石杉碱甲生物合成中N-甲基转移酶的分子克隆与催化机制研究

Huperzine A(HupA), one of the lycopodium alkaloids which extracted from Huperzia Serrata, is a potent, highly specific and reversible inhibitor of acetylcholinesterase (ACHE). The phase IV clinical trials in China have demonstrated that HupA significantly improved memory deficits in elderly people with benign senescent forgetfulness, and patients with Alzheimer disease (AD). .　　Because of the difficulty in total synthesis of Hup A, it is principally extracted from fresh plant. The content of HupA in plant is very low (about 0.01%). In recent years, with the development and vegetation deterioration, this plant is suffering exhaustion. The successful tissue culture or other good method has seldom been reported. Bio-engineering pathway to solve this problem is a whole new area of study..　　The potential biosynthetic pathways of several of the Lycopodium alkaloids have been proposed. Lycopodine has been proposed to be a central intermediate in the formation of other classes of alkaloids and the N-methyltransferase (N-MTase) might play the important roles in these pathways. We predict that the HupA N-MTase is specific for the methylation from HupA to huperzizine. Considering the reduced activity of ACHE for huperzizine, the HupA N-MTase should play an important role in the biosynthetic pathways of HupA. Low-expressing of HupA N-MTase might contribute to the accumulation of HupA. .　　We already cloned a novel methyltransferase from Huperzia Serrata and the preliminary experiment demonstrated that the cDNA encodes a novel SAM-dependent methyltransferase can catalyze a methylation of HupA. To elucidate the function of this enzyme, we will carry out the function identification and structure analysis. The substrate selectivity, methylation site and catalyze central will be studied..　　This study will lay the foundation of the follow-up work which to obtain the HupA accumulated plant via the inhibition of N-MTase, and also support the elucidation of whole biosynthesis pathway of HupA.

石杉碱甲（HupA）是用于治疗老年痴呆症（AD）的抗胆碱酯酶抑制剂，其化学合成困难，在植物中含量又极低，面临严重资源问题。在HupA生物合成途径中，N-甲基转移酶（N-MTase）催化HupA甲基化，生成Huperzinine，后者抗胆碱酯酶活性大大降低。阻断该甲基化途径可使HupA在植物体内累积。我们前期已通过分子克隆和重组技术，从千层塔中获得一种SAM（S-腺苷甲硫氨酸）依赖性N-MTase，并初步证实其能催化HupA甲基化。本项目拟对该N-MTase的催化活性和作用机制进行研究，包括底物选择性、催化动力学、甲基化位点、酶的空间结构以及通过突变株活性验证酶催化中心。该项目可为今后通过降低其表达水平或抑制其活性从而促使HupA累积获取高产HupA植株打下理论基础，同时也为HupA完整生物合成途径解析提供支持。

石杉碱甲是治疗早老性痴呆的重要药物，目前资源面临匮乏。研究石杉碱甲生物合成途径不仅能解决资源问题，还有利于发展生物合成药物的新思路。我们利用分子克隆和重组技术，通过大肠杆菌原核表达，获得高表达的重组酶—SAM依赖性石杉碱甲甲基转移酶（HupA-N MTase），并对该重组酶特异性催化HupA N-甲基化的催化活性进行研究，确定其天然底物和催化产物，对产物进行了结构确证，测定各动力学参数；我们还进行了同源建模，构建了突变株，突变株未显活性，推测其三维空间对活性具有影响。该研究通过阐明生物合成途径中从HupA到N-甲基HupA的过程，可以解释HupA在千层塔中的“去路”。为下一步采取基因敲除等技术获得高产HupA千层塔工作提供直接有力的理论支持。我们还额外对千层塔产石杉碱甲的内生真菌进行了筛选，并进行了培养。并对其表观遗传进行研究。我们通过化学抑制手段对其表观遗传进行了调控，得到了产物明显变化的结果。该研究为降低该酶表达水平或抑制其活性来阻断HupA甲基化过程获取高产Hup A植株提供实验基础，具有重要的转化意义。