Isolation, sequence, and bacterial expression of a cDNA for (S)-tetrahydroberberine oxidase from cultured berberine-producing Coptis japonica cells.
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.
Okada
N
N
Research Center for Cell and Tissue Culture, Faculty of Agriculture, Kyoto, Japan.
Koizumi
N
N
Tanaka
T
T
Ohkubo
H
H
Nakanishi
S
S
Yamada
Y
Y
D000595:Amino Acid Sequence; D001483:Base Sequence; D001599:Berberine / Q000096:biosynthesis*; D001600:Berberine Alkaloids / Q000096:biosynthesis*; D015153:Blotting, Western; D002478:Cells, Cultured; D002851:Chromatography, High Pressure Liquid; D004247:DNA / Q000235:genetics; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics; D005786:Gene Expression Regulation; D008969:Molecular Sequence Data; D009693:Nucleic Acid Hybridization; D010088:Oxidoreductases / Q000096:biosynthesis / Q000235:genetics*; D044925:Oxidoreductases Acting on CH-CH Group Donors; D010944:Plants; D010957:Plasmids; D012313:RNA / Q000235:genetics; D015183:Restriction Mapping
D001600:Berberine Alkaloids; D001599:Berberine; D012313:RNA; D004247:DNA; D010088:Oxidoreductases; D044925:Oxidoreductases Acting on CH-CH Group Donors; C056768:tetrahydroprotoberberine oxidase