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 14β‐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 14α‐demethylase as a cytochrome P450 and it is assigned to the CYP51 family together with the sterol 14α‐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 14α‐demethylases are orthologous enzymes. The sterol 14α‐demethylases catalyse an essential step in sterol biosynthesis as evidenced by the absence of a 14α‐methyl group in all known functional sterols. A functional sorghum obtusifoliol 14α‐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 14α‐demethylase catalyses the 14α‐demethylation of obtusifoliol to 4α‐methyl‐5α‐ergosta‐8,14,24(28)‐trien‐3β‐ol as evidenced by GC—MS. The isolation of a cDNA clone encoding the plant sterol 14α‐demethylase, combined with the previously isolated cDNA clones for fungal and mammalian sterol 14α‐demethylases, provides an important tool in the rational design of specific inhibitors towards the individual sterol 14α‐demethylases.