Gene cloning and overproduction of an aminopeptidase from Streptomyces septatus TH-2, and comparison with a calcium-activated enzyme from Streptomyces griseus.
An aminopeptidase secreted from Streptomyces septatus TH-2 (SSAP) was identified as a heat stable enzyme, and the Ssap gene was cloned and sequenced. The primary structure of SSAP showed 71% identity with that of a Streptomyces griseus aminopeptidase (SGAP), however, it lacked a unique calcium binding site. The recombinant SSAP was overexpressed in the culture supernatant of Escherichia coli harboring pET-KmS2. A comparison of recombinant SSAP and SGAP showed that both enzymes are different in terms of modulation by calcium and substrate specificity. The activity of SSAP was not modulated by calcium, while SGAP is a calcium-activated enzyme. SSAP catalyzed the hydrolysis of L-Lys-pNA efficiently whereas the reaction rate for L-Lys-pNA hydrolysis of SGAP was significantly low. Furthermore, in SGAP, the presence of Ca2+ decreased the reaction rate of L-Lys-pNA hydrolysis. SSAP also had different pKas s of reaction from that of SGAP, although almost all the residues which compose the active site were conserved in both enzymes. This result indicates that SSAP has a different environment of substrate binding and active sites from those of SGAP.
Arima
Jiro
J
Research Institute for Biological Sciences, Okayama (RIBS), Kayo-cho, Jyobo-gun, Okayama 716-1241, Japan.
Iwabuchi
Masaki
M
Hatanaka
Tadashi
T
D000595:Amino Acid Sequence; D000626:Aminopeptidases / Q000096:biosynthesis* / Q000737:chemistry* / Q000145:classification / Q000235:genetics; D002118:Calcium / Q000737:chemistry* / Q000378:metabolism*; D003001:Cloning, Molecular; D004789:Enzyme Activation; D004795:Enzyme Stability; D006863:Hydrogen-Ion Concentration; D008969:Molecular Sequence Data; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry; D017386:Sequence Homology, Amino Acid; D013302:Streptomyces / Q000201:enzymology* / Q000235:genetics; D013305:Streptomyces griseus / Q000201:enzymology* / Q000235:genetics; D013696:Temperature
D011994:Recombinant Proteins; D000626:Aminopeptidases; D002118:Calcium