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

Abstract

A β-peptidyl aminopeptidase, a peptidase belonging to the P1 family, catalyzes aminolysis in accordance with its hydrolytic activity. We specifically examined β-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 β-homo-Gly-p-nitroanilide (βhGly-pNA), but the products of the enzyme reaction differed completely from one another. Wild-type BapF showed βhGly-βhGly-pNA synthetic activity, but the product vanished in a few minutes and converted to free βhGly. In contrast, the product βhGly-βhGly-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, βhGly-cysteamine was synthesized only in the reaction using S237C BapF. In contrast, S237C BapF was unable to synthesize βhGly-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.

A common active site of polyhydroxyalkanoate synthase from Bacillus cereus YB-4 is involved in polymerization and alcoholysis reactions

“…PhaRC YB4 (C151S) is able to catalyze alcoholysis, as confirmed by NMR analysis, but may acquire hydrolysis activity as well since the molar ratio of the alcohol-capped terminus to the hydroxy terminus was significantly decreased (Table 3). Similar observation has been obtained from studies with β-peptidyl aminopeptidase from Pseudomonas aeruginosa PAO1 (Arima et al 2014). This enzyme has serine as the catalytic center and possesses both aminolysis and hydrolysis activities.…”

Purification and Characterization of a Novel β-Cypermethrin-Degrading Aminopeptidase from Pseudomonas aeruginosa GF31

“…There are some previous reports on the structure and catalytic mechanism of aminopeptidase. It is generally believed that the active center of the aminopeptidase contains a catalytic ternary structure consisting of Glu, Ser, and His. , Similarly, carboxylesterase has a typical “Ser-His-Asp/Glu” catalytic triad . Moreover, whether it is aminopeptidase or carboxylate esterase, the nucleophilic serine is located at a prominent position in the active center, and the oxygen on the serine residue engages in a nucleophilic attack on the carbonyl carbon of the amido bond or ester bond in the catalytic process.…”

Gene cloning and biochemical characterization of eryngase, a serine aminopeptidase of Pleurotus eryngii belonging to the family S9 peptidases

“…17) Herein, mutation of catalytic Ser to Cys or other amino acid to engineer the serine peptidase into "transpeptidase" for peptide bond formation has been well characterized for some serine peptidases. [26][27][28] Therefore, we compared the aminolysis activity between recombinant eryngase and its S524C mutant using L-Phe-NH 2 as a substrate.…”