酪氨酸裂解酶催化活性多样性及赛普霉素生物合成机制研究

发布时间：2018-06-29 22:21

本文选题：Radical + SAM酶　；参考：《兰州大学》2017年硕士论文

【摘要】：S-腺苷甲硫氨酸自由基酶参与生物体中很多重要的生化反应,其反应机制是裂解SAM产生5-Ado自由基攫取底物的H原子,但也有不同反应机理的酶如MqnE发生自由基加成反应,而很多含有核苷单元的化合物是具有良好的生物活性。对NosL酶的研究发现利用含有双键的色氨酸类似物能够改变酶的反应性发生自由基加成反应。本论文为研究酪氨酸裂解酶催化活性的多样性,选取的四种酪氨酸裂解酶FbiC、HydG、ThiHEc和ThiHCb,并合成了S-腺苷甲硫氨酸(SAM)类似物S-鸟苷甲硫氨酸(SGM)和S-胞苷甲硫氨酸(SCM)以及底物酪氨酸的烯烃类似物2-(4-羟基)丙烯酸(HBAA)和2-(4-羟基)丙烯酸甲酯(MHBA)进行反应,研究其催化活性。结果表明四种酶都具有识别HBAA和(或)MHBA并生成腺苷加成产物的能力,且FbiC能够生成鸟苷加成产物。本文的研究拓宽了我们对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单元的化合物成为可能。赛普霉素(Cypemycin)是一类具有抗哺乳动物白血病肿瘤细胞活性的核糖体合成翻译后修饰多肽(Ripps),同时还具有良好的抗微生物活性。自1993年cypemycin首次报道以来其生物合成基因簇已被广泛研究,推测其中CypH和Cyp L酶与cypemycin分子中苏氨酸脱水形成Dhb及半胱氨酸的脱水有关,但功能仍然有待表征。为研究CypH和Cyp L两个酶的功能,本论文克隆并成功表达了其前体肽CypA,设计体内及体外修饰实验,检测到了一段大小为3461Da的肽段,推测是由前体肽部分酶解并发生一个苏氨酸脱水而形成,在体外修饰时苏氨酸脱水导致前体肽易被降解,无法继续进行修饰。这一发现对cypemycin生物合成机制的研究提供了重要信息,其研究方法也可以应用到其他核糖体肽的生物合成研究中,同时也可以用于发现并组合生物合成更多结构新颖,活性良好的此类天然产物,为未来生物医药的发展提供重要的理论依据。
[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.
【学位授予单位】：兰州大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：O629.72;Q55

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