Substrate recognition and selection by the initiation module PheATE of gramicidin S synthetase

The initiation module of non-ribosomal peptide synthetases (NRPS) selects a nd activates the first amino acid and serves as the aminoacyl donor in the first peptide bond-forming step of the NRPS assembly line. The gramicidin S synthetase initiation module (PheATE) is a three-domain subunit, recognizi ng L-phenylalanine (L-Phe) and activating it (by adenylation domain) as tig htly bound L-phenylalanyl-adenosine-5'-monophosphate diester (L-Phe-AMP), t ransferring it to the HS-phosphopantetheine arm of the holo-thiolation (hol o-T) domain, and then epimerizing it (by epimetization domain) to the D-Phe -S-4'-Ppant-acyl enzyme. In this study, we have assayed the selectivity of the PheATE adenylation domain with a number of proteinogenic amino acids an d observed that three additional amino acids. L-Tyr. L-Trp, and L-Leu, were activated to the aminoacyl-AMPs and transferred to the HS-phosphopantethei ne arm of the holo-T domain. Hydrolytic editing of noncognate aminoacyl-AMP s and/or aminoacyl-S-4'-Ppant-acyl enzymes by the enzyme was not observed b y three different assays for adenylation domain function. The microscopic r eaction rates and thermodynamic equilibrium constants obtained from single- turnover studies of reactions Of L-Phe, L-Trp, L-Tyr, and L-Leu with holoPh eATE allowed us to construct free energy profiles for the reactions, reveal ing the kinetic and thermodynamic basis for substrate recognition and selec tion. In particular, the rates of epimerization of the L-aminoacyl-S-enzyme to the D-aminoacyl-S-enzyme intermediate showed reductions of 245-, 300, a nd 540-fold for L-Trp, L-Tyr, and L-Leu respectively, suggesting that the e pimerization domain is an important gatekeeper for generation of the D-Phe- S-enzyme that starts gramicidin S chain growth.