本文選題：蛇足石杉 + 石杉?jí)A甲　； 參考：《北京中醫(yī)藥大學(xué)》2017年博士論文

【摘要】：蛇足石杉Huperzia serrata(Thunb.)Trev.,俗名是千層塔、蛇足草、山芝,為石杉科Huperziaceae石杉屬Huperzia Bernh多年生草本蕨類(lèi)植物。全草可以散瘀消腫、解毒和止痛。從蛇足石杉中分離得到的生物堿-石杉?jí)A甲(HuperzineA,HupA)為高效、可逆、高選擇性的乙酰膽堿酯酶抑制劑,能夠提高學(xué)習(xí)、記憶效果,且其特異性強(qiáng)、毒性低,臨床上已成為國(guó)內(nèi)外治療阿爾茲海默癥最有效的藥物之—。植物聚酮合酶(plant polyketide synthases,Ⅲ PKSs)是植物中廣泛存在的一大類(lèi)能夠催化合成植物多酚類(lèi)成分的酶的總稱(chēng)。植物聚酮合酶通過(guò)催化起始底物(�；o酶A)與丙二酰輔酶A反復(fù)地縮合形成鏈狀聚酮中間體,再經(jīng)過(guò)克萊森、Aldol等環(huán)化方式生成查耳酮、二苯乙烯、間苯三酚等結(jié)構(gòu)多樣的天然產(chǎn)物和"非天然小分子"。在植物聚酮合酶催化合成多酚類(lèi)化合物的過(guò)程中,起始底物、鏈的增長(zhǎng)單位、起始底物與鏈增長(zhǎng)單位縮合的分子數(shù)、鏈狀聚酮中間體環(huán)合方式等對(duì)酶催化產(chǎn)物的結(jié)構(gòu)起決定性作用,因此,利用植物聚酮合酶作為工具酶,開(kāi)展以底物為導(dǎo)向的酶催化合成,構(gòu)建數(shù)目可觀(guān)、結(jié)構(gòu)新穎的"非天然小分子"群(庫(kù)),再結(jié)合藥理活性篩選,用于活性小分子的發(fā)現(xiàn),將對(duì)于新藥研發(fā)具有重要意義。HupA在原植物體內(nèi)的含量較低,因此傳統(tǒng)的提取分離方法難以滿(mǎn)足市場(chǎng)需求,且極容易造成野生植物資源的破壞;而有機(jī)合成的方法普遍存在著合成步驟多、最終產(chǎn)率低、尤其是合成光學(xué)純的Hup A的難度大等問(wèn)題,因此無(wú)法實(shí)現(xiàn)利用工業(yè)化生產(chǎn)來(lái)解決Hup A的來(lái)源問(wèn)題。近年來(lái),隨著生物技術(shù)的發(fā)展,一些與藥物生產(chǎn)緊密相關(guān)的活性中間體的生物合成調(diào)控基因或生物合成途徑被闡明,通過(guò)對(duì)相關(guān)調(diào)控基因的人為修飾,可以實(shí)現(xiàn)目標(biāo)產(chǎn)物的定向合成或規(guī)�；a(chǎn)。目前比較成功的化合物如來(lái)源于植物的紫杉醇、青蒿素、吲哚類(lèi)生物堿等,其在生物工程菌(或生物工程植株)中可實(shí)現(xiàn)規(guī)�；铣�。因此,闡明Hup A的生物合成途徑及關(guān)鍵酶,將為利用合成生物學(xué)的方法解決HupA的藥源問(wèn)題提供必要條件。根據(jù)文獻(xiàn)中同位素標(biāo)記結(jié)果,我們推測(cè)植物聚酮合酶可能參與石杉?jí)A甲的生物合成。因此,本課題針對(duì)蛇足石杉中的聚酮合酶進(jìn)行研究。一方面,克隆鑒定蛇足石杉中的植物聚酮合酶,鑒定其催化功能,并在此基礎(chǔ)上開(kāi)展組合合成研究,構(gòu)建"非天然小分子"化合物庫(kù),結(jié)合活性篩選,進(jìn)行活性化合物的發(fā)現(xiàn);另一方面,探討獲得的聚酮合酶和Hup A的生物合成相關(guān)性,力圖闡明其在Hup A生物合成中的作用。目前,課題組取得了以下成果:一、從蛇足石杉新鮮葉片中成功克隆并表達(dá)三個(gè)植物聚酮合酶HsPKS1、HsPKS2、HsPKS3,并對(duì)這三個(gè)酶在體外的催化活性進(jìn)行分析,發(fā)現(xiàn)HsPKS1、HsPKS2與HsPKS3體外活性明顯不同。