The Aminolysis Reaction of Streptomyces S9 Aminopeptidase Promotes the Synthesis of Diverse Prolyl Dipeptides

Prolyl dipeptide synthesis by S9 aminopeptidase from Streptomyces thermocyaneoviolaceus (S9AP-St) has been demonstrated. In the synthesis, S9AP-St preferentially used l-Pro-OBzl as the acyl donor, yielding synthesized dipeptides having an l-Pro-Xaa structure. In addition, S9AP-St showed broad specificity toward the acyl acceptor. Furthermore, S9AP-St produced cyclo (l-Pro-l-His) with a conversion ratio of substrate to cyclo (l-Pro-l-His) higher than 40%.Some proline-containing dipeptides and their cyclic analogs exhibit biological activity. For example, cyclo (l-arginyl-d-proline) c(lR-dP)] is known to act as a specific inhibitor of family 18 chitinase (4, 10). A cyclic peptide, c(lP-lH), produced by the cleavage of thyrotropin-releasing hormone protects against oxidative stress, promotes cytoprotection (6, 7), and exhibits antihyperglycemic activity (11).Some serine peptidases exhibit peptide bond formation (i.e., aminolysis of esters, thioesters, and amides) in accordance with their hydrolytic activity (2, 14). The exchange of catalytic Ser for Cys to engineer the serine endopeptidase into “transpeptidase” for peptide bond formation has been well characterized (3, 5). Our recent approach confirmed the wide distribution of family S9 aminopeptidases that have catalytic Ser in actinomycetes (12). Of them, we obtained S9 aminopeptidase from Streptomyces thermocyaneoviolaceus NBRC14271 (S9AP-St). The enzyme was engineered into “transaminopeptidase” by exchange of catalytic Ser for Cys, and its aminolytic activity was evaluated (13). The engineered enzyme, designated as aminolysin-S, can synthesize hydrophobic dipeptides through an aminolysis reaction. However, aminolysin-S was unable to synthesize peptides containing proline. Although the report of aminolysin-S demonstrated that S9AP-St shows no aminolysis reaction toward limited substrates, details of its characteristics remain unknown. This study verified the peptide synthetic activity of S9AP-St, demonstrating that S9AP-St can synthesize widely varied prolyl dipeptides through an aminolysis reaction. The report also shows that S9AP-St is applicable to the synthesis of a biologically active peptide—c(lP-lH).