title: Requirement of insertion sequence IS1 for thermal adaptation of Pro-Tk-subtilisin from hyperthermophilic archaeon.
abstract: Tk-subtilisin from the hyperthermophilic archaeon Thermococcus kodakarensis matures from Pro-Tk-subtilisin (Pro-TKS) upon autoprocessing and degradation of propeptide. Pro-TKS contains the insertion sequence (IS1) at the N-terminus of the mature domain as compared to bacterial pro-subtilisins. To analyze the role of IS1, the Pro-TKS derivative without IS1 (∆IS1-Pro-TKS) and its active-site mutants (∆IS1-Pro-S324A and ∆IS1-Pro-S324C) were constructed and characterized. ∆IS1-Pro-S324A and ∆IS1-Pro-TKS represent an unautoprocessed and autoprocessed form of ∆IS1-Pro-TKS, respectively. The CD and ANS fluorescence spectra of these proteins indicate that folding of ∆IS1-Pro-TKS is not completed by binding of Ca(2+) ions but is completed by the subsequent autoprocessing reaction. Thermal denaturation of these proteins analyzed by DSC and CD spectroscopy indicates that unautoprocessed ∆IS1-Pro-TKS is less stable than autoprocessed ∆IS1-Pro-TKS by 26.3 °C in T (m). The stability of autoprocessed ∆IS1-Pro-TKS is comparable to that of Pro-TKS, which is slightly lower than that of unautoprocessed Pro-TKS. These results suggest that ∆IS1-Pro-TKS is fully folded and greatly stabilized by autoprocessing. ∆IS1-Pro-TKS more slowly matured to ∆IS1-Tk-subtilisin than Pro-TKS did, due to a decrease in the autoprocessing rate. We propose that IS1 is required not only for hyperstabilization of Pro-TKS but also for its rapid maturation.
authors.affiliation: Department of Material and Life Science, Graduate School of Engineering, Osaka University, 2-1 Yamadaoka, Suita, Osaka, 565-0871, Japan.
authors.forename: Ryo
authors.initials: R
authors.lastname: Uehara
authors.forename: Shun-Ichi
authors.initials: S
authors.lastname: Tanaka
authors.forename: Kazufumi
authors.initials: K
authors.lastname: Takano
authors.forename: Yuichi
authors.initials: Y
authors.lastname: Koga
authors.forename: Shigenori
authors.initials: S
authors.lastname: Kanaya
mesh_terms: D000220:Adaptation, Biological; D000595:Amino Acid Sequence; D019843:Archaeal Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D002118:Calcium / Q000378:metabolism; D020134:Catalytic Domain; D004792:Enzyme Precursors / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D006358:Hot Temperature; D008969:Molecular Sequence Data; D016254:Mutagenesis, Insertional; D010446:Peptide Fragments / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D011489:Protein Denaturation; D055550:Protein Stability; D013381:Subtilisins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D019712:Thermococcus / Q000201:enzymology*
chemical_list: D019843:Archaeal Proteins; D004792:Enzyme Precursors; D010446:Peptide Fragments; C057107:prosubtilisin; D013381:Subtilisins; D002118:Calcium