Structural Insights into the Substrate Recognition Mechanism of an ATP-Grasp Peptide-Ligase Producing Diverse Dipeptides Containing Unnatural Amino Acids

ATP-grasp enzymes are involved in many different biological processes of primary and secondary metabolism and catalyze the production of many physiological or medicinally valuable peptides. Here, we report an ATP-grasp enzyme Alb28, from Streptomyces albogriseolus MGR072, which exhibits broad substrate promiscuity and strict stereoselectivity, thereby capable of producing at least 55 structurally diversified dipeptides, greatly expanding the existing dipeptide library. By combining crystallographic studies, molecular dynamics simulations, and mutagenesis assays, we identified the critical residues in Alb28 responsible for regulating the substrate recognition and enzymatic catalysis. Particularly, two structural motifs in Alb28 are involved in dictating the entry of substrate into the active-site via the opening/closing motions. Our work potentiates the future applications of Alb28 in generating structurally diversified dipeptides for therapeutic usages.