Okada N, Koizumi N, Tanaka T, Ohkubo H, Nakanishi S, Yamada Y
Research Center for Cell and Tissue Culture, Faculty of Agriculture, Kyoto, Japan.
Proc Natl Acad Sci U S A. 1989 Jan;86(2):534-8. doi: 10.1073/pnas.86.2.534.
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.
通过用可编码该酶氨基末端序列的合成核苷酸筛选黄连cDNA文库，分离出了培养的产黄连素黄连细胞中（S）-四氢小檗碱（H4Ber）氧化酶的cDNA克隆。对克隆的cDNA插入片段的核苷酸序列分析揭示了一个759个碱基对的开放阅读框，其编码一个253个氨基酸的多肽，分子量为27,089，通过微量测序方法确定其氨基末端和内部序列与（S）-H4Ber氧化酶的序列相同。用携带（S）-H4Ber氧化酶cDNA的表达载体转化大肠杆菌。诱导转化后的细菌过量产生一种与黄连（S）-H4Ber氧化酶特异性抗体发生反应的28 kDa蛋白。将（S）-H4Ber氧化酶的推导氨基酸序列与蛋白质研究基金会蛋白质数据库中的序列进行比较，结果显示（S）-H4Ber氧化酶与小鼠P1-450的氨基末端部分有显著相似性，小鼠P1-450由小鼠P1-450基因的一个外显子编码。（S）-H4Ber氧化酶克隆cDNA的可用性使得可以利用分子生物学方法来研究培养的黄连细胞中（S）-H4Ber氧化酶基因表达与黄连素生物合成的关系。
