Academic Thesis

Basic information

Name Koga Yuuichi
Belonging department
Occupation name
researchmap researcher code 5000076449
researchmap agency Okayama University of Science

Title

Accelerated maturation of Tk-subtilisin by a Leu→Pro mutation at the C-terminus of the propeptide, which reduces the binding of the propeptide to Tk-subtilisin.

Bibliography Type

 

Author

Ryo Uehara
Yasunori Ueda
Dong-Ju You
Yuichi Koga
Shigenori Kanaya

Summary

Tk-subtilisin, a subtilisin homologue (Gly70-Gly398) from Thermococcus kodakarensis, is matured from its precursor, Pro-Tk-subtilisin [Tk-subtilisin in a pro form (Gly1-Gly398)], by autoprocessing and degradation of propeptide [Tk-propeptide, a propeptide of Tk-subtilisin (Gly1-Leu69)]. The scissile peptide bond between Leu69 and Gly70 of Pro-Tk-subtilisin is first self-cleaved to produce an inactive Tk-propeptide:Tk-subtilisin complex, in which the C-terminal region of Tk-propeptide binds to the active-site cleft of Tk-subtilisin. Tk-propeptide is then dissociated from Tk-subtilisin and degraded by Tk-subtilisin to release active Tk-subtilisin. To examine whether the mutation of Leu69 to Pro, which is the most unfavourable residue in the P1 position for subtilisins, affects the maturation of Pro-Tk-subtilisin, the Pro-Tk-subtilisin and Tk-propeptide derivatives with this mutation (Pro-L69P and L69P-propeptide) were constructed and characterized. Pro-L69P was autoprocessed more slowly than Pro-Tk-subtilisin. Nevertheless, it matured to Tk-subtilisin more rapidly than Pro-Tk-subtilisin because L69P-propeptide was degraded by Tk-subtilisin more rapidly than Tk-propeptide. The chaperone function and stability of L69P-propeptide were comparable to those of Tk-propeptide, whereas the inhibitory potency and binding ability of L69P-propeptide were considerably reduced compared to those of Tk-propeptide. The crystal structure of the complex between L69P-propeptide and S324A-subtilisin (i.e. a protease activity-defective mutant) revealed that the C-terminal region of L69P-propeptide does not well fit into the substrate binding pockets of Tk-subtilisin (S1-S4 subsites) as a result of a conformational change caused by the mutation. These results suggest that the Leu→Pro mutation accelerates the maturation of Pro-Tk-subtilisin by reducing the binding ability of Tk-propeptide to Tk-subtilisin.

Magazine(name)

The FEBS journal

Publisher

WILEY-BLACKWELL

Volume

280

Number Of Pages

4

StartingPage

994

EndingPage

1006

Date of Issue

2013-02

Referee

Exist

Invited

Not exist

Language

English

Thesis Type

Research papers (academic journals)

ISSN

 

DOI

10.1111/febs.12091

NAID

 

PMID

 

J-GLOBAL ID

 

arXiv ID

 

ORCID Put Code

 

DBLP ID