倍半萜合酶C末端在其功能中的作用
发布时间：2018-11-25 10:59
【摘要】：萜类化合物数量众多,结构多样,普遍存在于植物和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,还有助于进一步研究其在药物、香精等方面的应用。以本实验室前期初步实验结果(ADS的C末端6个氨基酸截短活性下降)为基础,利用软件对催化机制不同的三种倍半萜合酶进行比较,在相应位置进行截短,明确C末端保留几个氨基酸能够保留其原有活性,以探讨此效应是否有普遍性,具体工作如下:1.为探讨C末端对倍半萜环化酶功能的影响,我们选择对黄花蒿紫穗槐二烯合酶(amorpha-4,11-diene synthase,ADS)C末端进行点突变,取纯化后的蛋白在相同酶浓度下对相同浓度底物进行体外催化,催化生成的产物经GC-FID分析,同时用MG法测定酶动力学参数Km和Kcat,发现:C1-C10截短不改变酶催化生成的产物,C1-C6截短从酶动力学参数上来看,其活性没有明显变化,C7-C10活性降低,没有用此方法测出动力学参数。2.为探讨C末端对非环化倍半萜合酶功能的影响,我们选择对非环化酶法呢烯合酶((E)-β-farnesene,BFS)C末端进行点突变,取纯化后的蛋白在相同酶浓度下对相同浓度底物进行体外催化,催化生成的产物经GC-FID分析,同时用MG法测定酶动力学参数Km和Kcat,发现:C1-C10截短不改变酶催化生成的产物,C1-C6截短从酶动力学参数上来看,其活性没有明显变化,C7-C10活性降低,没有用此方法测出动力学参数。3.为探讨C末端对反式倍半萜环化酶功能的影响,我们选择反式环化酶烟草马兜铃烯合酶(5-epi-aristolochene synthase,EAS)C末端进行点突变,取纯化后的蛋白在相同酶浓度下对相同浓度底物进行体外催化,催化生成的产物经GC-FID分析,同时用MG法测定酶动力学参数Km和Kcat,发现:C1-C10截短不改变酶催化生成的产物,C1-C6截短从酶动力学参数上来看,其活性没有明显变化,C7-C10活性降低,没有用此方法测出动力学参数。结合三种倍半萜合酶的结构信息,我们发现三种酶C末端1-10个氨基酸的截短不会改变产物,α螺旋上的氨基酸会降低酶活性,而α螺旋以外的氨基酸的截短与野生型相比,在活性上没有明显变化。
[Abstract]:Terpenoids are abundant in quantity and diverse in structure. They 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 is also helpful to further study its application in medicine, essence and so on. Based on the preliminary experimental results of our laboratory (the reduced activity of 6 amino acids at the C-terminal of ADS), three sesquiterpene synthase with different catalytic mechanisms were compared by software, and the corresponding sites were truncated. It is clear that several amino acids in C terminal can retain their original activity in order to explore the universality of the effect. The specific work is as follows: 1. In order to investigate the effect of C-terminal on the function of sesquiterpene cyclase, we selected the point mutation of C-terminal of Amorpha fruticosa diene synthase (amorpha-4,11-diene synthase,ADS) from Artemisia annua. The purified protein was used to catalyze the substrate of the same concentration at the same enzyme concentration in vitro. The product was analyzed by GC-FID, and the kinetic parameters of Km and Kcat, were determined by MG method. It was found that C1-C10 truncation did not change the enzyme catalyzed product, but C1-C6 truncation did not change the activity of the enzyme from the kinetic parameters of the enzyme, but the activity of C7-C10 decreased, and the kinetic parameters were not measured by this method. 2. In order to investigate the effect of C-terminal on the function of acyclic sesquiterpene synthase, we selected a point mutation on the C-terminal of (E)-尾-farnesene,BFS. The purified protein was used to catalyze the substrate of the same concentration at the same enzyme concentration in vitro. The product was analyzed by GC-FID, and the kinetic parameters of Km and Kcat, were determined by MG method. It was found that C1-C10 truncation did not change the product catalyzed by the enzyme, but C1-C6 truncation did not change the activity of the enzyme from the kinetic parameters of the enzyme, but the activity of C7-C10 decreased, and the kinetic parameters were not measured by this method. 3. In order to investigate the effect of C-terminal on the function of trans-sesquiterpene cyclase, we selected the trans-cyclase (5-epi-aristolochene synthase,EAS) for point mutation at the C-terminal of tobacco aristolochene synthase (5-epi-aristolochene synthase,EAS). The purified protein was used to catalyze the substrate of the same concentration at the same enzyme concentration in vitro. The product was analyzed by GC-FID, and the kinetic parameters of Km and Kcat, were determined by MG method. It was found that C1-C10 truncation did not change the enzyme catalyzed product, but C1-C6 truncation did not change the activity of the enzyme from the kinetic parameters of the enzyme, and the activity of C7-C10 decreased, and the kinetic parameters were not measured by this method. Combined with the structural information of the three sesquiterpene synthase, we found that the truncation of 1-10 amino acids at the C-terminal of the three enzymes did not change the products, the amino acids on the 伪 helix decreased the enzyme activity, but the truncation of the amino acids other than the 伪 helix was compared with the wild type. There was no obvious change in activity.