HsPKS1和HsPKS2是典型的查耳酮合酶,在體外可以催化一分子對(duì)羥基肉桂酰輔酶A和三分子的丙二酰輔酶A縮合,經(jīng)過(guò)克萊森環(huán)合生成柚皮素查耳酮,酸性條件下環(huán)合成二氫黃酮,該化合物是黃酮類(lèi)化合物生物合成的重要前體,為蛇足石杉中黃酮類(lèi)成分的生物合成機(jī)制提供參考。此外HsPKS1和HsPKS2還可以催化一分子的非天然底物2-N-甲基-苯甲酰輔酶A和三分子丙二酰輔酶A縮合生成1,3-二羥基-N-甲基吖啶酮。HspKS3是一個(gè)新穎的植物聚酮合酶,雖然HsPKS3與HsPKS1、HsPKS2序列相似度較高,但是HsPKS3的催化活性完全不同。HsPKS3在體外可以催化兩分子對(duì)羥基肉桂酰輔酶A與一分子丙二酰輔酶A進(jìn)行"頭碰頭"縮合生成雙去甲氧基姜黃素和副產(chǎn)物對(duì)羥基芐基丙酮;HsPKS3還可以催化非天然底物2-N-甲基-苯甲酰輔酶A與一分子丙二酰輔酶A縮合生成簡(jiǎn)單喹諾酮。目前,自然界中具備如此多樣催化功能的植物聚酮合酶尚屬首次發(fā)現(xiàn)。二、通過(guò)對(duì)已表達(dá)的植物聚酮合酶的結(jié)構(gòu)與底物以及催化活性的相關(guān)性進(jìn)行研究,發(fā)現(xiàn)三個(gè)酶具有廣泛的底物選擇性,因此開(kāi)展了初步的組合合成。HsPKS1和HsPKS2根據(jù)其催化活性,利用4CL酶法合成了一系列苯丙烯類(lèi)及其類(lèi)似物的有機(jī)酸硫酯如阿魏酰輔酶A、苯環(huán)上含有吸電子基團(tuán)的4-氟-肉桂輔酶A,含有五元芳雜環(huán)的2-呋喃苯丙烯輔酶A,并在體外組合HsPKS1和HsPKS2進(jìn)行催化反應(yīng),實(shí)現(xiàn)了完全酶催化的體外合成一系列黃酮、4-羥基-δ-在內(nèi)酯類(lèi)化合物。對(duì)于HsPKS3,利用其廣泛的底物選擇性,人工合成了芳香環(huán)、脂肪族、雜環(huán)等結(jié)構(gòu)多樣的起始底物,同時(shí)為了探討延長(zhǎng)單位的選擇性,還制備了丙二酰輔酶A以外的非天然的延長(zhǎng)單位如β-酮酸和β-酮酸的N-乙�；腚装�(N-acetylcysteamine,NAC)硫酯結(jié)構(gòu),初步開(kāi)展以底物為導(dǎo)向的組合合成,得到了一系列喹諾酮及1,3-二酮類(lèi)成分,并通過(guò)藥理活性篩選,發(fā)現(xiàn)部分分子具有較好的抗炎活性。本課題利用植物聚酮合酶作為工具酶,開(kāi)展酶催化的組合合成,構(gòu)建結(jié)構(gòu)新穎多樣的"非天然小分子"庫(kù)進(jìn)行藥理活性篩選,為活性小分子的發(fā)現(xiàn)提供酶法合成的新思路。三、植物聚酮合酶HsPKS3的催化機(jī)制及其與Hup A的生物合成相關(guān)性探討。通過(guò)同源建模的方法獲得了 HsPKS3的三維結(jié)構(gòu)數(shù)據(jù),且利用在線(xiàn)評(píng)價(jià)平臺(tái)系統(tǒng)對(duì)獲得的三維結(jié)構(gòu)進(jìn)行評(píng)價(jià),證明獲得的三維結(jié)構(gòu)合理。在此基礎(chǔ)上對(duì)HsPKS3的催化機(jī)制進(jìn)行了研究,發(fā)現(xiàn)Ser142是生成姜黃素類(lèi)化合物的一個(gè)重要氨基酸位點(diǎn),在此基礎(chǔ)上對(duì)該氨基酸殘基進(jìn)行定點(diǎn)突變后,突變體將不能生成姜黃素類(lèi)化合物。另一方面,將HsPKS3與小分子前體4PAA-CoA、phlegmarine類(lèi)似物分別進(jìn)行分子對(duì)接,從三維結(jié)構(gòu)上解釋了石榴堿和4PAA/4PAA-CoA在某種酶的催化作用下進(jìn)行縮合反應(yīng)的可能性,同時(shí)對(duì)實(shí)驗(yàn)過(guò)程中所遇到的問(wèn)題、后續(xù)酶催化中間體的反應(yīng)進(jìn)行了理論性的指導(dǎo)。
[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.

