Purification in an Active State and Properties of the 3-Step Phytoene Desaturase from Rhodobacter capsulatus Overexpressed in Escherichia coil
- 1 March 1996
- journal article
- research article
- Published by Oxford University Press (OUP) in The Journal of Biochemistry
- Vol. 119 (3) , 559-564
- https://doi.org/10.1093/oxfordjournals.jbchem.a021278
Abstract
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 ζ-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 ζ-carotene were determined to be 33.3 and 16.6 /iM, 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 μM. Inhibition of the desaturation reaction was observed with diphenyl-amineKeywords
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