【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
本文编号：2355858
[Abstract]:Terpenoids are abundant in quantity and diverse in structure. They 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 is also helpful to further study its application in medicine, essence and so on. Based on the preliminary experimental results of our laboratory (the reduced activity of 6 amino acids at the C-terminal of ADS), three sesquiterpene synthase with different catalytic mechanisms were compared by software, and the corresponding sites were truncated. It is clear that several amino acids in C terminal can retain their original activity in order to explore the universality of the effect. The specific work is as follows: 1. In order to investigate the effect of C-terminal on the function of sesquiterpene cyclase, we selected the point mutation of C-terminal of Amorpha fruticosa diene synthase (amorpha-4,11-diene synthase,ADS) from Artemisia annua. The purified protein was used to catalyze the substrate of the same concentration at the same enzyme concentration in vitro. The product was analyzed by GC-FID, and the kinetic parameters of Km and Kcat, were determined by MG method. It was found that C1-C10 truncation did not change the enzyme catalyzed product, but C1-C6 truncation did not change the activity of the enzyme from the kinetic parameters of the enzyme, but the activity of C7-C10 decreased, and the kinetic parameters were not measured by this method. 2. In order to investigate the effect of C-terminal on the function of acyclic sesquiterpene synthase, we selected a point mutation on the C-terminal of (E)-尾-farnesene,BFS. The purified protein was used to catalyze the substrate of the same concentration at the same enzyme concentration in vitro. The product was analyzed by GC-FID, and the kinetic parameters of Km and Kcat, were determined by MG method. It was found that C1-C10 truncation did not change the product catalyzed by the enzyme, but C1-C6 truncation did not change the activity of the enzyme from the kinetic parameters of the enzyme, but the activity of C7-C10 decreased, and the kinetic parameters were not measured by this method. 3. In order to investigate the effect of C-terminal on the function of trans-sesquiterpene cyclase, we selected the trans-cyclase (5-epi-aristolochene synthase,EAS) for point mutation at the C-terminal of tobacco aristolochene synthase (5-epi-aristolochene synthase,EAS). The purified protein was used to catalyze the substrate of the same concentration at the same enzyme concentration in vitro. The product was analyzed by GC-FID, and the kinetic parameters of Km and Kcat, were determined by MG method. It was found that C1-C10 truncation did not change the enzyme catalyzed product, but C1-C6 truncation did not change the activity of the enzyme from the kinetic parameters of the enzyme, and the activity of C7-C10 decreased, and the kinetic parameters were not measured by this method. Combined with the structural information of the three sesquiterpene synthase, we found that the truncation of 1-10 amino acids at the C-terminal of the three enzymes did not change the products, the amino acids on the 伪 helix decreased the enzyme activity, but the truncation of the amino acids other than the 伪 helix was compared with the wild type. There was no obvious change in activity.
【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
【参考文献】
相关硕士学位论文 前1条
1 高瑞平;青蒿紫穗槐二烯合酶C-末端截短和两个关键区域突变对其功能的影响[D];河北大学;2014年
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