Cloning of the astaxanthin synthase gene from Xanthophyllomyces dendrorhous (Phaffia rhodozyma) and its assignment as a beta-carotene 3-hydroxylase/4-ketolase.
A gene has been cloned from Xanthophyllomyces dendrorhous by complementation of astaxanthin formation in a beta-carotene accumulating mutant. It consists of 3,166 bp and contains 17 introns. For the beta-carotene mutant ATCC 96815, a single point mutation in the splicing sequence of intron 8 was found. The resulting improper splicing of the mRNA results in an inactive protein. The cDNA of this beta-carotene oxygenase encodes a cytochrome P450 monooxygenase belonging to the 3A subfamily. P450-specific domains were identified including a cytochrome P450 and an oxygen binding motif. Electrons are provided by a cytochrome P450 reductase. Functional characterization of the enzyme by genetic modification of X. dendrorhous demonstrated that this P450 monooxygenase is multifunctional catalyzing all steps from beta-carotene to astaxanthin formation by oxygenation of carbon 3 and 4. The reaction sequence is first 4-ketolation of beta-carotene followed by 3-hydroxylation. A hydroxylation mechanism at allylic carbon atoms has been proposed for the generation of 4-keto and 3-hydroxy groups at both beta-ionone ends.
Ojima
Kazuyuki
K
Department of Applied Microbiology, Nippon Roche Research Center, Kamakura, Kanagawa, Japan.
Breitenbach
Jürgen
J
Visser
Hans
H
Setoguchi
Yutaka
Y
Tabata
Kazuyuki
K
Hoshino
Tatsuo
T
van den Berg
Johan
J
Sandmann
Gerhard
G
D000595:Amino Acid Sequence; D001483:Base Sequence; D001487:Basidiomycota / Q000201:enzymology* / Q000235:genetics*; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000235:genetics; D004271:DNA, Fungal / Q000235:genetics; D005800:Genes, Fungal; D006899:Mixed Function Oxygenases / Q000145:classification / Q000235:genetics* / Q000378:metabolism; D008954:Models, Biological; D008969:Molecular Sequence Data; D017386:Sequence Homology, Amino Acid; D024341:Xanthophylls; D019207:beta Carotene / Q000031:analogs & derivatives* / Q000096:biosynthesis
D018076:DNA, Complementary; D004271:DNA, Fungal; D024341:Xanthophylls; D019207:beta Carotene; C005948:astaxanthine; D006899:Mixed Function Oxygenases; C079232:beta-carotene hydroxylase