Uraji_2007_Biochim.Biophys.Acta_1774_1462

A salt-tolerant prolyl aminopeptidase from Streptomyces aureofaciens TH-3 (TH-3PAP) was purified from a culture supernatant. The gene encoding TH-3PAP was cloned and sequenced. The primary structure of TH-3PAP showed 65% identity with that of PAP from Streptomyces lividans (SLPAP) and possessed a conserved catalytic motif, GxSxGG, which is conserved in the alpha/beta hydrolase fold family. The characterization of the recombinants TH-3PAP and SLPAP indicated a difference: in 4.0 M NaCl, TH-3PAP showed enzyme activity, whereas SLPAP was inactive. Next, we constructed chimeras between TH-3PAP and SLPAP using an in vivo DNA shuffling system and a sandwich chimera (sc-PAP), whose region from 63 to 78 amino acids of TH-3PAP was substituted with that of SLPAP. Comparison of the biochemical properties between TH-3PAP and the salt-sensitive sc-PAP suggested that the fine tuning of the N-terminal conformation of TH-3PAP by hydrophobic interaction is important for the salt tolerance mechanism of the enzyme.