A putative 8,7SI (sterol 8,7-isomerase) from Zea mays, termed Zm8,7SI, has been isolated from an EST (expressed sequence tag) library and subcloned into the yeast erg2 mutant lacking 8,7SI activity. Zm8,7SI restored endogenous ergosterol synthesis. An in vitro enzymatic assay in the corresponding yeast microsomal extract indicated that the preferred Δ8-sterol substrate possesses a single C4α methyl group, in contrast with 8,7SIs from animals and fungi, thus reflecting the diversity in the structure of their active site in relation to the distinct sterol biosynthetic pathways. In accordance with the proposed catalytic mechanism, a series of lipophilic ammonium-ion-containing derivatives possessing a variety of structures and biological properties, potently inhibited the Zm8,7SI in vitro. To evaluate the importance of a series of conserved acidic and tryptophan residues which could be involved in the Zm8,7SI catalytic mechanism, 20 mutants of Zm8,7SI were constructed as well as a number of corresponding mutants of the Saccharomyces cerevisiae 8,7SI. The mutated isomerases were assayed in vivo by sterol analysis and quantification of Δ5,7-sterols and directly in vitro by examination of the activities of the recombinant Zm8,7SI mutants. These studies have identified His74, Glu78, Asp107, Glu121, Trp66 and Trp193 that are required for Zm8,7SI activity and show that binding of the enzyme–substrate complex is impaired in the mutant T124I. They underline the functional homology between the plant and animal 8,7SIs on one hand, in contrast with the yeast 8,7SI on the other hand, in accordance with their molecular diversity and distinct mechanisms.
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Research Article|
August 12 2008
Identification of essential amino acid residues in a sterol 8,7-isomerase from Zea mays reveals functional homology and diversity with the isomerases of animal and fungal origin
Alain Rahier;
Alain Rahier
1
*Institut de Biologie Moléculaire des Plantes, CNRS, UPR2357, 28 rue Goethe, 67083 Strasbourg cedex, France
1To whom correspondence should be addressed (email alain.rahier@ibmp-ulp.u-strasbg.fr).
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Sylvain Pierre;
Sylvain Pierre
*Institut de Biologie Moléculaire des Plantes, CNRS, UPR2357, 28 rue Goethe, 67083 Strasbourg cedex, France
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Geneviève Riveill;
Geneviève Riveill
†UMR 1131, INRA, Université Louis Pasteur, F-68000 Colmar, France
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Francis Karst
Francis Karst
†UMR 1131, INRA, Université Louis Pasteur, F-68000 Colmar, France
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Publisher: Portland Press Ltd
Received:
February 04 2008
Revision Received:
April 25 2008
Accepted:
May 07 2008
Accepted Manuscript online:
May 07 2008
Online ISSN: 1470-8728
Print ISSN: 0264-6021
© The Authors Journal compilation © 2008 Biochemical Society
2008
Biochem J (2008) 414 (2): 247–259.
Article history
Received:
February 04 2008
Revision Received:
April 25 2008
Accepted:
May 07 2008
Accepted Manuscript online:
May 07 2008
Citation
Alain Rahier, Sylvain Pierre, Geneviève Riveill, Francis Karst; Identification of essential amino acid residues in a sterol 8,7-isomerase from Zea mays reveals functional homology and diversity with the isomerases of animal and fungal origin. Biochem J 1 September 2008; 414 (2): 247–259. doi: https://doi.org/10.1042/BJ20080292
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