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. |
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| 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] |
| Copyright of PEDS: Protein Engineering, Design & Selection is the property of Oxford University Press / USA and its content may not be copied or emailed to multiple sites without the copyright holder's express written permission. Additionally, content may not be used with any artificial intelligence tools or machine learning technologies. However, users may print, download, or email articles for individual use. This abstract may be abridged. No warranty is given about the accuracy of the copy. Users should refer to the original published version of the material for the full abstract. (Copyright applies to all Abstracts.) | |
| Database: | Engineering Source |
| FullText | Links: – Type: pdflink Text: Availability: 0 |
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| Header | DbId: egs DbLabel: Engineering Source An: 44357365 AccessLevel: 6 PubType: Academic Journal PubTypeId: academicJournal PreciseRelevancyScore: 0 |
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| Items | – Name: Title Label: Title Group: Ti Data: Directed evolution of Tk-subtilisin from a hyperthermophilic archaeon: identification of a single amino acid substitution responsible for low-temperature adaptation. – Name: Author Label: Authors Group: Au Data: <searchLink fieldCode="AR" term="%22Pulido%2C+M%2E+A%2E%22">Pulido, M. A.</searchLink><relatesTo>1</relatesTo><br /><searchLink fieldCode="AR" term="%22Koga%2C+Y%2E%22">Koga, Y.</searchLink><relatesTo>1</relatesTo><br /><searchLink fieldCode="AR" term="%22Takano%2C+K%2E%22">Takano, K.</searchLink><relatesTo>1,2</relatesTo><br /><searchLink fieldCode="AR" term="%22Kanaya%2C+S%2E%22">Kanaya, S.</searchLink><relatesTo>1</relatesTo><i> kanaya@mls.eng.osaka-u.ac.jp</i> – Name: TitleSource Label: Source Group: Src Data: <searchLink fieldCode="JN" term="%22PEDS%3A+Protein+Engineering%2C+Design+%26+Selection%22">PEDS: Protein Engineering, Design & Selection</searchLink>. Mar2007, Vol. 20 Issue 3, p143-153. 11p. 6 Diagrams, 2 Charts, 3 Graphs. – Name: Subject Label: Subjects Group: Su Data: <searchLink fieldCode="DE" term="%22Subtilisins%22">Subtilisins</searchLink><br /><searchLink fieldCode="DE" term="%22Peptides%22">Peptides</searchLink><br /><searchLink fieldCode="DE" term="%22Genes%22">Genes</searchLink><br /><searchLink fieldCode="DE" term="%22Mutagenesis%22">Mutagenesis</searchLink><br /><searchLink fieldCode="DE" term="%22Genetic+mutation%22">Genetic mutation</searchLink> – Name: Abstract Label: Abstract Group: Ab Data: 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] – Name: AbstractSuppliedCopyright Label: Group: Ab Data: <i>Copyright of PEDS: Protein Engineering, Design & Selection is the property of Oxford University Press / USA and its content may not be copied or emailed to multiple sites without the copyright holder's express written permission. Additionally, content may not be used with any artificial intelligence tools or machine learning technologies. However, users may print, download, or email articles for individual use. This abstract may be abridged. No warranty is given about the accuracy of the copy. Users should refer to the original published version of the material for the full abstract.</i> (Copyright applies to all Abstracts.) |
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| RecordInfo | BibRecord: BibEntity: Identifiers: – Type: doi Value: 10.1093/protein/gzm006 Languages: – Code: eng Text: English PhysicalDescription: Pagination: PageCount: 11 StartPage: 143 Subjects: – SubjectFull: Subtilisins Type: general – SubjectFull: Peptides Type: general – SubjectFull: Genes Type: general – SubjectFull: Mutagenesis Type: general – SubjectFull: Genetic mutation Type: general Titles: – TitleFull: Directed evolution of Tk-subtilisin from a hyperthermophilic archaeon: identification of a single amino acid substitution responsible for low-temperature adaptation. Type: main BibRelationships: HasContributorRelationships: – PersonEntity: Name: NameFull: Pulido, M. A. – PersonEntity: Name: NameFull: Koga, Y. – PersonEntity: Name: NameFull: Takano, K. – PersonEntity: Name: NameFull: Kanaya, S. IsPartOfRelationships: – BibEntity: Dates: – D: 01 M: 03 Text: Mar2007 Type: published Y: 2007 Identifiers: – Type: issn-print Value: 17410126 Numbering: – Type: volume Value: 20 – Type: issue Value: 3 Titles: – TitleFull: PEDS: Protein Engineering, Design & Selection Type: main |
| ResultId | 1 |
