托品烷生物碱的生物合成途径中铜胺氧化酶和聚酮合酶的研究

	托品烷生物碱的生物合成途径中铜胺氧化酶和聚酮合酶的研究; The study of copper amine oxidase and polyketide synthase involved in the biosynthesis of tropane alkaloid
	田恬
导师	黄胜雄
摘要	Tropane Alkaloids (Tropane Alkaloids，TAs) are a class of alkaloids sharing a N-methyl-8-azabicyclo[3.2.1]octane fragment and are mainly distributed in the Solanaceae, besides that they are also scattered in Erythroxylaceae, Proteaceae, Convolvulaceae, ect. Many of them are known for their pharmacological activity, and are widely used for clinical. For examples, hyoscyamine and scopolamine can effectively alleviate symptoms of nausea, vomiting, spasms, motion sickness, etc.; cocaine is approved by FDA as a local anesthesia of nasal cavities. The research on the biosynthesis of TAs began in the early 1900s. The biosynthetic pathways of scopolamine which belongs to Solanaceae, was stablished in 2020. Previous radio isotope labeling study show that Solanaceae and Erythroxylaceae share a similar TAs biosynthesis pathway. However, the biosynthetic pathway of cocaine has not yet been completely clarified with only three enzymes having been reported. In the present study, we handle with polyketide synthase and copper-amine oxidase to explore the fact of TAs’ biosynthesis. Here are the results: 1. Cloning and functional identification of MPO: The screening of a transcriptome database of Erythroxylum novogranatense, yield seven sequences with annotation of MPO functions. Phylogenetic analysis revealed that two of them converge with MPO (designated EnMPO8208 and EnMPO25921) which has been functionally identified. The enzymatic activities of the corresponding proteins confirmed by heterologous expression in E. coli. Both of them have the ability to catalyze the oxidation of putrescine and N-putrescine. 2. Cloning and functional identification of PKS: The screening of a transcriptome database of E. novogranatense yield eleven sequences with annotation of PKS functions. After analyzing the TAs’ tissues profiles and collinearity of transcriptome data of different tissues, which contain bud, young leaf, old leaf, root, stem and flower. The candidate genes scale down to 3 pieces of sequences. The enzymatic activities of the corresponding proteins confirmed by heterologous expression in E. coli. Two of them have the ability to condense the malonyl-CoA to generate the 3-oxo-glutaric acid. The EnPKS1 and EnPKS2 Km values for malonyl-CoA are 112.9 μM and 51.2 μM, respectively. Kcat/Km values for malonyl-CoA are 16768 M-1s-1 and 8897 M-1s-1. The crystal structure of EnPKS1 and EnPKS2 were determined. Based on the crystal structures and the site mutation experiments, we proposed a mechanism for EnPKS1 and EnPKS2: K138 and R212 residues inhibits further chain elongation through the formation of hydrogen bond and electrostatic interaction. And other aliphatic residues shape the pocket by hydrophobic interaction, leading to only one round condensation to produce 3-oxo-glutaric acid. The differences in PYKS of Solanaceae and that of Erythroxylum are as follows: the change of key amino acid residues; the different shape and size of pocket in both of them.
	2021-05
文献类型	学位论文
条目标识符	http://ir.kib.ac.cn/handle/151853/74597
专题	昆明植物所硕博研究生毕业学位论文
推荐引用方式 GB/T 7714	田恬. 托品烷生物碱的生物合成途径中铜胺氧化酶和聚酮合酶的研究, The study of copper amine oxidase and polyketide synthase involved in the biosynthesis of tropane alkaloid[D],2021.

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