蛇足石杉聚酮合酶的克隆鉴定及其在组合合成中的应用

发布时间：2018-04-23 11:06

本文选题：蛇足石杉 + 石杉碱甲　；参考：《北京中医药大学》2017年博士论文

【摘要】：蛇足石杉Huperzia serrata(Thunb.)Trev.,俗名是千层塔、蛇足草、山芝,为石杉科Huperziaceae石杉属Huperzia Bernh多年生草本蕨类植物。全草可以散瘀消肿、解毒和止痛。从蛇足石杉中分离得到的生物碱-石杉碱甲(HuperzineA,HupA)为高效、可逆、高选择性的乙酰胆碱酯酶抑制剂,能够提高学习、记忆效果,且其特异性强、毒性低,临床上已成为国内外治疗阿尔兹海默症最有效的药物之—。植物聚酮合酶(plant polyketide synthases,Ⅲ PKSs)是植物中广泛存在的一大类能够催化合成植物多酚类成分的酶的总称。植物聚酮合酶通过催化起始底物(酰基辅酶A)与丙二酰辅酶A反复地缩合形成链状聚酮中间体,再经过克莱森、Aldol等环化方式生成查耳酮、二苯乙烯、间苯三酚等结构多样的天然产物和"非天然小分子"。在植物聚酮合酶催化合成多酚类化合物的过程中,起始底物、链的增长单位、起始底物与链增长单位缩合的分子数、链状聚酮中间体环合方式等对酶催化产物的结构起决定性作用,因此,利用植物聚酮合酶作为工具酶,开展以底物为导向的酶催化合成,构建数目可观、结构新颖的"非天然小分子"群(库),再结合药理活性筛选,用于活性小分子的发现,将对于新药研发具有重要意义。HupA在原植物体内的含量较低,因此传统的提取分离方法难以满足市场需求,且极容易造成野生植物资源的破坏;而有机合成的方法普遍存在着合成步骤多、最终产率低、尤其是合成光学纯的Hup A的难度大等问题,因此无法实现利用工业化生产来解决Hup A的来源问题。近年来,随着生物技术的发展,一些与药物生产紧密相关的活性中间体的生物合成调控基因或生物合成途径被阐明,通过对相关调控基因的人为修饰,可以实现目标产物的定向合成或规模化生产。目前比较成功的化合物如来源于植物的紫杉醇、青蒿素、吲哚类生物碱等,其在生物工程菌(或生物工程植株)中可实现规模化合成。因此,阐明Hup A的生物合成途径及关键酶,将为利用合成生物学的方法解决HupA的药源问题提供必要条件。根据文献中同位素标记结果,我们推测植物聚酮合酶可能参与石杉碱甲的生物合成。因此,本课题针对蛇足石杉中的聚酮合酶进行研究。一方面,克隆鉴定蛇足石杉中的植物聚酮合酶,鉴定其催化功能,并在此基础上开展组合合成研究,构建"非天然小分子"化合物库,结合活性筛选,进行活性化合物的发现;另一方面,探讨获得的聚酮合酶和Hup A的生物合成相关性,力图阐明其在Hup A生物合成中的作用。目前,课题组取得了以下成果:一、从蛇足石杉新鲜叶片中成功克隆并表达三个植物聚酮合酶HsPKS1、HsPKS2、HsPKS3,并对这三个酶在体外的催化活性进行分析,发现HsPKS1、HsPKS2与HsPKS3体外活性明显不同。HsPKS1和HsPKS2是典型的查耳酮合酶,在体外可以催化一分子对羟基肉桂酰辅酶A和三分子的丙二酰辅酶A缩合,经过克莱森环合生成柚皮素查耳酮,酸性条件下环合成二氢黄酮,该化合物是黄酮类化合物生物合成的重要前体,为蛇足石杉中黄酮类成分的生物合成机制提供参考。此外HsPKS1和HsPKS2还可以催化一分子的非天然底物2-N-甲基-苯甲酰辅酶A和三分子丙二酰辅酶A缩合生成1,3-二羟基-N-甲基吖啶酮。HspKS3是一个新颖的植物聚酮合酶,虽然HsPKS3与HsPKS1、HsPKS2序列相似度较高,但是HsPKS3的催化活性完全不同。HsPKS3在体外可以催化两分子对羟基肉桂酰辅酶A与一分子丙二酰辅酶A进行"头碰头"缩合生成双去甲氧基姜黄素和副产物对羟基苄基丙酮;HsPKS3还可以催化非天然底物2-N-甲基-苯甲酰辅酶A与一分子丙二酰辅酶A缩合生成简单喹诺酮。目前,自然界中具备如此多样催化功能的植物聚酮合酶尚属首次发现。二、通过对已表达的植物聚酮合酶的结构与底物以及催化活性的相关性进行研究,发现三个酶具有广泛的底物选择性,因此开展了初步的组合合成。HsPKS1和HsPKS2根据其催化活性,利用4CL酶法合成了一系列苯丙烯类及其类似物的有机酸硫酯如阿魏酰辅酶A、苯环上含有吸电子基团的4-氟-肉桂辅酶A,含有五元芳杂环的2-呋喃苯丙烯辅酶A,并在体外组合HsPKS1和HsPKS2进行催化反应,实现了完全酶催化的体外合成一系列黄酮、4-羟基-δ-在内酯类化合物。对于HsPKS3,利用其广泛的底物选择性,人工合成了芳香环、脂肪族、杂环等结构多样的起始底物,同时为了探讨延长单位的选择性,还制备了丙二酰辅酶A以外的非天然的延长单位如β-酮酸和β-酮酸的N-乙酰基半胱胺(N-acetylcysteamine,NAC)硫酯结构,初步开展以底物为导向的组合合成,得到了一系列喹诺酮及1,3-二酮类成分,并通过药理活性筛选,发现部分分子具有较好的抗炎活性。本课题利用植物聚酮合酶作为工具酶,开展酶催化的组合合成,构建结构新颖多样的"非天然小分子"库进行药理活性筛选,为活性小分子的发现提供酶法合成的新思路。三、植物聚酮合酶HsPKS3的催化机制及其与Hup A的生物合成相关性探讨。通过同源建模的方法获得了 HsPKS3的三维结构数据,且利用在线评价平台系统对获得的三维结构进行评价,证明获得的三维结构合理。在此基础上对HsPKS3的催化机制进行了研究,发现Ser142是生成姜黄素类化合物的一个重要氨基酸位点,在此基础上对该氨基酸残基进行定点突变后,突变体将不能生成姜黄素类化合物。另一方面,将HsPKS3与小分子前体4PAA-CoA、phlegmarine类似物分别进行分子对接,从三维结构上解释了石榴碱和4PAA/4PAA-CoA在某种酶的催化作用下进行缩合反应的可能性,同时对实验过程中所遇到的问题、后续酶催化中间体的反应进行了理论性的指导。
[Abstract]:Huperzia serrata (Thunb.) Trev., commonly known as the pagoda, pagoda, and ganoderma, is a perennial fern of the Huperzia Bernh of the genus Huperziaceae in the family of taxidfamily. The whole grass can dissipate swelling, detoxify and relieve pain. The alkaloid - huperzine A (HuperzineA, HupA) isolated from the Sequoia serpidis (HuperzineA, HupA) is highly efficient, reversible and highly selective. Acetylcholinesterase inhibitors, which can improve learning, memory effect, and have a strong specificity and low toxicity, have become the most effective drugs for Alzheimer's disease both at home and abroad. Plant polyketone synthase (plant polyketide synthases, III PKSs) is a broad class of plant polyphenols that can catalyze the synthesis of plant polyphenols. The plant polyketone synthase is repeatedly condensed by catalytic initiating substrate (acyl coenzyme A) and propylene two acyl coenzyme A to form chain polyketone intermediates, and then through Claeson, Aldol and other cyclization methods to produce chalcone, two