Mutation of active site serine residue with cysteine displays change in acyl-acceptor preference of ß-peptidyl aminopeptidase from Pseudomonas aeruginosa PAO1.

A ß-peptidyl aminopeptidase, a peptidase belonging to the P1 family, catalyzes aminolysis in accordance with its hydrolytic activity. We specifically examined ß-peptidyl aminopeptidase of Pseudomonas aeruginosa PAO1 (BapF) to assess the effects of mutation of catalytic Ser with Cys or Thr on its catalytic ability. Recombinant BapF and its S237C mutant exhibited p-nitroaniline release activity toward ß-homo-Gly-p-nitroanilide (ßhGly-pNA), but the products of the enzyme reaction differed completely from one another. Wild-type BapF showed ßhGly-ßhGly-pNA synthetic activity, but the product vanished in a few minutes and converted to free ßhGly. In contrast, the product ßhGly-ßhGly-pNA was synthesized by S237C BapF efficiently without degradation, indicating that because of the mutation, the enzyme came to recognize only the amine group as an acyl acceptor instead of water. Furthermore, a difference in acyl acceptor preference between that of wild type and S237C BapF was observed. When using cysteamine as an acyl acceptor, ßhGly-cysteamine was synthesized only in the reaction using S237C BapF. In contrast, S237C BapF was unable to synthesize ßhGly-cystamine when using cystamine as an acyl acceptor, although it was synthesized by wild-type BapF. Such a dynamic change in the acyl acceptor by the mutation of catalytic Ser with Cys is regarded as a unique feature of family P1 peptidases.

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%.

Beta-alanyl peptide synthesis by Streptomyces S9 aminopeptidase.

Synthesis of beta-alanine (beta-Ala) containing dipeptide using S9 aminopeptidase from Streptomyces thermocyaneoviolaceus NBRC14271 (S9AP-St) was demonstrated with beta-Ala-benzyl ester (-OBzl) and various L-aminoacyl derivatives. For synthesis of beta-Ala-containing dipeptide, beta-Ala-OBzl was used preferentially as the acyl donor for S9AP-St, producing synthesized dipeptides having beta-Ala-Xaa structure. In contrast, engineering of S9AP-St into "transaminopeptidase" by substitution of catalytic Ser with Cys--designated as aminolysin-S--produced only dipeptides having Xaa-beta-Ala structure. Investigation of the specificity of S9AP-St toward acyl acceptors showed that S9AP has a broad substrate specificity toward various aminoacyl derivatives. Furthermore, S9AP-St produced carnosine methyl ester (-OMe) with a conversion ratio of beta-Ala-OBzl to carnosine-OMe that was greater than 30%.