乙�；{(diào)節(jié)大腸桿菌S-腺苷甲硫氨酸合成酶活性的機制研究
發(fā)布時間：2018-07-31 13:43
【摘要】：S-腺苷-L-甲硫氨酸(S-adenosyl-L-methionine,SAM)廣泛存在于動植物及微生物中,是甲硫氨酸在體內(nèi)的活性形式。其作為體內(nèi)主要的甲基供體和生理性硫基化合物前體,參與體內(nèi)眾多化合物的合成及其他重要生化反應(yīng),例如轉(zhuǎn)甲基、轉(zhuǎn)硫基與轉(zhuǎn)氨丙基等。SAM合成酶(S-adenosylmethionine synthase,MAT)是SAM在生物體內(nèi)合成的關(guān)鍵限速酶。SAM合成酶從原核到真核生物都高度保守,對于生物體維持正常生命活動是十分重要的。在大腸桿菌中,該酶的編碼基因為細菌生長所必需。之前,有相關(guān)乙酰化蛋白質(zhì)組學(xué)的文獻報道大腸桿菌SAM合成酶存在乙�；揎�,但此修飾的生物學(xué)功能還不明確。蛋白質(zhì)的賴氨酸乙�；揎椬鳛橐环N動態(tài)、可逆的蛋白質(zhì)翻譯后修飾,廣泛存在于原核及真核生物中,主要通過蛋白質(zhì)的乙�；D(zhuǎn)移酶和去乙酰化酶進行調(diào)控。目前對于細菌中的去乙酰化酶研究較多的是大腸桿菌中依賴于NAD+的CobB蛋白。雖然近些年來研究人員發(fā)現(xiàn)賴氨酸乙酰化修飾在細胞中發(fā)揮著諸多生物學(xué)功能,并對CobB蛋白的一些功能進行了研究,但關(guān)于乙�；揎椀脑敿毶頇C制及CobB蛋白在細菌中的更多功能還需要更深入的探索。本研究首先在體外成功表達并純化了大腸桿菌MAT蛋白,通過質(zhì)譜技術(shù)鑒定出大腸桿菌MAT蛋白的乙�；嚢彼嵛稽c,隨后對這些賴氨酸位點進行定點突變,結(jié)果表明這些位點對于MAT蛋白的酶活性是至關(guān)重要的。同時,體外實驗結(jié)果表明MAT蛋白可被大腸桿菌去乙�；窩obB催化去乙酰化。體外酶活性測定實驗表明,MAT蛋白的體外乙酰化修飾可降低其酶活性;而Cob B催化去乙�；�,MAT蛋白酶活性顯著回升。為了進一步在體內(nèi)分析Cob B蛋白對MAT蛋白的影響,我們對大腸桿菌cob B基因敲除株和野生型菌株中MAT蛋白的表達量、體內(nèi)SAM總量進行了比較。結(jié)果顯示,野生型菌株與cobB基因敲除株中MAT蛋白的表達量并無明顯區(qū)別,但cob B敲除株中體內(nèi)SAM總量明顯降低。這些結(jié)果表明,可逆的乙酰化修飾可以調(diào)節(jié)大腸桿菌MAT蛋白的生理活性。隨后,我們對cobB基因敲除株和野生型菌株細胞內(nèi)基因組DNA的5-mC甲基化進行了檢測,以探究大腸桿菌體內(nèi)SAM含量差異與DNA甲基化的關(guān)聯(lián)。在本研究中,我們還意外發(fā)現(xiàn)在體外MAT蛋白可以以乙�；姿�(Ac-Phosphate)和氨基甲酰磷酸作為磷酸基團供體發(fā)生自磷酸化;同時,這一過程可被堿性磷酸酶在體外進行去磷酸化,但具體的生理意義還需要進一步深入研究。綜上所述,本研究發(fā)現(xiàn)了可逆的賴氨酸乙�；揎椗c大腸桿菌MAT蛋白功能之間的關(guān)系,拓寬了我們對賴氨酸乙酰化修飾調(diào)節(jié)蛋白質(zhì)功能的認知。
[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.
【學(xué)位授予單位】：中國科學(xué)院研究生院(武漢病毒研究所)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2016
【分類號】：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.
【學(xué)位授予單位】：中國科學(xué)院研究生院(武漢病毒研究所)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2016
【分類號】：Q936
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