Putative "acylaminoacyl" peptidases from Streptomyces griseus and S. coelicolor display "aminopeptidase" activities with distinct substrate specificities and sensitivities to reducing reagent.
Aminopeptidases from Streptomyces griseus (SGRAP) and S. coelicolor (SCOAP) were cloned and characterized to clarify their biochemical characteristics. Although both enzymes had been annotated as putative oligopeptidases of family S9 enzymes, they showed "aminopeptidase" activities, not "oligopeptidase" activities. Although their deduced amino acid sequences showed high similarity (69% overall sequence homology), they showed distinct substrate specificities and sensitivities to the reducing reagent dithiothreitol (DTT). The reaction pH and addition of DTT dramatically affected the substrate preference of SGRAP. Furthermore, SCOAP selectively hydrolyzed phenyalanine p-nitroanilide (Phe-pNA) in the presence or absence of DTT. The chimera protein between SGRAP and SCOAP was constructed to identify the region responsible for the properties described above. Furthermore, Cys(409) of SCOAP was identified as a functional residue responsible for activation by reducing reagent DTT.
Usuki
Hirokazu
H
Research Institute for Biological Sciences (RIBS), Okayama, 7549-1 Kibichuo-cho, Kaga-gun, Okayama 716-1241, Japan.
Uesugi
Yoshiko
Y
Iwabuchi
Masaki
M
Hatanaka
Tadashi
T
D000595:Amino Acid Sequence; D000626:Aminopeptidases / Q000378:metabolism*; D003001:Cloning, Molecular; D004229:Dithiothreitol / Q000494:pharmacology; D006863:Hydrogen-Ion Concentration; D008969:Molecular Sequence Data; D010447:Peptide Hydrolases / Q000235:genetics / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D016415:Sequence Alignment; D048372:Streptomyces coelicolor / Q000201:enzymology* / Q000235:genetics; D013305:Streptomyces griseus / Q000201:enzymology* / Q000235:genetics; D013379:Substrate Specificity / Q000187:drug effects
D011993:Recombinant Fusion Proteins; D010447:Peptide Hydrolases; C028759:oligopeptidase; D000626:Aminopeptidases; D004229:Dithiothreitol