Substrate flexibility of the adenylation reaction in the Tyrocidine non-ribosomal peptide synthetase

The initiation module of the biosynthetic pathway of Tyrocidine A, a non-ribosomal peptide antibiotic, selectively binds l-phenylalanine along with ATP and Mg2+ catalyzing the formation of an aminoacyl adenylate. The adenylation (activating) reactions of non-ribosomal peptide synthetases are considered the first selectivity filter for correct processing of natural products. Although each adenylation domain is selective for a specific substrate, many of these domains have the ability to adenylate a number of substrates. The overall tolerance of the active site of these domains has not been extensively characterized in terms of substrate kinetics. Exploiting a recently developed ATP-PPi exchange assay protocol, we were able to screen and fully characterize a panel of amino acids and non-proteinogenic substrates to show the broad specificity of the first l-phenylalanine activating domain of Tyrocidine TycA A1. The results revealed unexpected flexibility in the substrate interactions thought necessary for activation. These results could be universal for adenylation domains and can be exploited for the biosynthesis of new and interesting “natural” products.