【作者】 王白帆；

【导师】 席真；

【作者基本信息】 南开大学 ， 化学生物学， 2013， 博士

【摘要】 原卟啉原Ⅸ氧化酶(PPO)催化氧化原卟啉原Ⅸ成为原卟啉Ⅸ,广泛存在于动物、植物、真菌和细菌中,是生物体内叶绿素和亚铁血红素合成途径中的一个关键酶。在人体中,PPO的缺损会造成显性遗传疾病混合型卟啉症(Variegated Porphyria,VP)。目前,VP患者的数量和发病地域在不断扩大。同时,PPO也被利用于光动力学对于癌症的治疗,因此对PPO的研究具有重要的医学意义。植物体内的PPO的活性被抑制剂所抑制会导致绿色植物组织的干枯与白化,最终使得植物死亡,因而PPO是一大类除草剂的作用靶标。因PPO非植物所特有,考虑到人畜及环境安全,种属选择性是针对该靶标设计、开发除草剂的关键问题。而且,开发抗PPO抑制剂除草剂的转基因作物,能够减少农药的使用,降低农业生产成本,具有重大的经济价值。因此对PPO的研究具有重要农学意义。另外,PPO是植物光信号转导过程中的重要因子；PPO的多底物特性、催化底物的电子转移功能,也使得底物的结合与催化机制的研究非常具有研究价值。鉴于PPO在生物体内的重要性,以及在医学和农学上的应用,本文选择了PPO作为主要的研究对象。在本论文中我们主要进行了以下五方面的工作：一、多种属的PPO生物信息学研究。通过对不同种属的PPO的序列和结构的比较,识别了PPO活性位点保守残基和差异残基,揭示导致不同种属PPO在细胞内的定位方式与聚集状态差异的序列因素和结构因素,从原子水平上认识了不同种属对抑制剂选择性及天然抗性的序列因素和结构因素,为选择性抑制剂的设计提供最直接的信息。二、PPO与底物相互作用的研究。运用计算化学中分子对接、分子动力学模拟等方法,系统研究了底物在三个种属PPO(烟草线粒体、枯草芽孢杆菌和人体)中的结合方式,为PPO催化机制的研究提供了结构基础,也为进一步的定点突变实验提供了指导。三、PPO功能的计算结构生物学的研究。结合定点突变实验、结构生物学研究和分子动力学模拟,识别出人体PPO和枯草芽孢杆菌PPO中,对于PPO活性的保持有重要作用的极性作用网络。发现这个极性作用网络通过稳定FAD的异咯嗪环的结合环境,进而稳定底物与FAD之间的相互作用以维持酶的催化活性。另外,还对人体PPO活性空腔的残基功能进行定义,阐述了这些残基对于底物与PPO的结合的贡献。这些结果为进一步改造PPO,利用PPO发展抗除草剂转基因作物提供了关键信息。四、卟啉症分子致病机制的研究。结合分子动力学模拟和统计学方法,阐释了PPO致病突变体酶功能的丧失与平衡态构象的关系,通过引入定量描述PPO平衡态构象的参数--优势构象概率,建立了预测PPO催化活性的方法(Prezyme),并准确预测了44个卟啉症突变体的催化活性,阐述了其分子致病机制。该方法也对不同种属PPO的催化活性有较好的预测。五、PPO与抑制剂相互作用的研究。我们获取了人体PPO与两个二苯醚类抑制剂除草剂,乙氧氟草醚和氟磺胺草醚复合物的晶体结构,其分辨率分别为2.3A和2.0A。针对抑制剂三氟羧草醚对于烟草线粒体PPO、人体PPO和枯草芽孢杆菌PPO抑制活性的差异进行了计算研究。我们发现,烟草PPO中的Phe392、 Leu372、Leu356、Phe353和Arg98等残基是抑制剂结合的重要位点,人体PPO在Phe392和Leu372残基上相对于烟草PPO的差异是使得抑制剂对人体PPO的抑制活性较差的主要因素；而枯草芽孢杆菌PPO在上述位点的残基性质相对于烟草PPO表现除了较大的差异,使得抑制剂不能结合到上述位点,从而产生抑制剂的抗性。计算模拟的结果得到了定点突变实验很好的验证。综上所述,本文结合生物化学和计算化学方法,对于PPO的分子机制进行了系统研究。识别不同种属PPO序列和结构上的差异之处,为种属间PPO性质的研究和定点突变体实验提供指导。获得了不同种属PPO与底物的结合模型,为PPO催化机理的研究和抑制剂的设计提供了基础和信息。结合定点突变实验和计算模拟方法,对PPO突变体进行了研究,为理解PPO结构与功能之间的关系提供了指导。发展了一套能够定量预测卟啉症相关PPO突变体的方法,阐述了卟啉症突变体的分子机制。我们还阐述了PPO对于抑制剂的选择性机制,为理解敏感性靶标生物特异性提供了依据,也为新型高效低毒的农药分子设计提供了结构基础。更多还原

