Utility of epimerization domains for the redesign of nonribosomal peptide synthetases

Many pharmacologically important agents are assembled on multimodular nonribosomal peptide synthetases (NRPSs) whose modules comprise a set of core domains with all essential catalytic functions necessary for the incorporation and modi. cation of one building block. Very often, D-amino acids are found in such products which, with few exceptions, are generated by the action of NRPS integrated epimerization ( E) domains that alter the stereochemistry of the corresponding peptidyl carrier protein ( PCP) bound (L)-intermediate. In this study we present a quantitative investigation of substrate specificity of four different E domains (two ' peptidyl-' and two ' aminoacyl-' E domains) derived from different NRPSs towards PCP bound peptides. The respective PCP-E bidomain apo-proteins (TycB(3)-, FenD(2)-, TycA- and GrsA- PCP- E) were primed with various peptidyl- CoA precursors by utilizing the promiscuous phosphopantetheinyl transferase Sfp. PCP bound peptidyl-S-Ppant epimerization products were chemically cleaved and analyzed for their L/D-ratios by LCMS. We were able to show that all four E domains tolerate a broad variety of peptidyl-S-Ppant-substrates as evaluated by k(obs) values and final L/D-product equilibria determined for each reaction. The two C-terminal amino acids of the substrate seem to be recognized by ' peptidyl-' E domains. Interestingly, the ' amino-acyl ' E domains GrsA- and TycA- E were also able to convert the elongated intermediates. All four E domains accepted an N-methylated precursor as well and epimerized this substrate with high efficiency. Finally, we could demonstrate that the condensation ( C) domain of TycB(1) is also able to process peptidyl substrates transferred by TycA. In conclusion, these findings are of great impact on future engineering attempts.