Two bacterial collagenolytic serine proteases have different topological specificities.
From among 2000 soil isolates, we purified a secreted serine protease from Streptomyces omiyaensis (SOT), which has extremely high gelatinolytic activity. Using sequence analysis, the primary structure of SOT showed 77% identity with that of S. griseus trypsin (SGT). We constructed recombinants SOT and SGT using S. lividans. They indicated similar properties on optimum pH and temperature, thermostability, and substrate preference using fluorescence energy transfer combinatorial libraries. SOT greatly hydrolyzed both type I and type IV collagens, but SGT has poor ability to hydrolyze type IV collagen. Furthermore, SOT exhibits higher hydrolytic activities toward other protein substrate such as gelatin and casein than SGT. These results suggest that these two enzymes have different topological specificities in spite of their similar primary structures. We also constructed chimeras between SOT and SGT to investigate which domain is associated with differences in their substrate specificity. In comparison to substrate specificities of chimeras, we found that the N-terminal domain contributes to the determination of topological specificity.
Uesugi
Yoshiko
Y
Research Institute for Biological Sciences, Okayama, 7549-1 Kibichuo-cho, Kaga-gun, Okayama 716-1241, Japan.
Arima
Jiro
J
Usuki
Hirokazu
H
Iwabuchi
Masaki
M
Hatanaka
Tadashi
T
D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D001483:Base Sequence; D002118:Calcium / Q000378:metabolism / Q000494:pharmacology; D002384:Catalysis / Q000187:drug effects; D003001:Cloning, Molecular; D031541:Fluorescence Resonance Energy Transfer; D006863:Hydrogen-Ion Concentration; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D016415:Sequence Alignment; D020539:Sequence Analysis, Protein; D012697:Serine Endopeptidases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D013302:Streptomyces / Q000201:enzymology; D013379:Substrate Specificity; D013696:Temperature; D014357:Trypsin / Q000378:metabolism
D001426:Bacterial Proteins; D011994:Recombinant Proteins; D012697:Serine Endopeptidases; D014357:Trypsin; D002118:Calcium