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.
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.
Bak
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Department of Plant Biology, Royal Veterinary and Agricultural University, Copenhagen, Denmark.
Kahn
R A
RA
Olsen
C E
CE
Halkier
B A
BA
D000595:Amino Acid Sequence; D000818:Animals; D003001:Cloning, Molecular; D017124:Conserved Sequence / Q000235:genetics; D003577:Cytochrome P-450 Enzyme System / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism*; D002523:Edible Grain / Q000201:enzymology / Q000235:genetics*; D004926:Escherichia coli / Q000235:genetics*; D005658:Fungi / Q000201:enzymology / Q000235:genetics; D015870:Gene Expression; D008322:Mammals / Q000235:genetics; D008969:Molecular Sequence Data; D010088:Oxidoreductases / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism*; D010802:Phylogeny; D011993:Recombinant Fusion Proteins / Q000302:isolation & purification / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D058886:Sterol 14-Demethylase; D013379:Substrate Specificity
D011993:Recombinant Fusion Proteins; D003577:Cytochrome P-450 Enzyme System; D010088:Oxidoreductases; D058886:Sterol 14-Demethylase