Alkaloid biosynthesis in Rauvolfia -: cDNA cloning of major enzymes of the ajmaline pathway
Alkaloid biosynthesis in Rauvolfia -: cDNA cloning of major enzymes of the ajmaline pathway
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DOI：
10.2174/138527205774370540
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发表时间：
2005-10-01
影响因子：
2.6
通讯作者：
Stöckigt, J
中科院分区：
文献类型：
--
作者：
Ruppert, M;Ma, XY;Stöckigt, J
在印度药用植物萝芙木的细胞悬浮培养物中，所有参与阿娇苷生物合成的主要反应现在都已在酶水平上建立。在充分研究的10个步骤中，6个涉及的酶已被功能性克隆。这些酶是野strictosidine合酶（STR）、野strictosidine葡糖苷酶（SG）、多聚神经苷醛酯酶（PNAE）、长春碱合酶（VS）、细胞色素P450还原酶（CPR）和乙酰拉马兰乙酰酯酶（AAE）。由于这些酶的cDNA现在是已知的，因此第一次可以进行详细的分子分析。其中一些酶如STR、SG、VS等在大肠杆菌中均能产生。大肠杆菌中的酶，得到mg量的纯酶。它们也被结晶化，最近发表了它们的初步X射线分析。它们的分子结构和催化反应的机理将被阐明，这只是一个时间问题。可溶性酶vomilenine还原酶和1.2-dihydrovomilenine还原酶仍异源表达。最近已经分离出合适的cDNA克隆。该途径中的膜结合酶的克隆目前还不能实现。我们未来的策略是首先纯化这些酶，并使用“反向遗传”方法，就像我们对可溶性酶所做的那样。sarpagine桥酶（SBE），长春碱羟化酶（VH）和norajlavine N-甲基转移酶（NAMT）是唯一的酶，仍然是表达研究的主要候选人，以异源表达完整的ajlavine生物合成途径。
All the major reactions taking part in the biosynthesis of ajmaline in cell suspension cultures of the Indian medicinal plant Rauvolfia have now been established at the enzyme level. Of the well investigated 10 steps six of the involved enzymes have been functionally cloned. These are strictosidine synthase (STR), strictosidine glucosidase (SG), polyneuridine aldehyde esterase (PNAE), vinorine synthase (VS), cytochrome P450 reductase (CPR) and acetylajmalan acetylesterase (AAE). Because the cDNAs of these enzymes are now known their detailed molecular analysis became attainable for the first time. Some of these enzymes such as STR, SG or VS could be produced in E. coli at a preparative scale resulting in mg amounts of pure enzymes. They also have been crystallized and their preliminary X-ray analyses were published recently. It is only a matter of time that their molecular structure and the mechanisms of the catalyzed reactions will be elucidated. Of the soluble enzymes vomilenine reductase and 1.2-dihydrovomilenine reductase remain to be heterologously expressed. Appropriate cDNA clones have recently been isolated. What membrane bound enzymes of this pathway is concerned cloning could not be achieved up to now. Our future strategy is to purify these enzymes first and to use the "reverse genetic" approach as we did for the soluble enzymes. The sarpagine bridge enzyme (SBE), vinorine hydroxylase (VH) and norajmaline N-methyltransferase (NAMT) are the only enzymes which remain as the major candidates for expression studies in order to express heterologously the-complete ajmaline biosynthetic pathway.