Structural characterization of l-aspartate oxidase and identification of an interdomain loop by limited proteolysis.
Structural characterization of l-aspartate oxidase and identification of an interdomain loop by limited proteolysis.
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L-天冬氨酸氧化酶的结构表征和通过有限蛋白水解鉴定域间环。
DOI：
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发表时间：
1999
期刊：
影响因子：
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通讯作者：
S. Ronchi
中科院分区：
文献类型：
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作者：
G. Tedeschi;A. Negri;F. Ceciliani;A. Mattevi;S. Ronchi
L-天冬氨酸氧化酶是兼性好氧生物中吡啶辅酶从头合成的第一个酶。该酶依赖于FAD，它与黄素蛋白中的氧化酶和富马酸还原酶类有共同的特征。在这份报告中，我们通过有限的蛋白质分解研究，将注意力集中在分子的超二级结构上。此外，还描述了蛋白质在不同pH下的聚合状态以及与NAD及其类似物的相互作用。结果表明，在pH<4.5时，L-天冬氨酸氧化酶为单体，在pH>6.5时为二聚体。该蛋白质由两个主要结构域组成，由位于120-140区域的柔性环连接。通过在有无底物(富马酸和甲苯二酮)、抑制剂(琥珀酸)和NAD的存在和不存在的情况下对Holo和apo形式的有限蛋白质分解获得的数据，允许这两个结构域都参与黄素辅酶的结合。此外，这篇手稿中报道的数据表明，NAD通过与黄素竞争与酶的结合来抑制L天冬氨酸氧化酶的活性。
l-Aspartate oxidase is the first enzyme in the de novo biosynthesis of pyridinic coenzymes in facultative aerobic organisms. The enzyme is FAD dependent and it shares common features with both the oxidase and the fumarate reductase classes of flavoproteins. In this report we focused our attention on the supersecondary structure of the molecule by means of limited proteolysis studies. Moreover the polymerization state of the protein at different pH and the interactions with NAD and its analogues are described. The results suggest that l-aspartate oxidase is a monomer at pH values lower than 4.5 and a dimer at pH values higher than 6.5. The protein is organized in two major domains connected by a flexible loop located in the 120-140 region. The data obtained by limited proteolysis of the holo and the apo form in the presence and in the absence of substrates (fumarate and menadione), inhibitors (succinate) and NAD allows the proposition that both domains are involved in the binding of the flavin coenzyme. Moreover the data reported in this manuscript suggest that NAD inhibits l-aspartate oxidase activity by competing with the flavin for the binding to the enzyme.