styrene, phenylene three phenol and other natural products and "non natural small molecules". In the process of polyphenols, the initiating substrate, the growth unit of the chain, the number of molecules condensed by the starting substrate and the chain growth unit, the chain polyketone intermediate cyclization mode and so on, play a decisive role in the structure of the enzyme catalyzed products. Therefore, the plant polyketone synthase is used as a tool enzyme to catalyze the enzyme catalyzed synthesis of the substrate, and the number can be constructed. The new structure of "non natural small molecules" group (Library), combined with pharmacological activity screening, used for the discovery of active small molecules, will be of great significance to the research and development of new drugs.HupA in the original plant content is low, so the traditional extraction and separation method is difficult to meet the market demand, and it is very easy to cause the destruction of wild plant resources; and organic There are many synthetic methods, such as many synthetic steps, low final yield, especially the difficulty of synthesizing optical pure Hup A, so it is impossible to realize the problem of using industrial production to solve the source problem of Hup A. In recent years, with the development of biological technology, the biosynthetic regulator of some active intermediates closely related to drug production has been developed. The cause or biosynthesis pathway is clarified, by the artificial modification of the related regulatory genes, the directional synthesis or large-scale production of target products can be realized. The more successful compounds are derived from taxol, artemisinin, indole alkaloids, etc., which can be synthesized in a biosynthetic Cheng Jun (or Bioengineering plant). Therefore, clarifying the biosynthesis pathway and key enzymes of Hup A will provide the necessary conditions for solving the drug source problem of HupA using synthetic biology methods. According to the results of isotopic markers in the literature, we speculate that plant polyketone may participate in the biosynthesis of huperzine A. On the one hand, cloning and identification of the plant polyketone synthase in Sequoia serrata, identification of its catalytic function, and on this basis to carry out combinatorial synthesis research, construction of a "non natural small molecule" compound library, combined with active screening, the discovery of active compounds, on the other hand, to explore the biosynthesis of polyketone synthase and Hup A, try to find the correlation of the biosynthesis of polyketone synthase and Hup. In order to clarify its role in the biosynthesis of Hup A, the following achievements have been made: 1, three plant polyketones HsPKS1, HsPKS2, HsPKS3 were successfully cloned and expressed from fresh leaves of Chinese fir, and the catalytic activity of these three enzymes in vitro was analyzed, and HsPKS1, the activity of HsPKS2 and HsPKS3 in vitro was significantly different from.HsPKS1 and Hs. PKS2 is a typical chalcone synthase, which can catalyze the condensation of a molecule of a molecule to the hydroxyl cinnamyl coenzyme A and the three molecule of the propane coenzyme A in vitro. Through the kleon cyclization of the naringin chalcone and the synthesis of two hydrogen flavones under acid conditions, the compound is an important precursor of the biosynthesis of flavonoids, which is a flavonoid in the fir of serpus opserus. In addition, HsPKS1 and HsPKS2 can also catalyze the