Cloning and functional expression in yeast of a cDNA coding for an obtusifoliol 14alpha-demethylase (CYP51) in wheat.
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.
Cabello-Hurtado
F
F
Department of Cellular and Molecular Enzymology, Institute of Plant Molecular Biology, CNRS UPR 406, Strasbourg, France.
Zimmerlin
A
A
Rahier
A
A
Taton
M
M
DeRose
R
R
Nedelkina
S
S
Batard
Y
Y
Durst
F
F
Pallett
K E
KE
Werck-Reichhart
D
D
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
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