PEDS Advance Access published online on March 9, 2007
Protein Engineering Design and Selection, doi:10.1093/protein/gzm006
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Directed evolution of Tk-subtilisin from a hyperthermophilic archaeon: identification of a single amino acid substitution responsible for low-temperature adaptation
1 Department of Material and Life Science, Graduate School of Engineering, Osaka University, 2-1 Yamadaoka, Suita, Osaka 565-0871, Japan 2 PRESTO, JST, 2-1 Yamadaoka, Suita, Osaka 565-0871, Japan
3 To whom correspondence should be addressed. E-mail: kanaya{at}mls.eng.osaka-u.ac.jp
Tk-subtilisin from the hyperthermophilic archaeon Thermococcus kodakaraensis is synthesized in a prepro-form (prepro-Tk-subtilisin), secreted in a pro-form (pro-Tk-subtilisin), and matured to an active form (mat-Tk-subtilisin*; a Ca2+-bound active form of matured domain of Tk-subtilisin) upon autoprocessing and degradation of the propeptide [Tk-propeptide; propeptide of Tk-subtilisin (Gly1-Leu69)]. Pro-Tk-subtilisin exhibited halo-forming activity only at 80°C, but not at 70 and 60°C, because Tk-propeptide is not effectively degraded by mat-Tk-subtilisin* and forms an inactive complex with mat-Tk-subtilisin* at <80°C. Random mutagenesis in the entire prepro-Tk-subtilisin gene, followed by screening for mutant proteins with halo-forming activity at 70 and 60°C, allowed us to identify single Gly56 
Ser mutation in the propeptide region responsible for low-temperature adaptation of pro-Tk-subtilisin. SDS–PAGE analyses and mat-Tk-subtilisin* activity assay of pro-G56S-subtilisin indicated more rapid maturation than pro-Tk-subtilisin. The resultant active form was indistinguishable from mat-Tk-subtilisin* in activity and stability, indicating that Gly56 Ser mutation does not seriously affect the folding of the mature domain. However, this mutation greatly destabilized the propeptide, making it unstructured in an isolated form. As a result, Tk-propeptide with Gly56 Ser mutation (G56S-propeptide) was more susceptible to proteolytic degradation and less effectively inhibited mat-Tk-subtilisin* activity than Tk-propeptide. These results suggest that pro-G56S-subtilisin is more effectively matured than pro-Tk-subtilisin at lower temperatures, because autoprocessed G56S-propeptide is unstructured upon dissociation from mat-Tk-subtilisin* and is therefore effectively degraded by mat-Tk-subtilisin*.
Keywords: directed evolution/low-temperature adaptation/propeptide/subtilisin/Thermococcus kodakaraensis
Received November 9, 2006; revised December 27, 2006; accepted January 10, 2007.
Abbreviations: Tk-subtilisin, subtilisin from Thermococcus kodakaraensis; prepro-Tk-subtilisin, Tk-subtilisin in a prepro-form [Met(-24)-Gly398]; pro-Tk-subtilisin, Tk-subtilisin in a pro-form (Gly1-Gly398); pro-G56S/T135S-subtilisin, pro-Tk-subtilisin with the Gly56
Ser and Thr135 Ser mutations; pro-G56S-subtilisin, pro-Tk-subtilisin with the Gly56 Ser mutation; pro-T135S-subtilisin, pro-Tk-subtilisin with the Thr135 Ser mutation; mat-Tk-subtilisin, mature domain of Tk-subtilisin (Gly70-Gly398) in a Ca2+-free inactive form; mat-Tk-subtilisin*, a Ca2+-bound active form of mat-Tk-subtilisin; Tk-propeptide, propeptide of Tk-subtilisin (Gly1-Leu69); G56S-propeptide, Tk-propeptide with the Gly56 Ser mutation; Suc-AAPF-pNA, N-succinyl-Ala-Ala-Pro-Phe-p-nitroanilide; bp, base pair(s); API, Achromobacter protease I.