3-羟基邻氨基苯甲酸3，4-双氧化酶（3HAO）的结构和功能研究

【作者】 李小武；

【导师】 牛立文； 滕脉坤；

【作者基本信息】 中国科学技术大学 ， 生物化学与分子生物学， 2006， 博士

【摘要】 3-羟基邻氨基苯甲酸3，4-双氧化酶（3HAO）是色氨酸犬尿氨酸代谢途径的一个非血红素Fe²⁺离子外二醇双氧化酶。其催化3-羟基邻氨基苯甲酸转化为喹啉酸。喹啉酸是NAD⁺生物合成的前体，同时也是N-甲基-D-天冬氨酸（NMDA）受体的激动剂从而引发神经毒。酵母3HAO的2.4（?）分辨率的晶体结构证实其是功能多样化的Cupin超家族的一员。酵母3HAO晶体结构是至今解析的第一个真核的3HAO的结构，形成同二聚体，每个分子有两个Ni²⁺离子的结合位点，其中一个Ni²⁺离子占据了活性中心非血红素Fe²⁺离子位置，此活性中心位于一个保守的β桶折叠为特征的结构域中。分析结构可看出参与催化的一系列残基有别于其它的外二醇双氧化酶，Asp120、Asn51、Glu111、和Arg114与两个配位活性中心Fe²⁺离子的残基（His49和Glu55）形成一氢键网络，此氢键网络在3HAO的催化过程中起重要作用。残基Arg101、Gln59和结合底物的疏水口袋决定了底物的特异性。与3HAO from Ralstonia metallidurans的结构比较，活性位点的相似说明原核和真核的3HAO催化机理是相同的。基于序列比较，在Cupin超家族中，我们推测3HAO是从低等生物的单cupin二体向后尘动物的双cupin的单体进化。另一金属离子结合位点我们推测仅仅起结构稳定的作用。基于底物与酵母3HAO模拟复合物的结构，我们提出3HAO可能的反应机理。同时我们认为4-Cl-3-羟基-邻氨基苯甲酸的基于机理的3HAO失活机制是由于Cl原子空间效应的缘故阻碍了底物C3与氧的结合。更多还原

【Abstract】 3-Hydroxyanthranilic acid 3,4-dioxygenase (3HA0) is a non-heme ferrousextradiol dioxygenase in the kynurenine pathway from tryptophan. It catalyzesthe conversion of 3-hydroxyanthranilate (HAA) to quinolinic acid (QUIN), anendogenous neurotoxin, via the activation of N-methyl-D-aspartate (NMDA)receptors and the precursor of NAD+ biosynthesis. The crystal structure of3HA0 from S. cerevisiae at 2.4 (?) resolution shows it to be a member of thefunctionally diverse cupin superfamily. The structure represents the firsteukaryotic 3HA0 to be resolved. The enzyme forms homodimers, with twonickel binding sites per molecule. One of the bound nickel atoms occupies theproposed ferrous-coordinated active site, which is located in a conserveddouble-strand β-helix domain. Examination of the structure reveals theparticipation of a series of residues in catalysis different from other extradioldioxygenases. Together with two iron-binding residues (His49 and Glu55),Asp120, Asn51, Glu111, and Arg114 form a hydrogen-bonding network; thishydrogen-bond network is key to the catalysis of 3HAO. Residues Arg101,Gln59, and the substratebinding hydrophobic pocket are crucial for substratespecificity. Structure comparison with 3HA0 from Ralstonia metalliduransreveals similarities at the active site and suggests the same catalyticmechanism in prokaryotic and eukaryotic 3HAO. Based on sequencecomparison, we suggest that bicupin of human 3HA0 is the first example ofevolution from a monocupin dimer to bicupin monomer in the diverse cupinsuperfamilies. A structural stabilization role is proposed for the other metalbinding site. Based on the model of the substrate HAA at the active site ofY3HAO, we propose a mechanism of catalysis for 3HAO. One possibility ofThe Mechanism of Inactivation of 3-Hydroxyanthranilate-3,4-dioxygenase by4-Chloro-3-hydroxyanthranilate is that the chloro substituent perturbs thesubstrate binding in such a way that the addition of oxygen of the C3 carbon ofthe substrate is blocked.更多还原