RESUMO
cDNA clones for the (S)-tetrahydroberberine (H4Ber) oxidase of cultured berberine-producing Coptis japonica cells were isolated by screening a C. japonica cDNA library with synthetic nucleotides that can encode the NH2-terminal sequence of this enzyme. Analyses of the nucleotide sequences of the cloned cDNA inserts revealed a 759-base-pair open reading frame that encoded a 253-amino acid polypeptide with a Mr of 27,089 and NH2-terminal and internal sequences identical with those of the (S)-H4Ber oxidase, as determined by microsequencing methods. Escherichia coli were transformed with an expression vector carrying (S)-H4Ber oxidase cDNA. The transformed bacteria were induced to overproduce a 28-kDa protein that reacted with Coptis (S)-H4Ber oxidase-specific antibody. A comparison of the derived amino acid sequence of (S)-H4Ber oxidase with sequences in the protein data base of the Protein Research Foundation showed a marked similarity between (S)-H4Ber oxidase and the NH2-terminal portion of mouse P1-450, which is encoded by a single exon of the mouse P1-450 gene. The availability of cloned cDNA for (S)-H4Ber oxidase allows use of the methods of molecular biology to study the regulation of (S)-H4Ber oxidase gene expression in cultured C. japonica cells in relation to berberine biosynthesis.
Assuntos
Alcaloides de Berberina/biossíntese , Berberina/biossíntese , Oxirredutases atuantes sobre Doadores de Grupo CH-CH , Oxirredutases/genética , Sequência de Aminoácidos , Sequência de Bases , Western Blotting , Células Cultivadas , Cromatografia Líquida de Alta Pressão , DNA/genética , Eletroforese em Gel de Poliacrilamida , Escherichia coli/genética , Regulação da Expressão Gênica , Dados de Sequência Molecular , Hibridização de Ácido Nucleico , Oxirredutases/biossíntese , Plantas , Plasmídeos , RNA/genética , Mapeamento por RestriçãoRESUMO
Tyrosine decarboxylase (EC 4.1.1.25) was induced in suspension cultures of Thalictrum rugosum by treatment with a yeast glucan elicitor. Maximum induction was observed at a carbohydrate concentration of 0.4 mg/g fresh weight of cells and maximum enzyme activity was reached 20 h after addition of elicitor. The enzyme was inducible in late exponential and early stationary growth phases. A good correlation between induced tyrosine decarboxylase activity and berberine biosynthesis has been established. It is suggested that tyrosine decarboxylase may be a key enzyme between primary and secondary metabolisms in the biosynthesis of norlaudanosoline-derived alkaloids.
Assuntos
Alcaloides de Berberina/biossíntese , Berberina/biossíntese , Plantas/enzimologia , Tirosina Descarboxilase/biossíntese , Células Cultivadas , Cromatografia Líquida de Alta Pressão , Eletroforese em Gel de Poliacrilamida , Indução Enzimática , CinéticaRESUMO
The formation of tetrahydro-isoquinoline alkaloids in human and animal metabolisms subsequent to alcohol consumption is reported. The spectrum of identified products ranges from simple structures such as salsolinol to the complicated skeleton of protoberberines. These substances, hitherto found exclusively in plants, interfere with the biosynthesis, biodegradation and neurotransmission of the structurally closely related biogenic amines from which they are formed, and thus affect the vegetative nervous system.
