Molecular characterization of the salutaridinol 7-O-acetyltransferase involved in morphine biosynthesis in opium poppy Papaver somniferum
Molecular characterization of the salutaridinol 7-O-acetyltransferase involved in morphine biosynthesis in opium poppy Papaver somniferum
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DOI：
10.1074/jbc.m102688200
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发表时间：
2001-08-17
影响因子：
4.8
通讯作者：
Kutchan, TM
中科院分区：
文献类型：
--
作者：
Grothe, T;Lenz, R;Kutchan, TM
Salutaridinol 7-O-乙酰基转移酶(EC 2.3.1.150)催化菲生物碱Salutaridinol转化为Salutaridinol-7-O-乙酸酯，它是茶碱在吗啡生物合成途径中的直接前体。我们从罂粟悬浮细胞培养物中分离到一个与天然酶的内部氨基酸序列相对应的cDNA克隆。该重组酶在乙酰辅酶A存在下乙酰化了丹参糖醇的7-羟基部分。测得丹参素的表观K值为9微米，乙酰辅酶A为54微米。除野罂粟外，东方罂粟和苞叶罂粟提取物中也检测到该基因的转录本。基因组DNA凝胶印迹分析表明，该基因在睡眠菌基因组中可能只有一个拷贝。该酶的氨基酸序列与长春花脱乙酰长春花乙酰转移酶的氨基酸序列最为相似(同源性为37%)。水杨梅素7-O-乙酰转移酶是第二个专用于吗啡生物合成的酶，我们已经克隆了该酶的基因。与其他克隆的去甲黄嘌呤6-O-甲基转移酶、(S)-N-甲基辅酶A-3‘-羟基酶、细胞色素P-450还原酶和可待酮还原酶基因一起，在积累这一途径的基因方面取得了重大进展，从而实现了生物技术生产吗啡生物碱的最终目标。
Salutaridinol 7-O-acetyltransferase (EC 2.3.1.150) catalyzes the conversion of the phenanthrene alkaloid salutaridinol to salutaridinol-7-O-acetate, the immediate precursor of thebaine along the morphine biosynthetic pathway. We have isolated a cDNA clone that corresponds to the internal amino acid sequences of the native enzyme purified from a cell suspension culture of opium poppy Papaver somniferum. The recombinant enzyme acetylated the 7-hydroxyl moiety of salutaridinol in the presence of acetyl-CoA. The apparent K. value for salutaridinol was determined to be 9 mum and 54 mum for acetyl-CoA. The gene transcript was detected in extracts from Papaver orientale and Papaver bracteatum in addition to P. somniferum. Genomic DNA gel blot analysis indicated that there is likely a single copy of this gene in the P. somniferum genome. The amino acid sequence of salutaridinol 7-O-acetyltransferase is most similar (37% identity) to that of deacetylvindoline acetyltransferase of Catharanthus roseus. Salutaridinol 7-O-acetyltransferase is the second enzyme specific to morphine biosynthesis for which we have isolated a cDNA. Taken together with the other cDNAs cloned encoding norcoclaurine 6-O-methyl-transferase, (S)-N-methylcoclaurine 3 ' -hydroxylase, the cytochrome P-450 reductase, and codeinone reductase, significant progress has been made toward accumulating genes of this pathway to enable the end goal of a biotechnological production of morphinan alkaloids.