Takeshita N, Fujiwara H, Mimura H, Fitchen J H, Yamada Y, Sato F
Department of Agricultural Chemistry, Faculty of Agriculture, Kyoto University, Japan.
Plant Cell Physiol. 1995 Jan;36(1):29-36.
S-Adenosyl-L-methionine:scoulerine-9-O-methyltransferase (SMT) catalyzes the transfer of the S-methyl group of S-adenosyl-L-methionine to the 9-hydroxyl group of scoulerine during the biosynthesis of berberine. We have isolated functionally active cDNA clones (pCJSMTs) from a cDNA library prepared from cultured cells of Coptis japonica. The longest cDNA insert (pCJSMT1) had an open reading frame that encoded 351 amino acids, but the calculated molecular mass (38,364 Da) of the deduced product was slightly lower than the experimentally determined molecular mass of purified SMT. Rapid amplification of the 5' end of the cDNA indicated that the full-length cDNA of SMT consisted of 1,458 nucleotides that encoded 381 amino acids. When the full-length cDNA was expressed in E. coli, the molecular mass of the expressed SMT was greater than that of native SMT in Coptis cells. This result suggests that SMT might be produced in a pre-mature form and processed post-translationally. SMT was also found to exhibit sequence homology to other O-methyltransferases from plants and N-terminal region of the SMT polypeptide appeared to be necessary for enzymatic activity.
S-腺苷-L-甲硫氨酸：小檗碱-9-O-甲基转移酶（SMT）在小檗碱生物合成过程中催化S-腺苷-L-甲硫氨酸的S-甲基转移至小檗碱的9-羟基上。我们从日本黄连培养细胞制备的cDNA文库中分离出了功能活性cDNA克隆（pCJSMTs）。最长的cDNA插入片段（pCJSMT1）有一个编码351个氨基酸的开放阅读框，但推导产物的计算分子量（38,364 Da）略低于纯化的SMT的实验测定分子量。cDNA 5'端的快速扩增表明，SMT的全长cDNA由1458个核苷酸组成，编码381个氨基酸。当全长cDNA在大肠杆菌中表达时，表达的SMT的分子量大于黄连细胞中天然SMT的分子量。这一结果表明，SMT可能以一种前体形式产生并在翻译后进行加工。还发现SMT与来自植物的其他O-甲基转移酶具有序列同源性，并且SMT多肽的N端区域似乎是酶活性所必需的。
