TAILIEUCHUNG - Báo cáo khoa học: The role of Tyr71 in Streptomyces trypsin on the recognition mechanism of structural protein substrates

Studies of substrate recognition by serine proteases have focused on speci-ficities at the primary S1–Sn sites, but topological specificities (. recogni-tion at distinct three-dimensional structural motifs) have not been established. This is the first report to identify the key amino acid residue conferring topological specificity. | ễFEBS Journal The role of Tyr71 in Streptomyces trypsin on the recognition mechanism of structural protein substrates Yoshiko Uesugi Hirokazu Usuki Masaki Iwabuchi and Tadashi Hatanaka Research Institute for BiologicalSciences Okayama Japan Keywords collagenolytic enzyme repeat-length independent and broad spectrum RIBS in vivo DNA shuffling serine protease Streptomyces topologicalspecificity Correspondence T. Hatanaka Research Institute for BiologicalSciences Okayama 7549-1 Kibichuo-cho Kaga-gun Okayama 716-1241 Japan Fax 81 866 56 9454 Tel 81 866 56 9452 E-mail hatanaka@ Present address Department of Biomaterials Field of Tissue Engineering Institute for Frontier Medical Sciences Kyoto University Japan Studies of substrate recognition by serine proteases have focused on specificities at the primary S1-Sn sites but topological specificities . recognition at distinct three-dimensional structural motifs have not been established. This is the first report to identify the key amino acid residue conferring topological specificity. A serine protease from Streptomyces omi-yaensis SOT which is a trypsin-like enzyme was chosen as a model enzyme to clarify the recognition mechanism of structural protein substrates in serine proteases. We have found previously that the topological specificities of SOT and S. griseus trypsin SGT for high molecular mass substrates differ greatly even though the enzymes have similar primary structures. In this study we constructed chimeras between SOT and SGT using an in vivo DNA shuffling system and several mutants to identify the key residues involved in topological specificities. By comparing the substrate specificities of chimeras and mutants we found that residue 71 of SOT which is separate from the catalytic triad contributes to the topological specificity. Using site-directed mutagenesis residue 71 of SOT was also found to be crucial for catalytic efficiency and enzyme conformation. Received 7 April2009 revised 26 July .

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