The Schmeing Lab

Visualizing and understanding nature’s biosynthetic macromolecular machines

NRPS termination domains

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NRPSs need specialized termination domains to release the final product. This is often the thioesterase (TE) domain. The TE catalytic mechanism proceeds through two half reactions: In the first step, the linear substrate made by the enzyme is transferred from the final PCP domain to the TE domain active site serine, forming a covalently attached acyl-enzyme intermediate. In the second step, the TE domain can catalyze one of three alternatives: 1). The acyl-enzyme intermediate can be attacked by a water nucleophile and hydrolyzed to release a linear product. 2) The acyl-enzyme intermediate can be attacked by nucleophile in the intermediate itself, for example a hydroxyl in a side chain or the free N-terminal amino group, to release a cyclic product. 3) The acyl-enzyme intermediate can wait for the upstream modules to produce a new peptide on the final PCP domain to oligomerize the peptide. The TE domain later catalyzes the release of the oligomerized peptide by cyclization or hydrolysis. The decision to hydrolyze, cyclize or oligomerize appears to depend on both the identity of the TE domain catalyzing the reaction and the identity of the substrate of the reaction, and has been difficult to understand or predict. The mode of release and thus the form of the product (linear, cyclic, multimer, cyclic multimer) has a huge impact on the activity of the product: A cyclic antibiotic will NOT have potency or antibiotic activity if it is released as a linear peptide. Thus, this is an extremely important step to characterize, for the fundamental understanding of megaenzymes.

Relevant Schmeing lab papers: Huguenin-Dezot et al, Nature, 2019; Argyropoulos et al, Biochim Biophys Acta, 2016