Structural Elucidation of the Bispecificity of A Domains as a Basis for Activating Non-natural Amino Acids

Many biologically active peptide secondary metabolites of bacteria are produced by modular enzyme complexes, the non-ribosomal peptide synthetases. Substrate selection occurs through an adenylation (A) domain, which activates the cognate amino acid with high fidelity. The recently discovered Adomain of an Anabaenopeptin synthetase from Planktothrix agardhii (ApnA A(1)) is capable of activating two chemically distinct amino acids (Arg and Tyr). Crystal structures of the Adomain reveal how both substrates fit into to binding pocket of the enzyme. Analysis of the binding pocket led to the identification of three residues that are critical for substrate recognition. Systematic mutagenesis of these residues created Adomains that were monospecific, or changed the substrate specificity to tryptophan. The non-natural amino acid 4-azidophenylalanine is also efficiently activated by a mutant Adomain, thus enabling the production of diversified non-ribosomal peptides for bioorthogonal labeling.