Cloning and expression in Escherichia coli of the obtusifoliol 14α‐demethylase of Sorghum bicolor (L.) Moench, a cytochrome P450 orthologous to the sterol 14α‐demethylases (CYP51) from fungi and mammals

Abstract

Obtusifoliol 14 alpha-demethylase from Sorghum bicolor (L.) Moench has been cloned using a gene-specific probe generated using PCR primers designed from an internal 14 amino acid sequence. The sequence identifies sorghum obtusifoliol 14 alpha-demethylase as a cytochrome P450 and it is assigned to the CYP51 family together with the sterol 14 alpha-demethylases from fungi and mammals. The presence of highly conserved regions in the amino acid sequences, analogous substrates and the same metabolic role demonstrate that the sterol 14 alpha-demethylases are orthologous enzymes. The sterol 14 alpha-demethylases catalyse an essential step in sterol biosynthesis as evidenced by the absence of a 14 alpha-methyl group in all known functional sterols. A functional sorghum obtusifoliol 14 alpha-demethylase was expressed at high levels in Escherichia coli and purified using an efficient method based on temperature-induced Triton X-114 phase partitioning. The recombinant purified enzyme produced a type I spectrum with obtusifoliol as substrate. Reconstitution of purified recombinant enzyme with sorghum NADPH-cytochrome P450 reductase in dilaurylphosphatidylcholine micelles confirms that obtusifoliol 14 alpha-demethylase catalyses the 14 alpha-demethylation of obtusifoliol to 4 alpha-methyl-5 alpha-ergosta-8, 14,24(28)-trien-3 beta-ol as evidenced by GC-MS. The isolation of a cDNA clone encoding the plant sterol 14 alpha-demethylase, combined with the previously isolated cDNA clones for fungal and mammalian sterol 14 alpha-demethylases, provides an important tool in the rational design of specific inhibitors towards the individual sterol 14 alpha-demethylases.

Biochemical analysis of a multifunctional cytochrome P450 (CYP51) enzyme required for synthesis of antimicrobial triterpenes in plants

“…The 1 H and 13 C NMR spectra of the purified compound were compared with available NMR data for β-amyrin (34,35). Characteristic signals for the olefinic carbons in β-amyrin (C12 δ 123; C13 δ 145) (34) were missing in the 13 C NMR spectrum of the analyzed compound, indicating the loss of the double bond. Further distortionless enhancement by polarization transfer, heteronuclear multiple bond correlation, and selective NOE experiments revealed an epoxide between C12 and C13 and a hydroxyl group attached to C16, leading to the identification of the compound as 12,13β-epoxy-3β,16β-dihydroxy-oleanane (12,13β-epoxy-16β-hydroxy-β-amyrin) ( Fig.…”

“…In contrast, the cytochrome P450 14α-sterol demethylase (CYP51) family is one of the most ancient of the P450 families, and the function of CYP51 enzymes is highly conserved across fungi, plants, and animals (7,8). These enzymes are sterol demethylases required for the synthesis of essential sterols (9)(10)(11)(12)(13)(14). Although different sterol substrates are used (e.g., lanosterol in mammals and yeast and obtusifoliol in plants), the reaction mechanism-14α-demethylation and subsequent formation of a Δ14-15 double bond-is preserved.…”

Characterization of Maize Cytochrome P450 Monooxygenases Induced in Response to Safeners and Bacterial Pathogens

“…CYP73A6 transcripts were not detected at either 2.5 or 6.5 d in the shoots, suggesting that CYP73A7 transcripts encode the major t-CAH species present in these developmental stages. Maize EST csu25 transcripts, which are highly homologous to sorghum and wheat CYP51 transcripts and presumed to have a function in obtusifoliol 14-demethylation (Bak et al, 1997;CabelloHurtado et al, 1997), are induced in response to NA treatment of young seedlings and repressed in older seedlings.…”

“…To further determine the range of other P450s induced by NA plus T, these northern blots were probed with maize EST clones agrc115 (encoding CYP71C1, the third P450 in the DIMBOA biosynthetic pathway; Frey et al, 1997), csu25 (encoding CYP51 potentially involved in sterol 14␣-demethylation; Bak et al, 1997), and 6c06b11 and 7c02c12 (encoding P450s of unknown function). CYP71C1 and its related transcripts are expressed at a significant and equal level in 2.5-and 6.5-d-old seedling shoots (Fig.…”

Impacts of Diversification of Cytochrome P450 on Plant Metabolism

“…CYP51G and CYP710A encode obtusifoliol 14α-demethylase and sterol 22-desaturase, respectively, which are essential for sterol biosynthesis. 5,6) The CYP97 family functions in xanthophyll biosynthesis, and Arabidopsis CYP97A3 and CYP97C1 catalyze the hydroxylation of β-and ε-rings, respectively, of carotenoids in the biosynthetic pathway of xanthophylls, which are key components in photosynthetic complexes for light-harvesting and photoprotection. 7,8) These reactions are likely essential for the normal growth and development of plant cells.…”