Skip to Main content Skip to Navigation
Journal articles

The prosequence of thermolysin acts as an intramolecular chaperone when expressed in trans with the mature sequence in Escherichia coli.

Abstract : The zinc metalloendopeptidase, thermolysin (EC 3.4.24.27) produced by Bacillus thermoproteolyticus serves as a model of important physiological enzymes such as neprilysin, angiotensin converting enzyme and endothelin converting enzyme. Thermolysin is synthesised as a pre-proenzyme, with an N-terminal prosequence of 204 residues and a mature sequence of 316 residues. The prosequence facilitates the folding of the denatured mature sequence in vitro and the cleavage of the peptide bond linking the pro and mature sequences occurs by an autocatalytic, intramolecular process. With the aim to study the role of the prosequence in vivo and to produce active mutants for structural studies, the mature sequence of thermolysin has now been expressed in Escherichia coli, either alone or with the prosequence as an independent polypeptide, i.e. in trans form. In addition, the mature sequence of an inactive mutant in which Glu143 involved in the catalytic process was replaced by Ala has also been expressed in trans with the prosequence. The results show that the pro-sequence is required to obtain active thermolysin and that a covalent link with the mature sequence is not necessary for the correct folding of the protease in vivo. Moreover, when expressed in E. coli (in trans with the prosequence), the yield of correctly folded E143A mutant was similar to that of the wild-type protease, whereas no mature enzyme was detected when it was expressed as a pre-proenzyme in Bacillus subtilis. These results demonstrate that the thermolysin prosequence acts as an intramolecular chaperone in vivo and open the way to structural studies of catalytic site mutants produced in large quantities in E. coli.
Document type :
Journal articles
Complete list of metadatas

Cited literature [27 references]  Display  Hide  Download

https://www.hal.inserm.fr/inserm-00171255
Contributor : Cynthia Marie-Claire <>
Submitted on : Wednesday, September 12, 2007 - 10:00:53 AM
Last modification on : Thursday, November 19, 2020 - 8:50:29 AM
Long-term archiving on: : Friday, November 25, 2016 - 7:15:21 PM

File

 Restricted access
To satisfy the distribution rights of the publisher, the document is embargoed until : jamais

Please log in to resquest access to the document

Identifiers

Collections

Citation

Cynthia Marie-Claire, Emmanuel Ruffet, Ann Beaumont, Bernard Roques. The prosequence of thermolysin acts as an intramolecular chaperone when expressed in trans with the mature sequence in Escherichia coli.. Journal of Molecular Biology, Elsevier, 1999, 285 (5), pp.1911-5. ⟨10.1006/jmbi.1998.2449⟩. ⟨inserm-00171255⟩

Share

Metrics

Record views

448