Università degli Studi di Milano, Dipartimento di Patologia Animale Igiene e Sanità Pubblica Veterinaria, via Caloria 10, I-20133 Milano, Italy.
Biochimie. 2010 Oct;92(10):1335-42. doi: 10.1016/j.biochi.2010.06.015. Epub 2010 Jun 23.
L-aspartate oxidase (LASPO) is a flavoenzyme catalyzing the first step in the de novo biosynthesis of NAD+. The enzyme oxidizes L-aspartate both under aerobic and anaerobic conditions using oxygen as well as fumarate as electron acceptor. In accordance with its catalytic activities, LASPO displays strong primary and tertiary structure similarity with the flavin containing subunit of the proteins belonging to the succinate dehydrogenase/fumarate reductase family. The similarity extends to the active site residues, with LASPO differing from the other enzymes of the family only for the presence of a conserved glutamate (E121), which is substituted by apolar amino acids in the other enzymes. Three complementary approaches have been used to define the role of E121 in LASPO: characterization of mutants (E121A, E121Q, E121D and E121K), investigation of the catalytic activities of WT and mutants towards substrates and substrate analogues and molecular docking studies. All mutants retain fumarate reductase activity. On the contrary, all mutants lack L-aspartate oxidase activity, although retaining the ability to bind L-aspartate (except for E121K). These results and investigations on the oxidase activity towards substrate analogues suggest that the roles of E121 in catalysis include orienting L-aspartate in a productive binding mode and favouring proton abstraction from C2 by an active site base. Molecular docking studies of the substrate (L-aspartate), inhibitor (D-aspartate) and product (imino aspartate) in the active site of LASPO confirm that (a) the substrate/product energetically favoured orientation in the active site supports the conclusions reported above, (b) E121 interacts favourably with the charged amino group of the substrate and (c) different ligands might assume different orientations in the active site of the enzyme.
L-天冬氨酸氧化酶（LASPO）是一种黄素酶，催化 NAD+从头生物合成的第一步。该酶在有氧和无氧条件下均使用氧气和延胡索酸盐作为电子受体氧化 L-天冬氨酸。根据其催化活性，LASPO 与属于琥珀酸脱氢酶/延胡索酸还原酶家族的蛋白质的含黄素亚基具有很强的一级和三级结构相似性。这种相似性延伸到活性位点残基，LASPO 与该家族的其他酶不同之处仅在于存在一个保守的谷氨酸（E121），该谷氨酸在其他酶中被非极性氨基酸取代。已经使用三种互补方法来定义 E121 在 LASPO 中的作用：突变体（E121A、E121Q、E121D 和 E121K）的表征、WT 和突变体对底物和底物类似物的催化活性的研究以及分子对接研究。所有突变体均保留延胡索酸还原酶活性。相反，所有突变体均缺乏 L-天冬氨酸氧化酶活性，尽管保留了与 L-天冬氨酸结合的能力（除 E121K 外）。这些结果以及对底物类似物的氧化酶活性的研究表明，E121 在催化中的作用包括将 L-天冬氨酸定向到产生活性的结合模式，并有利于活性位点碱从 C2 上提取质子。LASPO 活性位点中底物（L-天冬氨酸）、抑制剂（D-天冬氨酸）和产物（亚氨基天冬氨酸）的分子对接研究证实，（a）活性位点中底物/产物在能量上有利的取向支持了上述结论，（b）E121 与底物的带电荷氨基基团有利地相互作用，（c）不同的配体可能在酶的活性位点中呈现不同的取向。
