title: Modulation of Streptomyces leucine aminopeptidase by calcium: identification and functional analysis of key residues in activation and stabilization by calcium.
abstract: Streptomyces griseus leucine aminopeptidase (SGAP), which has two zinc atoms in its active site, is clinically important as a model for understanding the structure and mechanism of action of other metallopeptidases. SGAP is a calcium-activated and calcium-stabilized enzyme, and its activation by calcium correlates with substrate specificity. In our previous study, we found a non-calcium-modulated leucine aminopeptidase secreted by Streptomyces septatus, the primary structure of which showed 71% identity with SGAP. In this study, we constructed chimeras of SGAP and S. septatus aminopeptidase by using an in vivo DNA shuffling system and several mutant enzymes by site-directed mutagenesis to identify the key residues in this modulation by calcium. We identified the key residues Asp-173 and Asp-174 of SGAP associated with both SGAP activation and stabilization by calcium. We also showed that the known calcium-binding site, which is composed of Asp-3, Ile-4, Asp-262, and Asp-266 of SGAP, only contributes to SGAP stabilization by calcium. Furthermore, we identified an important residue, Glu-196, that functions in cooperation with Asp-173, Asp-174, and calcium to increase the catalytic activity of SGAP.
authors.affiliation: Research Institute for Biological Sciences, Okayama, 7549-1 Kibichuo-cho, Kaga-gun, Okayama 716-1241, Japan.
authors.forename: Jiro
authors.initials: J
authors.lastname: Arima
authors.forename: Yoshiko
authors.initials: Y
authors.lastname: Uesugi
authors.forename: Misugi
authors.initials: M
authors.lastname: Uraji
authors.forename: Shouki
authors.initials: S
authors.lastname: Yatsushiro
authors.forename: Seiji
authors.initials: S
authors.lastname: Tsuboi
authors.forename: Masaki
authors.initials: M
authors.lastname: Iwabuchi
authors.forename: Tadashi
authors.initials: T
authors.lastname: Hatanaka
mesh_terms: D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D001665:Binding Sites; D002118:Calcium / Q000378:metabolism*; D004789:Enzyme Activation; D004795:Enzyme Stability; D007931:Leucyl Aminopeptidase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D011487:Protein Conformation; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D016415:Sequence Alignment; D013302:Streptomyces / Q000201:enzymology*
chemical_list: D001426:Bacterial Proteins; D011993:Recombinant Fusion Proteins; D007931:Leucyl Aminopeptidase; D002118:Calcium