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

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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.

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Abbreviations

Tk-subtilisin:

A subtilisin homolog from Thermococcus kodakarensis (Gly70-Gly398)

Pro-TKS:

Tk-subtilisin in a pro-form (Gly1-Gly398)

Tkpro:

Propeptide of Tk-subtilisin (Gly1-Leu69)

IS1:

First insertion sequence (Gly70-Pro82)

Pro-S324A (S324C):

Pro-TKS with the Ser324 → Ala (Ser324 → Cys) mutation

∆IS1-Pro-TKS (S324A, S324C):

Pro-TKS (S324A, S324C) derivative with IS1 deleted

Suc-AAPF-pNA:

N-succinyl-Ala–Ala-Pro-Phe-p-nitroanilide

Tricine:

N-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycine

CD:

Circular dichroism

TCA:

Trichloroacetic acid

DSC:

Differential scanning calorimetry

GdnHCl:

Guanidine hydrochloride

DTT:

Dithiothreitol

ANS:

1-Anillino-8-napthalene sulfonic acid

CBB:

Coomassie Brilliant Blue

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Acknowledgments

This work was supported in part by a Grant (24380055) from the Ministry of Education, Culture, Sports, Science, and Technology of Japan, and by an Industrial Technology Research Grant Program from the New Energy and Industrial Technology Development Organization (NEDO) of Japan. One of the authors (R. Uehara) expresses his special thanks for the Global COE (center of excellence) Program “Global Education and Research Center for Bio-Environmental Chemistry” of Osaka University.

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Author notes

  1. Shun-ichi Tanaka

    Present address: Gifu R&D Center, Amano Enzyme Inc, 1-6, Kagamihara, Gifu, 509-0109, Japan

  2. Kazufumi Takano

    Present address: Graduate School of Life and Environmental Sciences, Kyoto Prefectural University, 1-5 Hangi-cho, Shimogamo, Sakyo-ku, Kyoto, 606-8522, Japan

Authors and Affiliations

  1. Department of Material and Life Science, Graduate School of Engineering, Osaka University, 2-1 Yamadaoka, Suita, Osaka, 565-0871, Japan

    Ryo Uehara, Shun-ichi Tanaka, Kazufumi Takano, Yuichi Koga & Shigenori Kanaya

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  1. Ryo Uehara
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  2. Shun-ichi Tanaka
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  3. Kazufumi Takano
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  4. Yuichi Koga
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  5. Shigenori Kanaya
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Correspondence to Shigenori Kanaya.

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Communicated by H. Atomi.

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Uehara, R., Tanaka, Si., Takano, K. et al. Requirement of insertion sequence IS1 for thermal adaptation of Pro-Tk-subtilisin from hyperthermophilic archaeon. Extremophiles 16, 841–851 (2012). https://doi.org/10.1007/s00792-012-0479-3

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  • DOI: https://doi.org/10.1007/s00792-012-0479-3

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