乙酰化调节大肠杆菌S-腺苷甲硫氨酸合成酶活性的机制研究
发布时间：2018-07-31 13:43
【摘要】：S-腺苷-L-甲硫氨酸(S-adenosyl-L-methionine,SAM)广泛存在于动植物及微生物中,是甲硫氨酸在体内的活性形式。其作为体内主要的甲基供体和生理性硫基化合物前体,参与体内众多化合物的合成及其他重要生化反应,例如转甲基、转硫基与转氨丙基等。SAM合成酶(S-adenosylmethionine synthase,MAT)是SAM在生物体内合成的关键限速酶。SAM合成酶从原核到真核生物都高度保守,对于生物体维持正常生命活动是十分重要的。在大肠杆菌中,该酶的编码基因为细菌生长所必需。之前,有相关乙酰化蛋白质组学的文献报道大肠杆菌SAM合成酶存在乙酰化修饰,但此修饰的生物学功能还不明确。蛋白质的赖氨酸乙酰化修饰作为一种动态、可逆的蛋白质翻译后修饰,广泛存在于原核及真核生物中,主要通过蛋白质的乙酰基转移酶和去乙酰化酶进行调控。目前对于细菌中的去乙酰化酶研究较多的是大肠杆菌中依赖于NAD+的CobB蛋白。虽然近些年来研究人员发现赖氨酸乙酰化修饰在细胞中发挥着诸多生物学功能,并对CobB蛋白的一些功能进行了研究,但关于乙酰化修饰的详细生理机制及CobB蛋白在细菌中的更多功能还需要更深入的探索。本研究首先在体外成功表达并纯化了大肠杆菌MAT蛋白,通过质谱技术鉴定出大肠杆菌MAT蛋白的乙酰化赖氨酸位点,随后对这些赖氨酸位点进行定点突变,结果表明这些位点对于MAT蛋白的酶活性是至关重要的。同时,体外实验结果表明MAT蛋白可被大肠杆菌去乙酰化酶CobB催化去乙酰化。体外酶活性测定实验表明,MAT蛋白的体外乙酰化修饰可降低其酶活性;而Cob B催化去乙酰化后,MAT蛋白酶活性显著回升。为了进一步在体内分析Cob B蛋白对MAT蛋白的影响,我们对大肠杆菌cob B基因敲除株和野生型菌株中MAT蛋白的表达量、体内SAM总量进行了比较。结果显示,野生型菌株与cobB基因敲除株中MAT蛋白的表达量并无明显区别,但cob B敲除株中体内SAM总量明显降低。这些结果表明,可逆的乙酰化修饰可以调节大肠杆菌MAT蛋白的生理活性。随后,我们对cobB基因敲除株和野生型菌株细胞内基因组DNA的5-mC甲基化进行了检测,以探究大肠杆菌体内SAM含量差异与DNA甲基化的关联。在本研究中,我们还意外发现在体外MAT蛋白可以以乙酰基磷酸(Ac-Phosphate)和氨基甲酰磷酸作为磷酸基团供体发生自磷酸化;同时,这一过程可被碱性磷酸酶在体外进行去磷酸化,但具体的生理意义还需要进一步深入研究。综上所述,本研究发现了可逆的赖氨酸乙酰化修饰与大肠杆菌MAT蛋白功能之间的关系,拓宽了我们对赖氨酸乙酰化修饰调节蛋白质功能的认知。
[Abstract]:S-adenosyl-L-methionine SAM (S-adenosyl-L-methionine SAM) exists widely in animals, plants and microorganisms and is the active form of methionine in vivo. As the main methyl donor and precursor of physiologic sulfur compounds in the body, it participates in the synthesis of many compounds in the body and other important biochemical reactions, such as transmethylation, S-adenosylmethionine synthase (S-adenosylmethionine synthase) is the key rate-limiting enzyme. SAM synthase is highly conserved from prokaryotic to eukaryote, and is very important for organism to maintain normal life activity. In Escherichia coli, the encoder of the enzyme is necessary for bacterial growth. Acetylation modification of Escherichia coli SAM synthase has been reported previously, but the biological function of this modification is not clear. As a dynamic reversible post-translational modification of protein lysine acetylation is widely found in prokaryotes and eukaryotes and is mainly regulated by acetyltransferase and deacetylase. At present, the deacetylase in bacteria is the NAD dependent CobB protein in Escherichia coli. Although in recent years researchers have found that lysine acetylation plays a number of biological functions in cells, and have studied some of the functions of the CobB protein, However, the detailed physiological mechanism of acetylation modification and the more functions of CobB protein in bacteria need to be further explored. In this study, E. coli MAT protein was successfully expressed and purified in vitro. The acetylated lysine sites of E. coli MAT protein were identified by mass spectrometry and then mutated by site-directed mutation. The results showed that these sites were important for the enzyme activity of MAT protein. At the same time, the results of in vitro experiments showed that MAT protein could be deacetylated by E. coli deacetylase CobB. The results of in vitro enzyme activity test showed that the acetylation modification of mat protein could decrease its enzyme activity, while the activity of Cob B catalyzed deacetylation increased significantly. In order to further analyze the effect of Cob B protein on MAT protein in vivo, we compared the expression of MAT protein and total SAM in E. coli cob B knockout strain and wild type strain. The results showed that there was no significant difference in the expression of MAT protein between wild-type strains and cobB knockout strains, but the total amount of SAM in cob B knockout strains was significantly decreased. These results suggest that reversible acetylation modification can regulate the physiological activity of Escherichia coli MAT protein. Subsequently, we detected the 5-mC methylation of genomic DNA of cobB knockout and wild-type strains in order to explore the relationship between SAM content difference and DNA methylation in Escherichia coli. In this study, we also found that in vitro MAT protein can be self-phosphorylated with acetylphosphoric acid (Ac-Phosphate) and carbamyl phosphoric acid as phosphate group, and this process can be dephosphorylated by alkaline phosphatase in vitro. But the specific physiological significance still needs to be further studied. In conclusion, the relationship between reversible lysine acetylation modification and the function of MAT protein in Escherichia coli was found, which broadened our understanding of the regulation of protein function by lysine acetylation modification.
