Ruiz-Hidalgo M J, Benito E P, Sandmann G, Eslava A P
Departamento de Microbiología y Genética, Universidad de Salamanca, Spain.
Mol Gen Genet. 1997 Feb 27;253(6):734-44. doi: 10.1007/s004380050378.
By using a polymerase chain reaction based cloning strategy we isolated the gene (carB) encoding the enzyme phytoene dehydrogenase from Phycomyces blakesleeanus. The deduced protein, a 583 residue polypeptide, showed great similarity to carotenoid dehydrogenases from other fungi and bacteria, especially in the amino-terminal region. The main conserved regions found in other phytoene dehydrogenases, which are thought to be essential for the enzymatic activity, are present in the sequence from Phycomyces. Heterologous expression of the Phycomyces gene in Escherichia coli showed that, as in other fungi and bacteria, a single polypeptide catalyzes the four dehydrogenations that convert phytoene to lycopene. RNA measurements indicated that the level of expression of the phytoene dehydrogenase gene in wild-type mycelia increased in response to blue light. The kinetics of this increase in transcription of the gene after blue light induction (0.1 and 0.4 W/m2) exhibit a two-step (biphasic) dependence on fluence rate, suggesting that there could be two separate components involved in the reception of the low and high blue light signal. The presence of vitamin A in the medium stimulated transcript accumulation in the wild type and in some carotenogenic mutant strains. Diphenylamine, a phytoene dehydrogenase inhibitor, did not affect the level of transcription of this gene.
通过基于聚合酶链反应的克隆策略，我们从布氏毛霉中分离出了编码八氢番茄红素脱氢酶的基因（carB）。推导的蛋白质是一个含有583个残基的多肽，与其他真菌和细菌的类胡萝卜素脱氢酶具有高度相似性，尤其是在氨基末端区域。在其他八氢番茄红素脱氢酶中发现的主要保守区域，被认为对酶活性至关重要，在布氏毛霉的序列中也存在。布氏毛霉基因在大肠杆菌中的异源表达表明，与其他真菌和细菌一样，单一多肽催化将八氢番茄红素转化为番茄红素的四次脱氢反应。RNA测量表明，野生型菌丝体中八氢番茄红素脱氢酶基因的表达水平在蓝光照射下会增加。蓝光诱导（0.1和0.4 W/m2）后该基因转录增加的动力学表现出对光通量率的两步（双相）依赖性，这表明可能有两个独立的组分参与低强度和高强度蓝光信号的接收。培养基中维生素A的存在刺激了野生型和一些产类胡萝卜素突变菌株中转录物的积累。二苯胺是一种八氢番茄红素脱氢酶抑制剂，对该基因的转录水平没有影响。
