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[摘要]：A putative 8,7SI (sterol 8,7-isomerase) from Zea mays, termed 7m8,7SI, 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 ill the corresponding yeast microsomal extract indicated that the preferred Delta(8)-sterol Substrate possesses a single C4 alpha methyl group, in contrast with 8,7SIs front animals and fungi, thus reflecting the diversity ill the structure of their active site ill 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 Zin8, 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 sterols and directly in vitro by examination of the activities of the recombinant Zm8.7SI Mutants. These studies have identified HiS(74). Glu(78), Asp(107), Gin(121), Trp(66) and Trp(193) that are required for Zm8.7SI activity and show that binding of the enzyme-substrate complex is impaired in the mutant T124I. They underlinee the functional homology between the plant and animal 8,7SIs oil one hand, in contrast with the yeast 8,7SI oil the other hand, in accordance with their molecular diversity and distinct mechanisms. |
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