Phytoene desaturase: heterologous expression in an active state, purification, and biochemical properties.
Conditions were developed for heterologous expression in Escherichia coli of the membrane-bound cyanobacterial/plant-type phytoene desaturase (PDS) from Synechococcus in an active form. Decrease of growth temperature for the transformant to 28 degrees C resulted in an increase of proteins in a supernatant fraction obtained after pressure disruption (20 MPa) of cells and centrifugation. This supernatant in which the highest PDS activity was found was used for purification to a homogeneous protein by ammonium sulfate precipitation and DEAE chromatography. The purified PDS was employed to determine substrate specificity and cofactor requirement. Substrates in addition to phytoene were phytofluene and 1,2-epoxy phytoene which were converted to zeta-carotene and the corresponding 1,2-epoxide. The reaction was stimulated by NAD, NADP, and oxygen. The K(m) values determined for phytoene and NADP were 3.5 microM and 14.3 mM, respectively.
Schneider
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Lehrstuhl Physiologie und Biochemie der Pflanzen, Universität Konstanz, Germany.
Böger
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Sandmann
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D002338:Carotenoids / Q000378:metabolism; D000458:Cyanobacteria / Q000201:enzymology / Q000235:genetics; D004789:Enzyme Activation; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics; D005822:Genetic Vectors; D007700:Kinetics; D010088:Oxidoreductases / Q000096:biosynthesis / Q000737:chemistry* / Q000302:isolation & purification*; D013379:Substrate Specificity
D002338:Carotenoids; C100185:(all-E) phytoene; D010088:Oxidoreductases; C022398:phytoene dehydrogenase