本文選題：Radical + SAM酶��； 參考：《蘭州大學》2017年碩士論文

【摘要】：S-腺苷甲硫氨酸自由基酶參與生物體中很多重要的生化反應,其反應機制是裂解SAM產(chǎn)生5-Ado自由基攫取底物的H原子,但也有不同反應機理的酶如MqnE發(fā)生自由基加成反應,而很多含有核苷單元的化合物是具有良好的生物活性。對NosL酶的研究發(fā)現(xiàn)利用含有雙鍵的色氨酸類似物能夠改變酶的反應性發(fā)生自由基加成反應。本論文為研究酪氨酸裂解酶催化活性的多樣性,選取的四種酪氨酸裂解酶FbiC、HydG、ThiHEc和ThiHCb,并合成了S-腺苷甲硫氨酸(SAM)類似物S-鳥苷甲硫氨酸(SGM)和S-胞苷甲硫氨酸(SCM)以及底物酪氨酸的烯烴類似物2-(4-羥基)丙烯酸(HBAA)和2-(4-羥基)丙烯酸甲酯(MHBA)進行反應,研究其催化活性。結(jié)果表明四種酶都具有識別HBAA和(或)MHBA并生成腺苷加成產(chǎn)物的能力,且FbiC能夠生成鳥苷加成產(chǎn)物。本文的研究拓寬了我們對Radical SAM酶催化活性的研究,使合成具有生物活性的含不同核苷單元的化合物成為可能。賽普霉素(Cypemycin)是一類具有抗哺乳動物白血病腫瘤細胞活性的核糖體合成翻譯后修飾多肽(Ripps),同時還具有良好的抗微生物活性。自1993年cypemycin首次報道以來其生物合成基因簇已被廣泛研究,推測其中CypH和Cyp L酶與cypemycin分子中蘇氨酸脫水形成Dhb及半胱氨酸的脫水有關(guān),但功能仍然有待表征。為研究CypH和Cyp L兩個酶的功能,本論文克隆并成功表達了其前體肽CypA,設計體內(nèi)及體外修飾實驗,檢測到了一段大小為3461Da的肽段,推測是由前體肽部分酶解并發(fā)生一個蘇氨酸脫水而形成,在體外修飾時蘇氨酸脫水導致前體肽易被降解,無法繼續(xù)進行修飾。這一發(fā)現(xiàn)對cypemycin生物合成機制的研究提供了重要信息,其研究方法也可以應用到其他核糖體肽的生物合成研究中,同時也可以用于發(fā)現(xiàn)并組合生物合成更多結(jié)構(gòu)新穎,活性良好的此類天然產(chǎn)物,為未來生物醫(yī)藥的發(fā)展提供重要的理論依據(jù)。
[Abstract]:S-adenosine methionine free radical enzyme is involved in many important biochemical reactions in organisms. The mechanism of the reaction is to break down the H atom of 5-Ado free radical to grab the substrate, but there are also some enzymes with different reaction mechanisms, such as MqnE, which produce free radical addition reaction. Many compounds containing nucleoside units have good biological activity. The study of NosL enzyme showed that the tryptophan analogue containing double bonds could change the reactivity of the enzyme by free radical addition reaction. In this paper, the diversity of tyrosine lyase catalytic activity was studied. Four tyrosine lytic enzymes, FBIC HydGnThiHEc and ThiHCb, were selected, and S- adenosine methionine (SAM) analogues, SGM and S- cytidine methionine (SCM), and the olefin analogues of tyrosine 2- (4-hydroxyacrylic acid) (HBAA) and 2- (4- hydroxy) acrylic acid (HBAA) were synthesized. Methyl acrylate (MHBA), Its catalytic activity was studied. The results showed that all four enzymes could recognize HBAA and / or MHBA and produce adenosine addition products, and FBIC could produce guanosine addition products. This study broadens our research on the catalytic activity of radical SAM and makes it possible to synthesize compounds with different nucleoside units with biological activity. Cypemycin (Cypemycin) is a kind of ribosomal synthetic post-translational modified polypeptide (Ripps) with antitumor activity in mammalian leukemia cells. Since cypemycin was first reported in 1993, its biosynthetic gene cluster has been extensively studied. It is speculated that CypH and Cyp L enzymes are related to the dehydration of threonine to DHB and cysteine in cypemycin molecule, but the function remains to be characterized. In order to study the function of CypH and Cyp L, we cloned and successfully expressed its precursor peptide CypA, designed in vivo and in vitro modification experiments, and detected a peptide fragment of 3461Da in size. It is assumed that the precursor peptide is hydrolyzed partly by enzyme and a threonine dehydration occurs. The dehydration of threonine in vitro leads to the degradation of the precursor peptide, which can not be further modified. This discovery provides important information for the study of the biosynthesis mechanism of cypemycin, and its research methods can also be applied to the biosynthesis of other ribosomal peptides, as well as to the discovery and combination of novel structures for biosynthesis. This kind of natural product with good activity provides important theoretical basis for the development of biomedicine in the future.
【學位授予單位】：蘭州大學
【學位級別】：碩士
【學位授予年份】：2017
【分類號】：O629.72;Q55

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