Purification in an active state and properties of the 3-step phytoene desaturase from Rhodobacter capsulatus overexpressed in Escherichia coli.
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.
Raisig
A
A
Botanisches Institut, J.W. Goethe Universität, Frankfurt, Germany.
Bartley
G
G
Scolnik
P
P
Sandmann
G
G
D000645:Ammonium Sulfate; D002338:Carotenoids / Q000378:metabolism; D011232:Chemical Precipitation; D002848:Chromatography, DEAE-Cellulose; D003001:Cloning, Molecular; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli; D005182:Flavin-Adenine Dinucleotide / Q000378:metabolism; D007700:Kinetics; D010088:Oxidoreductases / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D016354:Rhodobacter capsulatus / Q000201:enzymology* / Q000235:genetics
D011994:Recombinant Proteins; D005182:Flavin-Adenine Dinucleotide; D002338:Carotenoids; C100185:(all-E) phytoene; D010088:Oxidoreductases; C022398:phytoene dehydrogenase; D000645:Ammonium Sulfate