Purification in an active state and properties of the 3-step phytoene desaturase from Rhodobacter capsulatus overexpressed in Escherichia coli.
Purification in an active state and properties of the 3-step phytoene desaturase from Rhodobacter capsulatus overexpressed in Escherichia coli.
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DOI：
10.1093/oxfordjournals.jbchem.a021278
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发表时间：
1996-03
影响因子：
2.7
通讯作者：
A. Raisig;Glenn E. Bartley;P. A. Scolnik;Gerhard Sandmann
中科院分区：
文献类型：
--
作者：
A. Raisig;Glenn E. Bartley;P. A. Scolnik;Gerhard Sandmann
在大肠杆菌中表达了荚膜红杆菌植物烯去饱和酶基因，并纯化了该基因蛋白。经过硫酸铵沉淀和离子交换层析纯化，得到了57 kDa的蛋白，具有较高的酶活性。纯化酶在底物特异性和产物形成方面进行了表征。除植物烯外，中间产物植物荧光素和ζ -胡萝卜素都被转化为神经孢烯，即反应的最终产物。此外，1,2-环氧植物烯是合适的底物，而C30二噻吩烯则不是。植物烯和β -胡萝卜素的Km值分别为33.3和16.6微米。去饱和反应依赖于辅因子FAD。氧化的尼古丁核苷酸或ATP没有积极作用。FAD的Km值为4.9微米。二苯胺对脱饱和反应有抑制作用。
The phytoene desaturase gene from Rhodobacter capsulatus was expressed in Escherichia coli and the resulting protein was purified. The purification steps involved were ammonium sulfate precipitation and ion exchange chromatography, leading to a homogenous protein of 57 kDa with high specific enzymatic activity. The purified enzyme was characterized with respect to substrate specificity and product formation. In addition to phytoene, the intermediates, phytofluene and zeta-carotene, were both converted to neurosporene, the end product of the reaction. Furthermore, 1,2-epoxy phytoene was a suitable substrate whereas the C30 diapophytoene was not. The Km values for phytoene and zeta-carotene were determined to be 33.3 and 16.6 microM, respectively. The desaturation reaction is dependent on the cofactor FAD. Oxidized nicotine nucleotides or ATP had no positive effect. The Km value for FAD was 4.9 microM. Inhibition of the desaturation reaction was observed with diphenylamine.