本文選題：紫穗槐二烯合酶 + 馬兜鈴烯合酶　； 參考：《河北大學(xué)》2017年碩士論文

【摘要】：萜類化合物普遍存在于植物、微生物的次級代謝產(chǎn)物中。隨著越來越多的萜類化合物的發(fā)現(xiàn),對萜類合酶的研究也更加深入,這不僅可以幫助我們從理論上認(rèn)識萜類化合物的合成機制,理解萜類合酶結(jié)構(gòu)和功能的關(guān)系,還可以幫助在藥物、香精等應(yīng)用方面的研究更進(jìn)一步。萜類合酶不同的催化機理是揭示萜類化合物數(shù)量和結(jié)構(gòu)多樣性的關(guān)鍵,對其保守基序DDXXD、NSE/DTE、RXR的研究更是其中的重點。本文選取了倍半萜合酶保守基序RXR作為研究切入口,探討在兩種不同的倍半萜合酶催化機制中該保守基序?qū)Υ呋a(chǎn)物特異性的影響,并做了如下工作:1.為探討RXR基序?qū)Ρ栋胼骗h(huán)化酶產(chǎn)物特異性的影響,我們選擇對青蒿紫穗槐二烯合酶(Artemisia annua Amorpha-4,11-diene synthase,ADS)R262、R264進(jìn)行單突變,并將R262K和T296A/V/I進(jìn)行聯(lián)合突變,發(fā)現(xiàn):R262輔助farnesyl diphosphate(FPP)離子化釋放焦磷酸基團(tuán)需要側(cè)鏈氨基的存在,但是環(huán)化反應(yīng)中間產(chǎn)物nerolidyl diphosphate((R,E)-NPP)的離子化不需要R262的協(xié)助就可以實現(xiàn)。推測T296A/V/I可以和R262K通過其他氨基孫或底物碳骨架發(fā)生相互作用,而且這種相互作用的結(jié)果跟底物構(gòu)象有關(guān),雙突變不能作用于(E,E)-FPP,但是可以催化(R,E)-NPP、(S,E)-NPP分別生成對應(yīng)的amorpha-4,7(11)-diene和farnesene,以及二者共有的nerolidol、α-bisabolol,作用于(Z,E)-FPP時,雙突變可以抑制R262K環(huán)化反應(yīng)中陽離子中間體對水的捕獲。R264K對產(chǎn)物沒有影響。2.為探討RXR基序?qū)Ψ词奖栋胼骗h(huán)化酶產(chǎn)物特異性的影響,我們選擇反式環(huán)化酶煙草馬兜鈴烯合酶(Tobacco 5-epi-aristolochene synthase,TEAS)R264、R266進(jìn)行單突變,發(fā)現(xiàn):雖然TEAS催化(Z,E)-FPP經(jīng)歷順式異構(gòu),但是R264K并不能明顯增加產(chǎn)物中萜醇的生成;底物伯碳位焦磷酸基團(tuán)的釋放需要266位側(cè)鏈含氨基的殘基協(xié)助,但是叔碳位焦磷酸的基團(tuán)則不需要該位點發(fā)揮作用。R266K突變體使酶喪失活性。3.倍半萜合酶RXR基序中的第一個R可以輔助FPP離子化,并引入水分子猝滅環(huán)化過程形成的陽離子中間體生成萜醇,第二個R作用機制尚不明確。而且,RXR對底物構(gòu)象具有特異性。
[Abstract]:Terpenoids are commonly found in secondary metabolites of plants and microorganisms. With the discovery of more and more terpenoids, the research on terpenoids synthase is more and more in-depth, which can not only help us to understand the synthesis mechanism of terpenes theoretically, but also understand the relationship between the structure and function of terpenoids synthase. It can also help to further research in drug, flavor and other applications. The different catalytic mechanism of terpene synthase is the key to reveal the quantity and structural diversity of terpene compounds, and the research on its conserved motif DDXXDX NSE / DTERXR is the focus. In this paper, the conserved motif RXR of sesquiterpene synthase was selected as the entry point to investigate the effect of the conserved motif on the specificity of the product in two different mechanisms of sesquiterpene synthase, and the following work was done: 1. In order to investigate the effect of RXR motif on the specificity of sesquiterpene cyclase products, we selected the single mutation of (Artemisia annua Amorpha-4Amorpha-4Amorpha-4 (11-diene synthase) R264, and combined R262K and T296A / V / I mutation. It is found that the side chain amino group is needed to release pyrophosphate group from the ion release of farnesyl diphosphate (/ R262, but the ionization of the intermediate product of cyclization reaction, nerolidyl diphosphate (Rau E) -NPP) can be realized without the assistance of R262. It is assumed that T296A / V / I can interact with R262K through other amino grandchildren or substrate carbon skeletons, and the results of this interaction are related to the substrate conformation. The double mutation could not act on (Eau E) -FPP, but could catalyze (Rau E) -NPP, (Sino E) -NPP to produce corresponding amorpha-4n 7 (11) -diene and farnesenee respectively, and the nerolidolol, 偽 -bisabololl, which were shared by both of them, and when acting on (ZE) -FPP, the double mutation could inhibit the capture of water by cationic intermediates in R262K cyclization. R264K had no effect on the product. In order to investigate the effect of RXR motifs on the specificity of trans-sesquiterpene cyclase products, we selected tobacco 5-epi-aristolochene synthase R264TEAS for single mutation. However, R264K did not significantly increase the formation of terpene alcohols in the product. The release of the primary carbon pyrophosphate group from the substrate required the assistance of amino residues in the 266 side chain. However, the tertiary carbon pyrophosphoric acid group does not need this site to play a role. R266K mutants make the enzyme inactive. 3. The first R of the sesquiterpene synthase RXR motif can assist the ionization of FPP and introduce the cationic intermediate formed by the quenching cyclization of water molecules to form terpene alcohols. The mechanism of the second R is not clear. Moreover, RXR is specific to substrate conformation.
【學(xué)位授予單位】：河北大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：Q55

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本文編號：2075214