Prolyl aminopeptidase from Streptomyces thermoluteus subsp. fuscus strain NBRC14270 and synthesis of proline-containing peptides by its S144C variant.
We specifically examined an exopeptidase, prolyl aminopeptidase (PAP), as a target for synthesis of proline-containing peptides. A PAP from Streptomyces thermoluteus subsp. fuscus NBRC14270 (PAP14270) was obtained using sequence-based screening. From PAP14270, 144Ser was replaced by Cys (scPAP14270) to give aminolysis activity. In contrast to wild-type PAP14270, scPAP14270 produced a polymer of proline benzyl ester and cyclo[Pro-Pro]. The product mass was confirmed using liquid chromatography-mass spectrometry (LC/MS). Several factors affecting the reaction, such as the pH, concentration of the substrate, and reaction time, were measured to determine their effects. Furthermore, a correlation was found between substrate specificity in proline peptide synthesis and the log D value of acyl acceptors in aminolysis catalyzed by scPAP14270. Results showed that dipeptide synthesis proceeded in a weakly acidic environment and that cyclization and polymerization occurred under alkaline conditions. Furthermore, results suggest that almost all amino acid esters whose log D value is greater than 0, except hydroxyproline benzyl ester (Hyp-OBzl), can be recognized as acyl acceptors. These findings support the use of PAPs as a tool for production of physiologically active proline peptides.
Yamamoto
Yukihiro
Y
Okayama Prefectural Technology Center for Agriculture, Forestry and Fisheries, Research Institute for Biological Sciences (RIBS), Kaga-gun, Okayama 716-1241, Japan.
Usuki
Hirokazu
H
Iwabuchi
Masaki
M
Hatanaka
Tadashi
T
D000626:Aminopeptidases / Q000235:genetics / Q000378:metabolism*; D001593:Benzyl Compounds; D002853:Chromatography, Liquid; D003001:Cloning, Molecular; D017931:DNA Primers / Q000235:genetics; D004926:Escherichia coli; D006863:Hydrogen-Ion Concentration; D013058:Mass Spectrometry; D016296:Mutagenesis; D010452:Peptide Biosynthesis / Q000502:physiology*; D016133:Polymerase Chain Reaction; D011392:Proline / Q000031:analogs & derivatives / Q000096:biosynthesis / Q000378:metabolism* / Q000502:physiology; D013302:Streptomyces / Q000201:enzymology*; D013379:Substrate Specificity; D013997:Time Factors
D001593:Benzyl Compounds; D017931:DNA Primers; C052594:proline benzyl ester; D011392:Proline; D000626:Aminopeptidases; C022450:prolyl aminopeptidase