【学位授予单位】：中国科学院研究生院(武汉病毒研究所)
【学位级别】：博士
【学位授予年份】：2016
【分类号】：Q936
[Abstract]:S-adenosyl-L-methionine SAM (S-adenosyl-L-methionine SAM) exists widely in animals, plants and microorganisms and is the active form of methionine in vivo. As the main methyl donor and precursor of physiologic sulfur compounds in the body, it participates in the synthesis of many compounds in the body and other important biochemical reactions, such as transmethylation, S-adenosylmethionine synthase (S-adenosylmethionine synthase) is the key rate-limiting enzyme. SAM synthase is highly conserved from prokaryotic to eukaryote, and is very important for organism to maintain normal life activity. In Escherichia coli, the encoder of the enzyme is necessary for bacterial growth. Acetylation modification of Escherichia coli SAM synthase has been reported previously, but the biological function of this modification is not clear. As a dynamic reversible post-translational modification of protein lysine acetylation is widely found in prokaryotes and eukaryotes and is mainly regulated by acetyltransferase and deacetylase. At present, the deacetylase in bacteria is the NAD dependent CobB protein in Escherichia coli. Although in recent years researchers have found that lysine acetylation plays a number of biological functions in cells, and have studied some of the functions of the CobB protein, However, the detailed physiological mechanism of acetylation modification and the more functions of CobB protein in bacteria need to be further explored. In this study, E. coli MAT protein was successfully expressed and purified in vitro. The acetylated lysine sites of E. coli MAT protein were identified by mass spectrometry and then mutated by site-directed mutation. The results showed that these sites were important for the enzyme activity of MAT protein. At the same time, the results of in vitro experiments showed that MAT protein could be deacetylated by E. coli deacetylase CobB. The results of in vitro enzyme activity test showed that the acetylation modification of mat protein could decrease its enzyme activity, while the activity of Cob B catalyzed deacetylation increased significantly. In order to further analyze the effect of Cob B protein on MAT protein in vivo, we compared the expression of MAT protein and total SAM in E. coli cob B knockout strain and wild type strain. The results showed that there was no significant difference in the expression of MAT protein between wild-type strains and cobB knockout strains, but the total amount of SAM in cob B knockout strains was significantly decreased. These results suggest that reversible acetylation modification can regulate the physiological activity of Escherichia coli MAT protein. Subsequently, we detected the 5-mC methylation of genomic DNA of cobB knockout and wild-type strains in order to explore the relationship between SAM content difference and DNA methylation in Escherichia coli. In this study, we also found that in vitro MAT protein can be self-phosphorylated with acetylphosphoric acid (Ac-Phosphate) and carbamyl phosphoric acid as phosphate group, and this process can be dephosphorylated by alkaline phosphatase in vitro. But the specific physiological significance still needs to be further studied. In conclusion, the relationship between reversible lysine acetylation modification and the function of MAT protein in Escherichia coli was found, which broadened our understanding of the regulation of protein function by lysine acetylation modification.
【学位授予单位】：中国科学院研究生院(武汉病毒研究所)
【学位级别】：博士
【学位授予年份】：2016
【分类号】：Q936
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