title: Cloning and functional expression in yeast of a cDNA coding for an obtusifoliol 14alpha-demethylase (CYP51) in wheat.
abstract: Screening of a wheat cDNA library with an heterologous CYP81B1 probe from Helianthus tuberosus led to the isolation of a partial cDNA coding a protein with all the characteristics of a typical P450 with high homology (32-39% identity) to the fungal and mammalian CYP51s. Extensive screening of several wheat cDNA libraries isolated a longer cDNA (W516) coding a peptide of 453 amino acids. Alignment of W516 with other P450 sequences revealed that it was missing a segment corresponding to the N-terminal membrane anchor of the protein. The corresponding segment from the yeast lanosterol 14alpha-demethylase was linked to the partial wheat cDNA and the chimera expressed in Saccharomyces cerevisiae. Compared to microsomes from control yeasts, membranes of yeast expressing the chimera catalysed 14alpha-demethylation of obtusifoliol with an increased efficiency relative to lanosterol demethylase activity. W516 is thus a plant member of the most ancient and conserved P450 family, CYP51.
authors.affiliation: Department of Cellular and Molecular Enzymology, Institute of Plant Molecular Biology, CNRS UPR 406, Strasbourg, France.
authors.forename: F
authors.initials: F
authors.lastname: Cabello-Hurtado
authors.forename: A
authors.initials: A
authors.lastname: Zimmerlin
authors.forename: A
authors.initials: A
authors.lastname: Rahier
authors.forename: M
authors.initials: M
authors.lastname: Taton
authors.forename: R
authors.initials: R
authors.lastname: DeRose
authors.forename: S
authors.initials: S
authors.lastname: Nedelkina
authors.forename: Y
authors.initials: Y
authors.lastname: Batard
authors.forename: F
authors.initials: F
authors.lastname: Durst
authors.forename: K E
authors.initials: KE
authors.lastname: Pallett
authors.forename: D
authors.initials: D
authors.lastname: Werck-Reichhart
mesh_terms: D000595:Amino Acid Sequence; D001483:Base Sequence; D002775:Cholestadienols / Q000378:metabolism; D003001:Cloning, Molecular; D003577:Cytochrome P-450 Enzyme System / Q000096:biosynthesis* / Q000737:chemistry / Q000378:metabolism; D017931:DNA Primers; D018076:DNA, Complementary; D019143:Evolution, Molecular; D015723:Gene Library; D007810:Lanosterol / Q000378:metabolism; D008861:Microsomes / Q000201:enzymology; D008969:Molecular Sequence Data; D010088:Oxidoreductases / Q000096:biosynthesis* / Q000737:chemistry / Q000378:metabolism; D010802:Phylogeny; D010840:Phytosterols; D016133:Polymerase Chain Reaction; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000378:metabolism; D012441:Saccharomyces cerevisiae; D017386:Sequence Homology, Amino Acid; D058886:Sterol 14-Demethylase; D013379:Substrate Specificity; D014908:Triticum / Q000201:enzymology* / Q000235:genetics
chemical_list: D002775:Cholestadienols; D017931:DNA Primers; D018076:DNA, Complementary; D010840:Phytosterols; D011993:Recombinant Fusion Proteins; D007810:Lanosterol; C008693:obtusifoliol; D003577:Cytochrome P-450 Enzyme System; D010088:Oxidoreductases; D058886:Sterol 14-Demethylase