【學(xué)位授予單位】：北京中醫(yī)藥大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2017
【分類(lèi)號(hào)】：R915

【相似文獻(xiàn)】

相關(guān)期刊論文 前10條

1 李娜;陳鈞;承曦;朱大元;;微生物對(duì)蛇足石杉扦插生根及若干生理生化指標(biāo)的影響[J];中國(guó)中藥雜志;2008年08期

2 張君誠(chéng);邢建宏;宋育紅;王錚敏;張杭穎;;藥用植物蛇足石杉研究新進(jìn)展[J];中國(guó)野生植物資源;2008年02期

3 曾漢元;張伍佰;;藥用植物蛇足石杉的營(yíng)養(yǎng)繁殖[J];懷化學(xué)院學(xué)報(bào)(自然科學(xué));2008年03期

4 韓邦興;陳乃富;姚勇;代劍平;徐驊;李媛媛;朱大元;陳鈞;;相對(duì)濕度對(duì)蛇足石杉生理生化指標(biāo)的影響[J];中華中醫(yī)藥雜志;2009年09期

5 周毅;黃衡宇;李菁;;湘西地區(qū)蛇足石杉資源調(diào)查研究[J];中藥材;2010年02期

6 黃馨鳳;李萬(wàn)成;;蛇足石杉的生態(tài)環(huán)境研究進(jìn)展[J];中國(guó)林副特產(chǎn);2011年02期

7 袁帶秀;鐘飛;恩特馬克·布拉提白;;蛇足石杉過(guò)氧化物酶和酯酶同工酶比較研究[J];時(shí)珍國(guó)醫(yī)國(guó)藥;2011年05期

8 王德立;馮錦東;朱殿龍;;蛇足石杉的地理分布[J];現(xiàn)代中藥研究與實(shí)踐;2011年05期

9 武蕓;楊婷;吳娟;楊蘭;田代學(xué);鄭小江;;珍稀瀕危藥材蛇足石杉的研究概況及開(kāi)發(fā)前景[J];湖北民族學(xué)院學(xué)報(bào)(自然科學(xué)版);2013年01期

10 林如輝;劉美龍;;中草藥蛇足石杉的研究概況[J];海峽藥學(xué);2013年09期

相關(guān)會(huì)議論文 前10條

1 王德立;馮錦東;甘炳春;;蛇足石杉生境及與真菌共生關(guān)系[A];第八屆全國(guó)藥用植物及植物藥學(xué)術(shù)研討會(huì)論文集[C];2009年

2 周毅;黃衡宇;;蛇足石杉生態(tài)適應(yīng)性研究Ⅰ莖葉解剖特征的比較[A];第九屆全國(guó)藥用植物及植物藥學(xué)術(shù)研討會(huì)論文集[C];2010年

3 張憲權(quán);田旗;王鳳英;蔣云;;蛇足石杉(Huperzia Serrata)在中國(guó)分布規(guī)律的初步研究[A];中國(guó)植物學(xué)會(huì)七十五周年年會(huì)論文摘要匯編（1933-2008）[C];2008年

4 王德立;齊耀東;馮錦東;魏建和;;海南蛇足石杉天然居群與環(huán)境因子[A];全國(guó)第9屆天然藥物資源學(xué)術(shù)研討會(huì)論文集[C];2010年

5 劉祝祥;賀樂(lè);劉艷;瞿專(zhuān);陳功錫;;蛇足石杉內(nèi)生真菌的分離及產(chǎn)石杉?jí)A甲菌株的篩選與鑒定[A];第八屆全國(guó)藥用植物及植物藥學(xué)術(shù)研討會(huì)論文集[C];2009年

6 殷秀梅;牛云云;張?chǎng)?羅紅梅;;蛇足石杉鯊烯環(huán)氧酶HsSE1基因克隆及表達(dá)分析[A];中藥與天然藥高峰論壇暨第十二屆全國(guó)中藥和天然藥物學(xué)術(shù)研討會(huì)論文集[C];2012年

