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

Florian Mayerthaler, Anna Lena Feldberg, Jonas Alfermann, Xun Sun, Wieland Steinchen, Haw Yang, Henning D. Mootz

Research output: Contribution to journalArticlepeer-review

10 Scopus citations


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.

Original languageEnglish (US)
Pages (from-to)843-854
Number of pages12
JournalRSC Chemical Biology
Issue number3
StatePublished - Jun 2021

All Science Journal Classification (ASJC) codes

  • Chemistry (miscellaneous)
  • Molecular Biology
  • Biochemistry, Genetics and Molecular Biology (miscellaneous)
  • Biochemistry


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