The specificity-conferring code of adenylation domains in nonribosomal peptide synthetases

Background: Many pharmacologically important peptides are synthesized nonri bosomally by multimodular peptide synthetases (NRPSs). These enzyme templat es:consist of iterated modules that, in their number and organization, dete rmine the primary structure of the corresponding peptide products. At the c ore of-each module is an adenylation domain that recognizes the cognate sub strate and activates it as its aminoacyl adenylate. Recently, the,crystal : structure of the phenylalanine-activating adenylation domain PheA was solv ed with phenylalanine and AMP, illustrating the structural basis for substr ate recognition, Results: By comparing the residues that line the phenylalanine-binding pock et in PheA with the corresponding moieties in other adenylation domains, ge neral rules for deducing substrate specificity were developed. We tested th ese in silico 'rules' by mutating specificity-conferring residues within Ph eA, The substrate specificity of most mutants was altered or relaxed. Gener alization of the selectivity determinants also allowed the targeted specifi city switch of an aspartate activating adenylation domain, the crystal stru cture of which has not yet been solved, by introducing a single mutation. Conclusions: In silico studies and structure-function mutagenesis have defi ned general rules for the structural basis of substrate recognition in aden ylation domains of NRPSs, These rules can be used to rationally alter the s pecificity of adenylation domains and to predict from the primary sequence the specificity of biochemically uncharacterized adenylation domains. Such efforts Could enhance the structural diversity of peptide antibiotics such as penicillins, cyclosporins and vancomycins by allowing synthesis of 'unna tural' natural products.