Crystal structure of the adenylation domain from an ε-poly-l-lysine synthetase provides molecular mechanism for substrate specificity.

ABSTRACT

ε-poly-l-lysine (ε-PL) synthetase (Pls) is a membrane protein that possesses both adenylation and thiolation domains, characteristic of non-ribosomal peptide synthetases (NRPSs). Pls catalyzes the polymerization of l-Lys molecules in a highly specific manner within proteinogenic amino acids. However, this enzyme accepts certain l-Lys analogs which contain small substituent groups at the middle position of the side chain. From the crystal structures of the adenylation domain from NRPSs, the amino acid residues involved in substrate binding can be assumed; however, the precise interactions for better understanding the Pls recognition of l-Lys and its analogs have not yet been fully elucidated. Here, we determined the crystal structure of the adenylation domain of Pls in complex with the intermediate lysyl adenylate at 2.3 Å resolution. This is the first structure determination of the l-Lys activating adenylation domain. The crystal structure reveals that the shape of the substrate-binding pocket determines the specific recognition of l-Lys and its analogs and the electrostatic and hydrogen-bonding interactions further strengthen substrate binding. This study helps us understand the ε-PL synthesis mechanism and contributes to improving our knowledge of the molecular mechanism of NRPS adenylation domains towards their successful application in bioengineering.