7 羅紅梅;李榕濤;李標(biāo);徐曉蘭;林余霖;胡志剛;姚輝;王德立;何柳;孫超;宋經(jīng)元;殷秀梅;牛云云;向麗;陳士林;;蛇足石杉1-脫氧-D-木酮糖5-磷酸還原異構(gòu)酶(HsDXR1)基因克隆及表達(dá)分析[A];第十五屆中國(guó)科協(xié)年會(huì)第21分會(huì)場(chǎng)：中藥與天然藥物現(xiàn)代研究學(xué)術(shù)研討會(huì)論文集[C];2013年

8 羅紅梅;孫超;何柳;李標(biāo);宋經(jīng)元;;蛇足石杉HsDXR1基因克隆與生物信息學(xué)分析[A];2013全國(guó)中藥與天然藥物高峰論壇暨第十三屆全國(guó)中藥和天然藥物學(xué)術(shù)研討會(huì)論文集[C];2013年

9 涂藝聲;吉枝單;丁明華;陳雄;蔣秀芳;;蛇足石杉離體培養(yǎng)產(chǎn)生石杉?jí)A甲研究[A];第十五屆中國(guó)科協(xié)年會(huì)第21分會(huì)場(chǎng)：中藥與天然藥物現(xiàn)代研究學(xué)術(shù)研討會(huì)論文集[C];2013年

10 鄒艷輝;邱亞利;滕海英;余宇燕;;蛇足石杉中石杉?jí)A甲ELISA檢測(cè)方法的建立[A];2013年中國(guó)藥學(xué)大會(huì)暨第十三屆中國(guó)藥師周論文集[C];2013年

相關(guān)重要報(bào)紙文章 前2條

1 記者 肖軍邋通訊員 周圣華 向滿(mǎn)紅;蛇足石杉人工繁殖獲突破[N];湖南日?qǐng)?bào);2008年

2 記者　 肖軍 通訊員　 楊志鎳 袁喜良;人工繁殖“蛇足石杉”項(xiàng)目啟動(dòng)[N];湖南日?qǐng)?bào);2006年

相關(guān)博士學(xué)位論文 前3條

1 趙昕梅;產(chǎn)石杉?jí)A甲蛇足石杉內(nèi)生真菌Colletotrichum gloeosporioides ES026研究[D];華中農(nóng)業(yè)大學(xué);2013年

2 王娟;蛇足石杉聚酮合酶的克隆鑒定及其在組合合成中的應(yīng)用[D];北京中醫(yī)藥大學(xué);2017年

3 李娜;蛇足石杉內(nèi)生真菌的促寄主生長(zhǎng)作用及發(fā)酵產(chǎn)物研究[D];江蘇大學(xué);2007年

相關(guān)碩士學(xué)位論文 前10條

1 林如輝;蛇足石杉引種馴化及其品質(zhì)的初步研究[D];福建農(nóng)林大學(xué);2009年

2 包日雙;蛇足石杉人工繁殖過(guò)程中的限制因素研究[D];西北大學(xué);2013年

3 孫紅敏;蛇足石杉內(nèi)生菌與沙坡頭沙漠環(huán)境放線(xiàn)菌的多樣性研究[D];北京協(xié)和醫(yī)學(xué)院;2015年

4 徐曉苗;產(chǎn)石杉?jí)A甲蛇足石杉內(nèi)生菌的篩選和鑒定[D];華中農(nóng)業(yè)大學(xué);2011年

5 劉江海;蛇足石杉的離體培養(yǎng)及內(nèi)生真菌的分離與鑒定[D];中南林業(yè)科技大學(xué);2016年

6 駱冰潔;蛇足石杉的芽胞繁殖及其生物堿的組織化學(xué)定位與活體釋放研究[D];吉首大學(xué);2016年

7 彭思露;蛇足石杉內(nèi)生真菌Shiraia sp.Slf14中LDC和CAO基因的克隆及表達(dá)[D];江西師范大學(xué);2016年

8 李沛玲;浙江及鄰近地區(qū)蛇足石杉種內(nèi)變異式樣及其與石杉?jí)A甲含量關(guān)系的研究[D];浙江師范大學(xué);2005年

9 劉敬寶;廣西藥源植物蛇足石杉資源調(diào)查與評(píng)價(jià)[D];廣西大學(xué);2008年

10 張慧;高溫脅迫對(duì)蛇足石杉生理生化特性的影響[D];中南林業(yè)科技大學(xué);2012年

，

本文編號(hào)：1791684