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 homogenous 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 mu M and 14.3 mM, respectively. (C) 1997 Academic Press.