Carotenogenic enzymes from Neurospora
In the fungus Neurospora crassa, the carotenogenic enzyme system, catalyzing both the synthesis of phytoene and that of more un saturated carotenoids, is membrane—bound. Using geranylgeranyl pyrophosphate as a substrate no co—factors are required for the conversion to carotenoids, but Mg2+ions stimulate the activity. Carotenogenic enzymes have been solu-. bilized partly from membranes by detergent treatment. Only by treatment with sodium cholate and CHAPS was most of the enzyme activity maintained, whereas almost full activity could be restored only by the addition of lipids. Photoinduction of de novo synthesis of carotenogenic enzymes is indicated by studies using inhibitors of protein synthesis and by light— mediated changes in the pattern of membrane proteins. INTRODUCTION Although the biosynthesis of carotenoids and its regulation by light have been intensively studied, we are still far from a complete understanding (for review Ref. 1 and also Rau this vol.). In particular, our knowledge of carotenogenic enzymes is very limited. In the fungus Neurospora crassa — as in some other microorganisms — carotenoid formation in the mycelium is light dependent (Ref. i). Consequently it has been concluded that the enzymes might be absent in dark—grown mycelia and present only after photoinduction. Therefore, these organisms seem to be well suited not only for investigations on the mechanism of photoregulation but also for studies on the properties of the carotenogenic enzymes. Cell—free systems from Neurospora capable •of in vitro carotenogenesis have been elaborated (Ref. 3—5). This report presents more recent findings on carotenogenic enzymes from Neurospora. Most of the methods and experimental procedures used have been described elsewhere (Ref. 2 & 5). LOCALISATION OF CAROTENOGENIC ENZYME ACTIVITY Subcellular fractionation of crude cell extracts has shown that in fungi the steps of the carotenoid biosynthesis pathway are compartmentalised: The enzyme(s) converting mevalonic acid to geranylgeranyl pyrophosphate (prenyltransferase(s)) are soluble, whereas carotenogenic enzymes, i.e. phytoene synthase, isomerase, dehydro— genase(s) and cyclase(s) are membrane—bound (Ref. 3). Maximum carotenogenic activity (ca. 80 ) was localized in two membrane fractions shown to contain plasma membranes and in particular membranes of the endoplasmic reticulum (Ref. 6). These two membrane fractions also contain some carotenoid pigments, whereas the bulk of the carotenoid is in lipid globules (Ref. 6). PROPERTIES OF THE MEMBRANE-BOUND ENZYME SYSTEM For further purification and characterization of membrane—bound carotenogenic enzymes it seemed necessary to optimize the in vitro assay for carotenoid synthesis. Therefore, several of the conditions, including the illumination regime for photoinduction of carotenogenic enzymes, centrifugation procedures, mevalonic acid concentration, requirement and concentrations of cofactors, have been varied (Ref. 2 & unpublished results).