Intermediary conformations linked to the directionality of the aminoacylation pathway of nonribosomal peptide synthetases

Abstract

Nonribosomal peptide synthetases (NRPSs) are multifunctional megaenzymes that govern the stepwise biosynthesis of pharmaceutically important peptides. In an ATP-dependent assembly-line mechanism dedicated domains are responsible for each catalytic step. Crystal structures have provided insight into several conformations of interacting domains. However, the complete picture in solution of how domain dynamics and the timing of conformational changes effect a directional biosynthesis remains only poorly understood and will be important for the efficient reprogramming of NRPSs. Here we dissect the multiple conformational changes associated with the adenylation and thiolation reactions of the aminoacylation pathway under catalytic conditions. We used pyrophosphate (PPi) to biochemically drive the conformational changes backward and forward while performing an online monitoring with a Förster resonance energy transfer (FRET) didomain sensor, consisting of adenylation (A) and peptidyl-carrier protein (PCP) domains. Notably, we found aminoacyl thioester formation to efficiently occur in the presence of PPi even at millimolar concentrations, despite the chemically and conformationally reversing effect of this metabolite and by-product. This finding settles conflicting reports and explains why intracellular PPi concentrations do not impair NRP biosynthesis. A conserved amino acid was identified to be important for the mechanism under these conditions. FRET time-course analyses revealed that the directionality of the aminoacylation catalysis is correlated with conformational kinetics. Complemented by equilibrium hydrogen–deuterium exchange (HDX) analyses, our data uncovered the existence of at least one new intermediary conformation that is associated with the rate-determining step. We propose an expanded model of conformational changes in the NRPS aminoacylation pathway.

Graphical abstract: Intermediary conformations linked to the directionality of the aminoacylation pathway of nonribosomal peptide synthetases

Supplementary files

Article information

Article type
Paper
Submitted
02 dec 2020
Accepted
18 feb 2021
First published
04 mar 2021
This article is Open Access
Creative Commons BY license

RSC Chem. Biol., 2021,2, 843-854

Intermediary conformations linked to the directionality of the aminoacylation pathway of nonribosomal peptide synthetases

F. Mayerthaler, A. Feldberg, J. Alfermann, X. Sun, W. Steinchen, H. Yang and H. D. Mootz, RSC Chem. Biol., 2021, 2, 843 DOI: 10.1039/D0CB00220H

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