【作者】 向福；

【导师】 余龙江；

【作者基本信息】 华中科技大学 ， 生物制药工程， 2007， 博士

【摘要】 分子进化是从分子水平上追溯生物演变的轨迹,重建其类缘关系。从结构的角度来探讨分子进化的问题近年来逐渐受到人们的关注。碳酸酐酶(Carbonic Anhydrase, CA)广泛分布于自然界,是一种很古老的含锌金属酶,双向催化二氧化碳的可逆水合反应,与人类健康密切相关,具有重要的生理作用,其不同的同工酶已成为不同疾病的重要靶点。考虑到蛋白质结构在分子进化过程中表现得比序列更为保守,因此能提供更为详细的进化信息。因此,本文首先探讨了α,β和γ三类碳酸酐酶的结构进化,其中包括进化机制、药物设计信息和分类信息等。这主要是通过对各类碳酸酐酶结构域结构进行多重结构比对,构建结构系统树;根据结构系统树,利用结构QR算法(QR factorization)构建非冗余结构数据集;利用非冗余结构数据集对蛋白质数据库进行blast搜索,从而引入序列相似性较高的同源序列;在剔除同源序列结构域以外的序列部分后,用序列QR算法来构建同源序列的非冗余数据集。以非冗余结构数据集的结构比对结果来引导同源序列的非冗余数据集的多重序列比对,从而构建非冗余结构数据集的结构系统树和非冗余同源序列数据集基于结构的系统发育树,完成各类碳酸酐酶基于结构的系统发育重建。同样从分子进化的角度,基于分子钟假设检验探讨了细胞培养对中国红豆杉细胞18S rRNA基因的影响。最后利用分子对接技术,基于两种青光眼药物对结构进化的功能类群进行了验证。本文结论如下:(1)γ-CA处于一个结构同源性较低的远缘超家族;α-CA则是一个从序列到结构都很保守的超家族,具有很高的结构同源性;β-CA超家族的结构同源性位于二者之间,比α-CA的结构更分化,比γ-CA的结构保守。(2)结构、序列和功能具有密切的进化关系,为碳酸酐酶的活性改造和药物设计提供了理论基础。蛋白质结构对其功能的保持非常重要,但在满足特定结构的前提下,一些关键残基的存在,才是蛋白质功能得以实现的重要因素。(3)不同碳酸酐酶的结构具有独特的进化保守性和特异性结构区域,为碳酸酐酶的活性改造和药物设计提供了重要线索。功能活性的差异在于保守区域的关键残基的变化,结构特异性区域主要在疏水结构部分。通过不同残基的取代可以改变其活性,而根据特异性结构也可以设计具有高度专一性的药物。(4)γ-CA的进化机制可能是:在进化早期通过基因复制事件形成金属络合结构,后来受到环境的压力,发生结构趋异事件,逐渐形成质子转移结构,才具备高效的催化活性。(5)为CsoSCA作为β-CA的一个新亚类提供了有力的证据。(6)细胞培养能在种的水平影响保守的18S rRNA基因进化,使得HG-1和Taxus chinensis的分歧时间相差约7Ma。更多还原

【Abstract】 The studies on the evolution of structures have been around for a long time, but only recently have significant progress been made.Carbonic anhydrase (CA, EC 4.2.1.1) are zinc-containing enzymes catalyzing the reversible hydration of CO2. They are ubiquitous in Eukarya, Archaea and Bacteria domains. The enzyme is important to many physiological processes such as respiration, CO2 transport and photosynthesis. CAs had been used as the important target proteins for drug designs.Protein structure is more highly conserved than sequence and it can give more detailed evolutionary information, the comparative analysis of structures allows the investigation of evolutionary events. So, this dissertation studied the molecular evolution ofα-,β-, andγ-class carbonic anhydrases based on their domain structures.To obtain a reliable analysis, we defined a subset that contains all specificities and organisms as the nonredundant structure set using QR factorization based on the multiple structural alignment of the known crystallographic structures ofα-,β-, andγ-CAs with QH and Qres as the structural homology measure. Then, we applied unweighted pair group method with arithmetic averages (UPGMA) to reconstruct structural phylogeny with 1-QH as the distance. Further analysis of the catalytic domain requires importing the sequences as well. This dissertation did so by performing a BLAST search of a carbonic anhydrase domain structure from the nonredundant structure set agaist the Swiss-Prot database one at a time. The over-represented sequences were eliminated. Using ClustalW to align the sequences and use MultiSeq to keep only the portions of the sequences that aligned to the catalytic domain in the structures. The sequence QR factorization was used to select the nonredundant sequence set. Finally, the structural alignment of the nonredundant structure set was used to guide the sequence alignment of the noredundant sequence set. The phylogenetic trees were reconstructed using neighbor-joining method with poisson distances as the metic.Then, this dissertation discussed the effect of cell culture on the Taxus chinensis cells 18S rRNA gene based on the phylogenetic analysis and molecular clock hypothsis. At last, we validated the functional group from evolution of structures via virtual screening based on molecular docking.The results suggest as follow:1.α-CAs are from a superfamily with highly structural homology, with QH > 0.4 and Sc > 5.5. Only the core structure of active site ofβ-CAs is highly conserved, and theγ-CA belongs to a very diverse superfamily of proteins that share the left-handedβ-helix (LβH).2. The protein moleculars with different sequences either can be in possession of the similar structures with the same function, or can bear different functions with some semblable structures. It is worth to note that the key residues in sequence have more important influence on protein function than structure. The relationships among sequence, structure and function provide theoretical principle for applied research of CA, such as the alteration of CA activity, and the drug design etc.3. The substitution of resides in active site can change the activity of CA, and the specificity of active site can help for the specific-drug design. The conserved and specific regions of active site give highlight to the applied research of CAs, such as the alteration of CA activity, and the drug design etc.4. The domain ofγ-CA underwent a process ofα-helical content from amino- terminal end to carboxyl-terminal end of the left-handedβ-helix; the capacity ofγ-CA to bind Zn occurred early in evolution and only later included the ability to catalyze the reversible hydration of CO2 efficiently for the occurrence of two loops involving Glu 62 and Glu 84 respectively and a long helix at the carboxyl-terminal end of the LβH.5. The structural similarities are sufficient to allow recognition of CsoSCA as a member of theβ-class of carbonic anhydrase. More than that, the structural analysis strongly surpports for the CsoSCA should qualifiy as a distinct subclass ofβ-CAs.6. Cell cultures make the evolution of Taxus chinensis cells 18S rRNA gene faster, and the divergence time between HG-1 and T. chinensis is about 7Ma.更多还原