The enzyme CPI (cyclopropylsterol-cycloisomerase) from the plant sterol pathway catalyses the cleavage of the 9β,19-cyclopropane ring of the 4α-methyl-cyclopropylsterol cycloeucalenol to produce the Δ8-sterol obtusifoliol. Randomly mutated plasmids carrying the Arabidopsis thaliana cpi gene were screened for inactive CPI mutant enzymes on the basis of their ability to genetically complement a Saccharomyces cerevisiae erg7 (defective in oxidosqualene cyclase) ergosterol auxotroph grown in the presence of exogenous cycloeucalenol, and led to the identification of four catalytically important residues. Site-directed mutagenesis experiments confirmed the role of the identified residues, and demonstrated the importance of selected acidic residues and a conserved G108NYFWTHYFF117 motif. The mutated isomerases were assayed both in vivo by quantification of cycloeucalenol conversion into ergosterol in erg7 cells, and in vitro by examination of activities of recombinant AtCPI (A. thaliana CPI) mutants. These studies show that Gly28, Glu29, Gly108 and Asp260 are crucial for CPI activity and that an hydroxy function at residue 113 is needed for maximal substrate affinity and CPI activity. CPI is inactive on upstream 4α,β-dimethyl-cyclopropylsterol precursors of phytosterols. The single mutation W112L generates a CPI with an extended substrate specificity, that is able to convert 4α,β-dimethyl-cyclopropylsterols into the corresponding Δ8 products. These findings provide insights into the molecular basis of CPI activity and substrate specificity.
Skip Nav Destination
Article navigation
April 2014
-
Cover Image
Cover Image
- PDF Icon PDF LinkFront Matter
- PDF Icon PDF LinkTable of Contents
- PDF Icon PDF LinkEditorial Board
Research Article|
March 28 2014
Plant cyclopropylsterol-cycloisomerase: key amino acids affecting activity and substrate specificity
Alain Rahier;
Alain Rahier
1
*Institut de Biologie Moléculaire des Plantes, Centre National de la Recherche Scientifique, UPR 2357, 28 rue Goethe, 67083 Strasbourg, France
1To whom correspondence should be addressed (email alain.rahier@ibmp-cnrs.unistra.fr).
Search for other works by this author on:
Francis Karst
Francis Karst
†UMR 1131, Institut National de Recherche Agronomique, Université Louis Pasteur, F-68000 Colmar, France
Search for other works by this author on:
Publisher: Portland Press Ltd
Received:
September 19 2013
Revision Received:
January 21 2014
Accepted:
February 03 2014
Accepted Manuscript online:
February 03 2014
Online ISSN: 1470-8728
Print ISSN: 0264-6021
© The Authors Journal compilation © 2014 Biochemical Society
2014
Biochem J (2014) 459 (2): 289–299.
Article history
Received:
September 19 2013
Revision Received:
January 21 2014
Accepted:
February 03 2014
Accepted Manuscript online:
February 03 2014
Citation
Alain Rahier, Francis Karst; Plant cyclopropylsterol-cycloisomerase: key amino acids affecting activity and substrate specificity. Biochem J 15 April 2014; 459 (2): 289–299. doi: https://doi.org/10.1042/BJ20131239
Download citation file:
Sign in
Don't already have an account? Register
Sign in to your personal account
You could not be signed in. Please check your email address / username and password and try again.
Captcha Validation Error. Please try again.