The phytoene dehydrogenase gene of Phycomyces: regulation of its expression by blue light and vitamin A.
By using a polymerase chain reaction based cloning strategy we isolated the gene (carB) encoding the enzyme phytoene dehydrogenase from Phycomyces blakesleeanus. The deduced protein, a 583 residue polypeptide, showed great similarity to carotenoid dehydrogenases from other fungi and bacteria, especially in the amino-terminal region. The main conserved regions found in other phytoene dehydrogenases, which are thought to be essential for the enzymatic activity, are present in the sequence from Phycomyces. Heterologous expression of the Phycomyces gene in Escherichia coli showed that, as in other fungi and bacteria, a single polypeptide catalyzes the four dehydrogenations that convert phytoene to lycopene. RNA measurements indicated that the level of expression of the phytoene dehydrogenase gene in wild-type mycelia increased in response to blue light. The kinetics of this increase in transcription of the gene after blue light induction (0.1 and 0.4 W/m2) exhibit a two-step (biphasic) dependence on fluence rate, suggesting that there could be two separate components involved in the reception of the low and high blue light signal. The presence of vitamin A in the medium stimulated transcript accumulation in the wild type and in some carotenogenic mutant strains. Diphenylamine, a phytoene dehydrogenase inhibitor, did not affect the level of transcription of this gene.
Ruiz-Hidalgo
M J
MJ
Departamento de Microbiología y Genética, Universidad de Salamanca, Spain.
Benito
E P
EP
Sandmann
G
G
Eslava
A P
AP
D000595:Amino Acid Sequence; D001483:Base Sequence; D003001:Cloning, Molecular; D004271:DNA, Fungal; D004159:Diphenylamine / Q000494:pharmacology; D004926:Escherichia coli; D015966:Gene Expression Regulation, Fungal / Q000187:drug effects / Q000528:radiation effects; D005800:Genes, Fungal; D008027:Light; D008969:Molecular Sequence Data; D010088:Oxidoreductases / Q000235:genetics*; D010800:Phycomyces / Q000187:drug effects / Q000201:enzymology* / Q000235:genetics; D012333:RNA, Messenger / Q000378:metabolism; D014158:Transcription, Genetic / Q000187:drug effects; D014801:Vitamin A / Q000494:pharmacology*
D004271:DNA, Fungal; D012333:RNA, Messenger; D014801:Vitamin A; D004159:Diphenylamine; D010088:Oxidoreductases; C022398:phytoene dehydrogenase