Unfolding intermediate in the peroxisomal flavoprotein D-amino acid oxidase
Unfolding intermediate in the peroxisomal flavoprotein D-amino acid oxidase
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DOI：
10.1074/jbc.m403489200
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发表时间：
2004-07-02
影响因子：
4.8
通讯作者：
Pollegioni, L
中科院分区：
文献类型：
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作者：
Caldinelli, L;Iametti, S;Pollegioni, L
来自纤细红酵母的黄素酶D-氨基酸氧化酶（DAAO）是过氧化物酶体酶，并且是黄素蛋白的谷胱甘肽还原酶家族的原型成员。DAAO是一种稳定的同源二聚体，FAD分子与每个40-kDa亚基紧密结合。在这项工作中，将尿素诱导的二聚体DAAO解折叠与相同蛋白质的单体形式（缺失的二聚环突变体）进行了比较。通过使用圆二色谱，蛋白质和黄素荧光，1，8-苯胺基萘磺酸结合和活性测定，我们证明了尿素诱导的DAAO的展开是一个三态过程，产生一个中间体，并且这个过程是可逆的。中间体物质缺乏天然DAAO的催化活性和特征性三级结构，但具有显著的二级结构并保留黄素结合。DAAO的解折叠通过形成膨胀的、部分解折叠的无活性中间体进行，其特征在于低溶解度、疏水表面的暴露增加以及属于FAD结合结构域的β链F5对胰蛋白酶的敏感性增加。寡聚状态不改变推断的折叠过程。链F5与含有对应于1型过氧化物酶体靶向信号的Ser-Lys-Leu序列的C-末端α-螺旋接触，并且该结构元件与N-末端β α黄素结合基序（Rossmann折叠）相互作用。折叠中间体的扩展构象（特别是所提到的二级结构元件的更高无序度）可以匹配DAAO通过过氧化物酶体膜的体内运输所需的无活性全酶的结构。
The flavoenzyme D-amino acid oxidase ( DAAO) from Rhodotorula gracilis is a peroxisomal enzyme and a prototypical member of the glutathione reductase family of flavoproteins. DAAO is a stable homodimer with a FAD molecule tightly bound to each 40-kDa subunit. In this work, the urea-induced unfolding of dimeric DAAO was compared with that of a monomeric form of the same protein, a deleted dimerization loop mutant. By using circular dichroism spectroscopy, protein and flavin fluorescence, 1,8-anilinonaphtalene sulfonic acid binding and activity assays, we demonstrated that the urea-induced unfolding of DAAO is a three-state process, yielding an intermediate, and that this process is reversible. The intermediate species lacks the catalytic activity and the characteristic tertiary structure of native DAAO but has significant secondary structure and retains flavin binding. Unfolding of DAAO proceeds through formation of an expanded, partially unfolded inactive intermediate, characterized by low solubility, by increased exposure of hydrophobic surfaces, and by increased sensitivity to trypsin of the beta-strand F5 belonging to the FAD binding domain. The oligomeric state does not modify the inferred folding process. The strand F5 is in contact with the C-terminal alpha-helix containing the Ser-Lys-Leu sequence corresponding to the type 1 peroxisomal targeting signal, and this structural element interacts with the N-terminal betaalphabeta flavin binding motif (Rossmann fold). The expanded conformation of the folding intermediate (and in particular the higher disorder of the mentioned secondary structure elements) could match the structure of the inactive holoenzyme required for in vivo trafficking of DAAO through the peroxisomal membrane.