Directed evolution of Tk-subtilisin from a hyperthermophilic archaeon: identification of a single amino acid substitution responsible for low-temperature adaptation.

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Title:	Directed evolution of Tk-subtilisin from a hyperthermophilic archaeon: identification of a single amino acid substitution responsible for low-temperature adaptation.
Authors:	Pulido, M. A.¹, Koga, Y.¹, Takano, K.^1,2, Kanaya, S.¹ kanaya@mls.eng.osaka-u.ac.jp
Source:	PEDS: Protein Engineering, Design & Selection. Mar2007, Vol. 20 Issue 3, p143-153. 11p. 6 Diagrams, 2 Charts, 3 Graphs.
Subjects:	Subtilisins, Peptides, Genes, Mutagenesis, Genetic mutation
Abstract:	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*. [ABSTRACT FROM PUBLISHER]
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Database:	Engineering Source

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Abstract:	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*. [ABSTRACT FROM PUBLISHER]
ISSN:	17410126
DOI:	10.1093/protein/gzm006