PHYTOENE DESATURASE - HETEROLOGOUS EXPRESSION IN AN ACTIVE STATE, PURIFICATION, AND BIOCHEMICAL-PROPERTIES

Conditions were developed for heterologous expression in Escherichia c oli of the membrane-bound cyanobacterial/plant-type phytoene desaturas e (PDS) from Synechococcus in an active form. Decrease of growth tempe rature 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 h ighest PDS activity was found was used for purification to a homogenou s protein by ammonium sulfate precipitation and DEAE chromatography, T he purified PDS was employed to determine substrate specificity and co factor requirement. Substrates in addition to phytoene were phytofluen e 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 m u M and 14.3 mM, respectively. (C) 1997 Academic Press.