Exploitation of the Selectivity-Conferring Code of Nonribosomal Peptide Synthetases for the Rational Design of Novel Peptide Antibiotics

Recently, the solved crystal structure of a phenylalanine-activating adenylation (A) domain enlightened the structural basis for the specific recognition of the cognate substrate amino acid in nonribosomal peptide synthetases (NRPSs). By adding sequence comparisons and homology modeling, we successfully used this information to decipher the selectivity-conferring code of NRPSs. Each codon combines the 10 amino residues of a NRPS A domain that are presumed to build up the substrate-binding pocket. In this study, the deciphered code was exploited for the first time to rationally alter the substrate specificity of whole NRPS modules in vitro and in vivo. First, the single-residue Lys239 of the l-Glu-activating initiation module C−AGlu−PCP of the surfactin synthetase A was mutated to Gln239 to achieve a perfect match to the postulated l-Gln-activating binding pocket. Biochemical characterization of the mutant protein C−AGlu−PCP(Lys239 → Gln) revealed the postulated alteration in substrate specificity from l-Glu to l-Gln without decrease in catalytic efficiency. Second, according to the selectivity-conferring code, the binding pockets of l-Asp and l-Asn-activating A domains differs in three positions: Val299 versus Ile, His322 versus Glu, and Ile330 versus Val, respectively. Thus, the binding pocket of the recombinant A domain AspA, derived from the second module of the surfactin synthetases B, was stepwisely adapted for the recognition of l-Asn. Biochemical characterization of single, double, and triple mutants revealed that His322 represents a key position, whose mutation was sufficient to give rise to the intended selectivity-switch. Subsequently, the gene fragment encoding the single-mutant AspA(His322 → Glu) was introduced back into the surfactin biosynthetic gene cluster. The resulting Bacillus subtilis strain was found to produce the expected so far unknown lipoheptapeptide [Asn5]surfactin. This indicates that site-directed mutagenesis, guided by the selectivity-conferring code of NRPS A domains, represents a powerful alternative for the genetic manipulation of NRPS biosynthetic templates and the rational design of novel peptide antibiotics.