【作者】 侯晓玮；

【导师】 龚为民；

【作者基本信息】 中国科学技术大学 ， 生化与分子生物学， 2008， 博士

【摘要】 第一部分钩端螺旋体腺苷甲硫氨酸依赖型氧甲基化酶的晶体学研究对所有的生物而言,甲基化是一个重要而又普遍存在的生物学过程。绝大部分的甲基化酶采用腺苷甲硫氨酸作为甲基供体,甲基化完成后腺苷甲硫氨酸转化为腺苷高胱氨酸。在致病性细菌问号型钩端螺旋体全基因组测序完成后,基因LA0415的产物被注释为腺苷甲硫氨酸依赖型氧甲基化酶（LiOMT）。经序列分析LiOMT被归为Methyltransf₃蛋白家族（Pfam PF01596）,在这个家族中细菌来源的所有已知功能的5个甲基化酶均被报道参与抗生素合成。我们用大肠杆菌大量表达了LiOMT并得到晶体。在晶体结构中LiOMT结合有腺苷高胱氨酸配基,展示出非常保守的腺苷甲硫氨酸（腺苷高胱氨酸）结合区域和独特的金属离子依赖的催化区域。LiOMT分子通过N端的交换形成同二聚体,据文献报道这种二聚的形式能够在没有底物结合的情况下促进底物结合区预折叠。LiOMT的催化区域和底物结合区域的一级序列和三维结构预示它的底物是一种酚类化合物,这种酚类化合物可能带有一个大环形状的尾巴。这是Methyltransf₃家族中的第一个细菌来源的结构,LiOMT晶体结构也给这个家族中其他细菌来源的抗生素合成相关蛋白提供了可供参考的结构信息。第二部分人线粒体外膜蛋白mitoNEET的晶体学研究mitoNEET是新近被发现的线粒体外膜蛋白,它能够特异性结合抗Ⅱ型糖尿病的药物吡格列酮。我们对mitoNEET的细胞质可溶部分(mitoNEET_33-108)进行了结构解析。mitoNEET_33-108拥有一个全新的折叠类型,并含有一个[2Fe-2S]铁硫中心。这个铁硫中心与mitoNEET_33-108的Cys72,Cys74,Cys83和His87配位,相对于已知的4个Cys配位和2个Cys、2个His配位的[2Fe-2S]铁硫中心来说,这种3个Cys、1个His的配位类型也是在自然界中第一次发现。mitoNEET_33-108在溶液中和在晶体结构中都形成同二聚体,两个单体通过界面间的疏水相互作用以及两个水分子介导的氢键相互作用结合在一起。与[2Fe-2S]铁硫中心配位的His-87暴露在二体的表面,对铁硫中心的功能起着重要作用。我们推测mitoNEET在线粒体外膜上形成二体,并通过二体表面上靠近[2Fe-2S]铁硫中心的区域与其它蛋白相互作用。第三部分大肠杆菌膜蛋白二酯酰甘油激酶的初步晶体学研究大肠杆菌膜蛋白二酯酰甘油激酶（DAGK）是一个分子量为13 kDa的小蛋白,它能催化脂分子二酯酰甘油（DAG）和MgATP磷酸化反应生成另一种脂分子卵磷脂（PA）和MgADP。它在膜上形成三体,每个单体三次跨膜,很可能三个单体共同参与磷酸转移反应。大肠杆菌DAGK代表了一类微生物DAGK,它们与其它所有已知的DAGK都没有序列同源性,却和其它DAGK一样催化复杂的磷酸转移反应。得到大肠杆菌的晶体结构有利于我们了解这类独特的激酶的分子机理。我们对DAGK进行了表达、纯化和晶体筛选,目前已经得到可以衍射的晶体,衍射分辨率为20（?）的衍射点。更多还原

【Abstract】 PartⅠCrystal structure of SAM-dependent O-methyltransferase from pathogenic bacterium Leptospira interrogansThe S-adenosylmethionine（SAM）-dependent O-methyltransferase from Leptospira interrogans（LiOMT）expressed by gene LA0415 belongs to the Methyltransf₃ family（Pfam PF01596）.In this family all of the five bacterial homologues with known function are reported as SAM-dependent O-methylstransferases involved in antibiotic production.The crystal structure of LiOMT in complex with S-adenosylhomocysteine reported here is the first bacterial protein structure in this family.The LiOMT structure shows a conserved SAM-binding region and a probable metal-dependent catalytic site.The molecules of LiOMT generate homodimers by N-terminal swapping,which assists the pre-organization of the substrate-binding site. Based on the sequence and structural analysis,it is implied by the catalytic and substrate-binding site that the substrate of LiOMT is a phenolic derivative,which probably has a large ring-shaped moiety.PartⅡCrystallographic studies of human mitoNEETMitoNEET was identified as an outer mitochondrial membrane protein that can bind the anti-diabetes drug pioglitazone.The crystal structure of the cytoplasmic mitoNEET（residue 33-108）is determined in this study.The structure presents a novel protein fold and contains a[2Fe-2S]cluster-binding domain.The[2Fe-2S] cluster is coordinated to the protein by Cys72,Cys74,Cys83 and His87 residues.This coordination is also novel compared to the traditional[2Fe-2S]cluster coordinated by four cysteines or two cysteines and two histidines.The cytoplasmic mitoNEET forms homodimers in solution and in crystal.The dimerization is mainly mediated by hydrophobic interactions as well as hydrogen bonds coordinated by two water molecules binding at the interface.His87 residue,which plays an important role in the coordination of the[2Fe-2S]cluster,is exposed to the solvent on the dimer surface.It is proposed that mitoNEET dimer may interact with other proteins via the surface residues in close proximity to the[2Fe-2S]cluster.PartⅢPriliminary crystallographic study on E.coli diacylglyceroi kinase Diacylglycerol kinase（DAGK）from E.coli is a small（13 kDa）enzyme which catalyzes the phosphorylation the lipid diacylglycerol by MgATP to form another lipid（phosphatidic acid）and MgADP.It forms homotrimer in the membrane.Every monomer has three transmembrane segments,and the active site is likely shared between subunits.DAGK is representative of an entire class of microbial enzymes that share no similarity with other known kinases but catalyze the complicated phosphate transfer as other kinases.To determine the crystal structure of E.coli DAGK could be facilitated to study molecular mechanism of these distinct enzymes. We have over-expressed and purified the DAGK from E.coli.The crystal we have got now can diffract to 20（?）.更多还原