NDLI: Requirement of insertion sequence IS1 for thermal adaptation of Pro-Tk-subtilisin from hyperthermophilic archaeon

Content Provider	Springer Nature Link
Author	Uehara, Ryo Tanaka, Shun ichi Taka, Kazufumi Koga, Yuichi Kanaya, Shigeri
Copyright Year	2012
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 Ca2+ 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.
Starting Page	841
Ending Page	851
Page Count	11
File Format	PDF
ISSN	14310651
Journal	Extremophiles
Volume Number	16
Issue Number	6
e-ISSN	14334909
Language	English
Publisher	Springer Japan
Publisher Date	2012-09-21
Publisher Place	Japan
Access Restriction	One Nation One Subscription (ONOS)
Subject Keyword	Subtilisin Thermococcus kodakarensis Maturation Folding Insertion sequence Biotechnology Ecology Microbiology
Content Type	Text
Resource Type	Article
Subject	Medicine Molecular Medicine Microbiology

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