Cloning and characterization of terpenoid hydroxylase gene form Croton stellatopilosus Ohba

Abstract

Plant cytochrome P450 (CYP or P450) is a heme-containing enzyme located on the membrane of endoplasmic recticulum (ER) as a microsomal type. It plays an important role in catalyzing a vast variety if metabolic reactions. P450 are closely associated with the cytochrome P450 reductases (CPRs), another group of membrane-bound enzymes required for electron transfer to P450s. Among various groups of secondary metabolites, the terpenoids, which represent the largest class of characterizes natural plant compounds, are often substrates for the P450s hydroxylation reactions. Since Croton stellatopilosus Ohba (Euphorbiaceae), the Thai medicinal plant producing the acyclic diterpenoid plaunotol, has been shown to involve the P450-dependent hydroxylation in the final step of the biosynthetic pathway of plaunotol, the plant was used in this study as a model for searching the involved P450 and CPR genes. Through the steps of gene cloning, characterization, sequence analysis, and phylogenetic analysis, one candidate of P450, namely CsCYP97 and one of the associated CPR, namely CsCPR were found from the plant. All the three genes could be expressed in Escherichia Coli (BL21(DE3)) in pE32a. The microsomal fractions of the P450 protein CsCYP97 appeared It have their enzyme activity only in the presence of CsCPR The CsCYP97 could catalyze the conversion, presumably hydroxylation, of geranylgeraniol to an unknown compound co- chromatographed closely with plaunotol on TLC plate. The CsCYP97 was proved to be P450 as characterized by rate limiting state of the carbon monoxide, with shifting of the absorbance peak from 413 to 426 nm. For the CsCPR, its activity assay at the steady-state for cytochrome c and NADPH was determined to have its kinetic values for KmctyC, KmNADPH and Vmax of 10.32±2.11 µM, 44.77±6.047 µM and 0.099±0.005 µM. min-1, respectively. Real-time PCR analysis of transcripts of CsCYP97 and CsCPR from the shoot to leaves in each position indicated that the expression pattern of CsCPR gene is correlated to the expression of the genes involved in the biosynthesis of plaunotol Until the current report, the whole genome elucidation of C. stellatopilosus Ohba is not available in database. Therefore, the cloning and functional studies of the P450 and CPR enzymes will lead to a better understanding of the metabolic control of plaunotol biosynthetic pathway in the future