A putative sterol 8,7-isomerase (8,7SI) from Zea mays termed Zm8,7SI has been isolated from an EST library and subcloned in 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 Delta8-sterol substrate possesses a single C4alpha methyl group, in contrast to 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 Zm8,7SI catalytical mechanism, 20 mutants of Zm8,7-SI were constructed as well as a number of corresponding mutants of the S.cerevisiae 8,7-SI. The mutated isomerases were assayed in vivo by sterol analysis and quantification of Delta5,7-sterols and directly in vitro by examination of the activities of the recombinant Zm8,7-SI 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 mutant T124I. They underline the functional homology between the plant and animal 8,7SIs on one hand, in contrast to the yeast 8,7SI on the other hand, in accord with their molecular diversity and distinct mechanisms.
|Evidence ID||Analyze ID||Interactor||Interactor Systematic Name||Interactor||Interactor Systematic Name||Type||Assay||Annotation||Action||Modification||Phenotype||Source||Reference||Note|
|Evidence ID||Analyze ID||Gene||Gene Systematic Name||Gene Ontology Term||Gene Ontology Term ID||Qualifier||Aspect||Method||Evidence||Source||Assigned On||Annotation Extension||Reference|
|Evidence ID||Analyze ID||Gene||Gene Systematic Name||Phenotype||Experiment Type||Experiment Type Category||Mutant Information||Strain Background||Chemical||Details||Reference|
|Evidence ID||Analyze ID||Regulator||Regulator Systematic Name||Target||Target Systematic Name||Experiment||Assay||Construct||Conditions||Strain Background||Reference|