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

Reference ID

9020

Title

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

Source

The Plant journal : for cell and molecular biology, 1997, vol. 11, pp. 191-201

Authors (4)

Bak-S	Kahn-R-A
Olsen-C-E	Halkier-B-A

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.