title: Analysis of al-2 mutations in Neurospora.
abstract: The orange pigmentation of the fungus Neurospora crassa is due to the accumulation of the xanthophyll neurosporaxanthin and precursor carotenoids. Two key reactions in the synthesis of these pigments, the formation of phytoene from geranylgeranyl pyrophosphate and the introduction of β cycles in desaturated carotenoid products, are catalyzed by two domains of a bifunctional protein, encoded by the gene al-2. We have determined the sequence of nine al-2 mutant alleles and analyzed the carotenoid content in the corresponding strains. One of the mutants is reddish and it is mutated in the cyclase domain of the protein, and the remaining eight mutants are albino and harbor different mutations on the phytoene synthase (PS) domain. Some of the mutations are expected to produce truncated polypeptides. A strain lacking most of the PS domain contained trace amounts of a carotenoid-like pigment, tentatively identified as the squalene desaturation product diapolycopene. In support, trace amounts of this compound were also found in a knock-out mutant for gene al-2, but not in that for gene al-1, coding for the carotene desaturase. The cyclase activity of the AL-2 enzyme from two albino mutants was investigated by heterologous expression in an appropriately engineered E. coli strain. One of the AL-2 enzymes, predictably with only 20% of the PS domain, showed full cyclase activity, suggesting functional independence of both domains. However, the second mutant showed no cyclase activity, indicating that some alterations in the phytoene synthase segment affect the cyclase domain. Expression experiments showed a diminished photoinduction of al-2 transcripts in the al-2 mutants compared to the wild type strain, suggesting a synergic effect between reduced expression and impaired enzymatic activities in the generation of their albino phenotypes.
authors.affiliation: Department of Genetics, Faculty of Biology, University of Seville, Seville, Spain.
authors.forename: Violeta
authors.initials: V
authors.lastname: Díaz-Sánchez
authors.forename: Alejandro F
authors.initials: AF
authors.lastname: Estrada
authors.forename: Danika
authors.initials: D
authors.lastname: Trautmann
authors.forename: M Carmen
authors.initials: MC
authors.lastname: Limón
authors.forename: Salim
authors.initials: S
authors.lastname: Al-Babili
authors.forename: Javier
authors.initials: J
authors.lastname: Avalos
mesh_terms: D019883:Alkyl and Aryl Transferases / Q000378:metabolism; D000483:Alleles; D000595:Amino Acid Sequence; D053898:Biosynthetic Pathways / Q000235:genetics / Q000528:radiation effects; D002338:Carotenoids / Q000096:biosynthesis / Q000378:metabolism; D004926:Escherichia coli / Q000378:metabolism; D005656:Fungal Proteins / Q000737:chemistry / Q000235:genetics; D015966:Gene Expression Regulation, Fungal / Q000528:radiation effects; D005800:Genes, Fungal / Q000235:genetics*; D051232:Geranylgeranyl-Diphosphate Geranylgeranyltransferase; D008027:Light; D000077276:Lycopene; D008969:Molecular Sequence Data; D050505:Mutant Proteins / Q000737:chemistry / Q000378:metabolism; D009154:Mutation / Q000235:genetics*; D009492:Neurospora crassa / Q000166:cytology / Q000201:enzymology / Q000235:genetics* / Q000528:radiation effects; D010455:Peptides / Q000378:metabolism; D010641:Phenotype; D010858:Pigmentation / Q000235:genetics / Q000528:radiation effects; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism
chemical_list: D005656:Fungal Proteins; D050505:Mutant Proteins; D010455:Peptides; D012333:RNA, Messenger; D002338:Carotenoids; D019883:Alkyl and Aryl Transferases; D051232:Geranylgeranyl-Diphosphate Geranylgeranyltransferase; D000077276:Lycopene