PURIFICATION OF THE CYTOCHROME-P-450 ENZYME GERANIOL 10-HYDROXYLASE FROM CELL-CULTURES OF CATHARANTHUS-ROSEUS

The cytochrome P-450 enzyme geraniol 10-hydroxylase (G10H) was purified from a suspension culture of Catharanthus roseus, grown on an alkaloid production medium. The cholate-solubilized G10H was purified in a four-step procedure, consisting of chromatography on DEAE-Sephacel, hydroxyapatite Ultrogel, omega-aminooctylagarose and TSK Phenyl-5PW. On DEAE-Sephacel a virtually complete separation of the cytochrome P-450 enzyme from NADPH:cytochrome P-450 (cytochrome c) reductase (EC 1.6.2.4) was achieved. The G10H activity of P-450-containing fractions was reconstituted by addition of the reductase and a lipid extract. Although a substantial loss of G10H activity occurred on solubilisation and the activity was much lower in the reconstituted system compared with solubilized preparations, the enzyme was remarkably stable during the later stages of its purification. An efficient separation of G10H from contaminating proteins was achieved by hydrophobic interaction chromatography on a high-performance TSK Phenyl-5PW column that was presaturated with non-ionic detergent. The G10H peak fractions from this column showed a single band on sodium dodecyl sulphate polyacrylamide gel electrophoresis with silver staining, corresponding to an M(r) of 56 000. The purified enzyme catalyses the hydroxylation of both geraniol and nerol, and has a specific cytochrome P-450 content of 4.7 nmol/mg protein.