【Abstract】 Protoporphyrinogen IX oxidase (PPO; EC1.3.3.4) catalyzes protoporphyrinogen IX to protoporphyrin IX. PPO is the key enzyme for the biosynthesis of protoporphyrin IX, which is the last check point of heme and chlorophyll common biosynthesis.Hence, PPO has been identified as one of the most importance action targets for the treatment of some important diseases including cancer and variegated porphyria (VP). In the agriculture, PPO inhibitors have been used as herbicides for many years. Inhibition of PPO in plants leads the accumulation of protogen, which exports to the cytoplasm and forms porphyrm through nonenzymatic oxidation. The photosensitizing porphyrin can produce singlet oxygen which can cause peroxidation of membrane lipids and cell death. PPO inhibitors herbicides are highly effective, low toxic to human and friendly to environment. The development of the new PPO inhibitors herbicides is the hot topic of the field. In humans, defects in the PPO gene, resulting in approximately50%decreased activity of PPO, is responsible for the dominantly inherited disorder VP. VP is a type of acute hepatic porphyria, which is characterized by an abnormal pattern of porphyrin excretion. VP has been found worldwide, and is particularly prevalent in the white population of South Africa.Since PPO is essential for the biological process, and has widely application in the agricultural field and medicine, we chose PPO as research object. We hope to gain understanding about the molecular mechanism of the PPO. In this dissertation we focus primary on the following aspects:1. Applying bioinformatics studies on PPO. We had identified the conserved and noconserved residues of the active site of PPO from different species by sequence alignments. We studied the contributions of these to the properties of PPO from different species, which provided information for the site directed mutagenesis study on PPO. We compared the structural domains of PPO to reveal the sequence and structural difference contribution to the location and assembly of PPO from different species. These studies provided important information for the further studies on the difference on the property of PPO and the relationship between structure and function of PPO.2. Applying studies on the interaction between substrate and PPO. We docked substrate into PPO from tobacco mitochondria, human and Bacillus subtilis. Molecular dynamics simulation were then performed on docked substrate-PPO complex. We access the the correct substrate-PPO binding models based on the molecular dynamics simulation results, with the site directed mutagenesis studies and quantum mechanics calculation. We also compared the binding mode of substrate in PPO from different species, in which we identified the origin of the difference catalytic activity on PPOs.3. Applying studies on the function of PPO based on structure. In this part of work, we applied site directed mutagenesis and computational studies on PPO, which including:(1) Study on the active site residues of PPO. We combined the site directed mutagenesis and molecular dynamics simulation to identified the function of the residues of human PPO.(2) Study on the residues cluster which regulate the activity of PPO. We applied structural biology, biochemical and simulation studies to identify a polar interaction network surrounding Arg59and Asp65in human and Bacillus subtilis PPO which can regulate the activity of PPO. This interaction network help to stabilize the environment of the isoalloxazine ring of FAD, in which stabilize the interaction between substrate and FAD to maintain the activity of PPO. By compared the sequence and structure of PPO, we discover this interaction network in conserved in PPO from different species.4. Applying studies on the molecular mechanism of the variegated porphyria. We applied structural study on the variegated porphyria, which indicated that the44VP-causing mutants distributed on all of the three structure domains of human PPO. We categorized these mutants into5potential categories, i.e., affecting the substrate binding, FAD binding, hydrophobic core, secondary structure and surface of human PPO. We then combined molecular dynamics simulation and statistical study to quantitative insight into the molecular mechanism of VP. In light of the collision theory, we develop a descriptor which can quantitative describe the catalytic activity of PPO, which called probability of privileged conformations. We successfully predicted the catalytic activity of44VP-causing mutants by calculating their probability of privileged conformations, and we elucidated the detailed molecular mechanism of these mutants. Based on these results we develop a protocol which can predict the catalytic activity of PPO. This protocol can be used to predict the activity of human PPO mutants and other species of PPO.5. Applying studies on the interaction between PPO and inhibitors. We applied structural biology study and computational study on the interaction between PPO and inhibitors, which mainly on:(1) The crystal structures of the complex of PPO and inhibitor. We obtained the crystal structures of human PPO and the inhibitors, oxyfluorfen and fomesafen, in which the resolution was2.3A and2.0A, respectively. We compared the binding mode of the acifluorofen, oxyfluorfen and fomesafen to human PPO and their activity, in which we found that the Arg97in human PPO is a important binding site for inhibitors.(2) The selectivity to inhibitor in PPO from different species. We performed molecular dynamics simulation and MM-GBSA energy calculation on the complex of acifluorofen and PPO from tobacco mitochondria, human and Bacillus subtilis. We found that, the residues Phe392, Leu372, Leu356, Phe353and Arg98is the "hot spots" for the selectivity to inhibitor of PPO. PPO regulated the binding of inhibitors by altering the size and electro-property of the side chain of these residues, which was validated by the mutagenesis study.In total, we combined the biochemical and computational methods to study the molecular mechanism of PPO. We identified the differences in the sequence and structure of PPO, which provides guidance for the study of the property of PPO and mutagenesis experiments. We obtained the binding mode of substrate in PPO from different species, which help to elucidate the catalytic mechanism of PPO and provide information for the design of new PPO inhibitor. We applied study on the mutant of PPO, which help to understand the relationship of the structure and function of PPO. We developed a protocol to quantitative predict the catalytic of VP-causing mutants, and make detailed description on the molecular mechanism of VP. And we also elucidated the selectivity of PPO, which provides the structural basis for the design of PPO inhibitor.更多还原