ABSTRACT
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.
Subject(s)
Berberine Alkaloids/biosynthesis , Berberine/biosynthesis , Oxidoreductases Acting on CH-CH Group Donors , Oxidoreductases/genetics , Amino Acid Sequence , Base Sequence , Blotting, Western , Cells, Cultured , Chromatography, High Pressure Liquid , DNA/genetics , Electrophoresis, Polyacrylamide Gel , Escherichia coli/genetics , Gene Expression Regulation , Molecular Sequence Data , Nucleic Acid Hybridization , Oxidoreductases/biosynthesis , Plants , Plasmids , RNA/genetics , Restriction MappingABSTRACT
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.