condensation of non natural substrates, 2-N- methyl benzoyl coenzyme A and three molecular prop two acyl coenzyme A, to form 1,3- two hydroxyl -N- methylacridone.HspKS3 is a novel plant polyketone, although HsPKS3 and HsPKS1, HsPKS2 sequence is higher, but H The catalytic activity of sPKS3 is completely different from.HsPKS3 in vitro, which can catalyze the "head collision" of two molecules of hydroxyl cinnamyl coenzyme A and a molecular prop two acyl coenzyme A to produce dimethoxy curcumin and by-product pair hydroxybenzyl acetone; HsPKS3 can also catalyze the non natural substrate 2-N- methylbenzoyl concoenzyme A and a molecular propane two acyl coenzyme. A condensate to produce simple quinolone. At present, the plant polyketone synthase with so many catalytic functions is first discovered in nature. Two, through the study of the relationship between the structure and the substrate and the catalytic activity of the expressed plant polyketone synthase, it is found that three enzymes have extensive substrate selectivity, so a preliminary combination has been carried out. According to the catalytic activity of.HsPKS1 and HsPKS2, a series of benzene and its analogues are synthesized by 4CL enzyme method, such as feruloyl coenzyme A, 4- fluoro cinnamyl coenzyme A, containing five yuan aromatic heterocyclic acid, 2- furan benzene coenzyme A, and the catalytic reaction of HsPKS1 and HsPKS2 in vitro. A complete enzyme catalyzed synthesis of a series of flavonoids, 4- hydroxyl Delta - in the lactone in vitro, has been developed. For HsPKS3, a variety of initiating substrates such as aromatic rings, aliphatic and heterocyclic rings have been synthesized by using its extensive substrate selectivity. In addition, in order to explore the selectivity of the extended unit, the non natural other than the propane two acyl coenzyme A is also prepared. The structure of N- acetyl cysteamine (N-acetylcysteamine, NAC) thioester, such as beta ketoacid and beta ketoacid, was extended, and a series of quinolones and 1,3- two ketones were synthesized. As a tool enzyme, a combination of enzyme catalysis is carried out, a novel and diverse "non natural small molecule" library is constructed for pharmacological activity screening, which provides a new idea for enzymatic synthesis for the discovery of small active molecules. Three, the catalytic mechanism of plant polyketone synthase HsPKS3 and its correlation with Hup A synthesis. The three-dimensional structure of HsPKS3 is obtained, and the 3D structure obtained by the online evaluation platform is evaluated to prove that the obtained three-dimensional structure is reasonable. On the basis of this, the catalytic mechanism of HsPKS3 is studied. It is found that Ser142 is a heavy essential amino acid site for the formation of curcumin compounds. On this basis, the amino acid is used for the amino acid. The mutants will not produce curcumin compounds after the site is mutagenesis. On the other hand, the HsPKS3 is butted with the small molecule precursor 4PAA-CoA and phlegmarine analogues respectively. The possibility of the condensation reaction of pomegranate and 4PAA/4PAA-CoA under the catalysis of some enzymes is explained in a three-dimensional structure, and the experiment has been carried out at the same time. The problems encountered in the process are followed by theoretical guidance for the subsequent reactions of enzyme catalyzed intermediates.

【学位授予单位】：北京中医药大学
【学位级别】：博士
【学位授予年份】：2017
【分类号】：R915

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