Biochemical and Kinetic Characterization of Geranylgeraniol 18-Hydroxylase (CYP97C27) from Croton stellatopilosus Ohba

Abstract

Geranylgeraniol 18-hydroxylase (EC 1.14.13.110) that exists solely in Croton stellatopilosus Ohba catalyses the last committed step of plaunotol biosynthetic pathways by conversion of geranylgeraniol (GGOH) to plaunotol. This enzyme and its gene are an attractive target for development of plaunotol production and its detailed biochemical properties need to be understood.   Recently, even though the gene (CYP97C27) coding for GGOH 18-hydroxylase has been identified, cloned, and expressed in Escherichia coli system, the enzyme activity has been detected mainly in the insoluble fraction (20,000 g). This means that biochemical and kinetic studies could not be undertaken. However, our previous study indicated that this enzyme activity was easily and specifically detected in the microsomal fraction (100,000 g) of a crude enzyme extract. Therefore, in this report we describe a comprehensive biochemical characterization of GGOH 18-hydroxylase activity in the microsomal fraction from C. stellatopilosus Ohba. The oxygen-dependent enzyme activity of GGOH 18-hydroxylase was inhibited by carbon monoxide and the inhibition was partially reversible upon illumination with white light. Kinetic studies of the GGOH 18-hydroxylase showed high affinity to GGOH and NADPH with apparent K_m values of 0.8 and 53 µM, respectively. Furthermore, the enzyme activity was inhibited by P450 inhibitors, including ancymidol, metyrapone, miconazole, potassium cyanide and cytochrome c, with the IC₅₀ values of 428, 65, 75, 66 and 8 µM, respectively. Based on the biochemical and kinetic characteristics, the GGOH 18-hydroxylase in the microsomal fraction is likely a P450 encoded by CYP97C27 gene as previously described.

Keywords:  Plaunotol, Cytochrome P450, Enzyme Activity, Enzyme Inhibitor, Microsome

Abbreviation: GGPP, Geranylgeranyl diphosphate; GGOH, geranylgeraniol; PMSF, phenylmethylsulfonyl fluoride; KCN, potasium cyanide; CO, carbon monoxide