RXR motif在倍半萜合酶催化产物特异性中的作用

发布时间：2018-06-27 20:38

本文选题：紫穗槐二烯合酶 + 马兜铃烯合酶　；参考：《河北大学》2017年硕士论文

【摘要】：萜类化合物普遍存在于植物、微生物的次级代谢产物中。随着越来越多的萜类化合物的发现,对萜类合酶的研究也更加深入,这不仅可以帮助我们从理论上认识萜类化合物的合成机制,理解萜类合酶结构和功能的关系,还可以帮助在药物、香精等应用方面的研究更进一步。萜类合酶不同的催化机理是揭示萜类化合物数量和结构多样性的关键,对其保守基序DDXXD、NSE/DTE、RXR的研究更是其中的重点。本文选取了倍半萜合酶保守基序RXR作为研究切入口,探讨在两种不同的倍半萜合酶催化机制中该保守基序对催化产物特异性的影响,并做了如下工作:1.为探讨RXR基序对倍半萜环化酶产物特异性的影响,我们选择对青蒿紫穗槐二烯合酶(Artemisia annua Amorpha-4,11-diene synthase,ADS)R262、R264进行单突变,并将R262K和T296A/V/I进行联合突变,发现:R262辅助farnesyl diphosphate(FPP)离子化释放焦磷酸基团需要侧链氨基的存在,但是环化反应中间产物nerolidyl diphosphate((R,E)-NPP)的离子化不需要R262的协助就可以实现。推测T296A/V/I可以和R262K通过其他氨基孙或底物碳骨架发生相互作用,而且这种相互作用的结果跟底物构象有关,双突变不能作用于(E,E)-FPP,但是可以催化(R,E)-NPP、(S,E)-NPP分别生成对应的amorpha-4,7(11)-diene和farnesene,以及二者共有的nerolidol、α-bisabolol,作用于(Z,E)-FPP时,双突变可以抑制R262K环化反应中阳离子中间体对水的捕获。R264K对产物没有影响。2.为探讨RXR基序对反式倍半萜环化酶产物特异性的影响,我们选择反式环化酶烟草马兜铃烯合酶(Tobacco 5-epi-aristolochene synthase,TEAS)R264、R266进行单突变,发现:虽然TEAS催化(Z,E)-FPP经历顺式异构,但是R264K并不能明显增加产物中萜醇的生成;底物伯碳位焦磷酸基团的释放需要266位侧链含氨基的残基协助,但是叔碳位焦磷酸的基团则不需要该位点发挥作用。R266K突变体使酶丧失活性。3.倍半萜合酶RXR基序中的第一个R可以辅助FPP离子化,并引入水分子猝灭环化过程形成的阳离子中间体生成萜醇,第二个R作用机制尚不明确。而且,RXR对底物构象具有特异性。
[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, 伪 -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.
【学位授予单位】：河北大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：Q55

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