Structural bases of the molecular recognition between syringomycin synthetase adenylation domains and their amino acid substrates.

Syringomycin is an antifungal lipodepsinonapeptide containing six modified amino acid residues, produced by many strains of the phytopathogenic bacterium Pseudomonas syringae pv. syringae. It is formed by the nonribosomal mechanism of peptide synthesis where the ATP-dependent activation of the amino acid residues is catalyzed by adenylation domains (A). Previous studies on the structural bases of the molecular recognition between the adenylation domains and their amino acidic substrates led to the discovery of the nonribosomal code, namely a set of ten residues in an adenylation domain substrate binding pocket that are responsible for the interaction with the amino acid. These investigations relied on the structural data deriving from the crystal structure of the PheA, an adenylation domain of gramicidin synthetase cocrystallized with its substrate, and in silico studies of 160 adenylation domains of known substrate specificity. Here we present the results of our studies on the substrate specificity of the adenylation (A) domains of syringomycin synthetase, carried out by using an in silico approach and enzymic assays. Multiple sequence alignment of the A domains of syringomycin synthetase and PheA allowed to identify the residues that delimitate the active site pocket. The percentage of identity between the A domains of SR synthetase and PheA (between 29% and 39%), was sufficiently high to allow the construction of spatial models of SR synthetase adenylation domains which were used for simulations of substrate docking into the active sites of the binding pockets, both in L and D forms. These studies evidenced the stabilizing interactions between the active sites and the L-form of their substrate amino acids, The adenylation domains were cloned, overexpressed, purified and their enzymic activity assayed in the ATP/32PPi exchange aminoacyl adenylation assay. In accordance of the prediction in silico, all the tested domains activated efficiently only the L-isomer of the substrate amino acid. This work is the first study on the structural bases of the stereospecificity of NRPS adenylation domains.