title: Molecular dissection of Streptomyces trypsin on substrate recognition.
abstract: We recently identified residue 71 of two homologous serine proteases from Streptomyces omiyaensis (SOT) and Streptomyces griseus (SGT) as a crucial residue for differences in their topological specificities, i.e. recognition of a distinct three-dimensional structure. To study the role of this key residue in substrate recognition, we used surface plasmon resonance analysis to evaluate the affinities of inactive mutants, in which residues 71 of SOT and SGT were substituted respectively with Leu and Tyr, toward different types of collagens. We identified another amino acid residue involved in the interaction with collagens from analyses of inactive chimeras between SOT and SGT using an in vivo DNA shuffling system. Results showed that residue 72 contributes to collagen binding. By substituting Leu71 and Gln72 with Tyr and Arg, respectively, SGT mutant showed a change in topological specificity and high hydrolytic activity toward type IV collagen comparable to SOT. We demonstrated that the neighboring residues 71 and 72 in the N-terminal β-barrel domain of the enzyme synergistically play an important role in substrate recognition.
authors.affiliation: Research Institute for Biological Sciences, Okayama, Japan.
authors.forename: Yoshiko
authors.initials: Y
authors.lastname: Uesugi
authors.forename: Hirokazu
authors.initials: H
authors.lastname: Usuki
authors.forename: Jiro
authors.initials: J
authors.lastname: Arima
authors.forename: Masaki
authors.initials: M
authors.lastname: Iwabuchi
authors.forename: Tadashi
authors.initials: T
authors.lastname: Hatanaka
mesh_terms: D000595:Amino Acid Sequence; D003094:Collagen / Q000378:metabolism; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D050505:Mutant Proteins / Q000737:chemistry / Q000378:metabolism; D011485:Protein Binding; D011487:Protein Conformation; D020539:Sequence Analysis, Protein; D017386:Sequence Homology, Amino Acid; D013302:Streptomyces / Q000201:enzymology* / Q000235:genetics / Q000378:metabolism; D013305:Streptomyces griseus / Q000201:enzymology / Q000235:genetics / Q000378:metabolism; D013379:Substrate Specificity; D014357:Trypsin / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*
chemical_list: D050505:Mutant Proteins; D003094:Collagen; D014357:Trypsin