Chamovitz D, Sandmann G, Hirschberg J
Department of Genetics, Hebrew University of Jerusalem, Israel.
J Biol Chem. 1993 Aug 15;268(23):17348-53.
Mutant strains of the cyanobacterium Synechococcus sp. PCC 7942 that are resistant to the herbicides norflurazon and fluridone were analyzed. These herbicides inhibit phytoene desaturase, a key enzyme in the carotenoid biosynthetic pathway. In three mutants the phenotype was related to specific point mutations in pds, the gene encoding phytoene desaturase. Since the resistance was manifested in a cell-free carotenogenic assay, it is evident that the predicted amino acid changes in the target protein alter the enzyme-herbicide interactions. In addition, the mutations also reduced the in vitro activity of phytoene desaturase. Increased levels of the substrate phytoene, accompanied by a reduction in colored carotenoids, were detected in cells of each of the mutant strains. A correlation was established between the degree of increase in the steady-state levels of phytoene and the extent of reduction in total carotenoid content in the cells. These two phenomena in turn are correlated with the rate of enzymatic activity of phytoene desaturase that was measured in vitro. Hence we deduce that phytoene desaturation is a rate-limiting step in carotenogenesis in cyanobacteria. Support for this conclusion is obtained from analysis of cells of an additional mutant strain, which overexpress phytoene desaturase due to a deletion mutation in the promoter region of pds. Cells of this mutant contained more colored carotenoids than the wild-type and were resistant to herbicides that inhibit phytoene desaturase.
对蓝藻聚球藻属PCC 7942的抗除草剂去甲草净和氟啶酮的突变菌株进行了分析。这些除草剂抑制八氢番茄红素去饱和酶，这是类胡萝卜素生物合成途径中的一种关键酶。在三个突变体中，表型与编码八氢番茄红素去饱和酶的基因pds中的特定点突变有关。由于在无细胞类胡萝卜素生成测定中表现出抗性，很明显目标蛋白中预测的氨基酸变化改变了酶与除草剂的相互作用。此外，这些突变还降低了八氢番茄红素去饱和酶的体外活性。在每个突变菌株的细胞中都检测到底物八氢番茄红素水平升高，同时有色类胡萝卜素减少。八氢番茄红素稳态水平的增加程度与细胞中总类胡萝卜素含量的减少程度之间建立了相关性。这两种现象又与体外测定的八氢番茄红素去饱和酶的酶活性速率相关。因此我们推断八氢番茄红素去饱和是蓝藻中类胡萝卜素生成的限速步骤。对这一结论的支持来自对另一个突变菌株细胞的分析，该菌株由于pds启动子区域的缺失突变而过表达八氢番茄红素去饱和酶。该突变体的细胞比野生型含有更多的有色类胡萝卜素，并且对抑制八氢番茄红素去饱和酶的除草剂具有抗性。
