Stoltz M, Brandsch R
Institut für Biochemie und Molekularbiologie, Hermann-Herder-Str. 7, D-79104 Freiburg, Germany.
J Biochem. 1998 Mar;123(3):445-9. doi: 10.1093/oxfordjournals.jbchem.a021957.
The contribution of (8alpha)-(N3)histidyl bond formation to the conformation of covalently flavinylated proteins was investigated by trypsin treatment of wild type and mutant versions of a model enzyme, 6-hydroxy-D-nicotine oxidase (6-HDNO) of Arthrobacter nicotinovorans. In the absence of FAD, apo-6-HDNO exhibited a conformation exposing a protease accessible site. Holoenzyme formation through FAD-attachment to His71 induced a conformational change in the protein that shielded the trypsin recognition site. This conformational change, however, did not require FAD-histidyl bond formation since trypsin resistance was also exhibited by a 6-HDNO.Cys71 mutant protein which was unable to bind FAD covalently. Replacement of Arg67, an amino acid residue supposed to be essential in flavinylation, by Ala rendered the protein protease sensitive as did replacement of Pro73 by Ala. These amino acids apparently play an essential role in stabilizing the native protein conformation. The inability to reach the native conformation also prevented FAD attachment, indicating that a specific conformation of the protein is a prerequisite for FAD-histidyl bond formation. Deletion of Phe448 and Arg449 from the 458 amino acid residues-containing enzyme resulted in complete protease sensitivity, demonstrating that flavinylation takes place posttranslationally.
通过用胰蛋白酶处理食尼古丁节杆菌的模型酶6-羟基-D-尼古丁氧化酶（6-HDNO）的野生型和突变体版本，研究了(8α)-(N3)组氨酰键形成对共价黄素化蛋白构象的贡献。在没有FAD的情况下，脱辅基6-HDNO呈现出一种暴露出蛋白酶可及位点的构象。通过FAD与His71连接形成全酶，会诱导蛋白质发生构象变化，从而屏蔽胰蛋白酶识别位点。然而，这种构象变化并不需要FAD-组氨酰键的形成，因为无法共价结合FAD的6-HDNO.Cys71突变蛋白也表现出对胰蛋白酶的抗性。将假定在黄素化中起关键作用的氨基酸残基Arg67替换为Ala，以及将Pro73替换为Ala，都会使蛋白质对蛋白酶敏感。这些氨基酸显然在稳定天然蛋白质构象中起着至关重要的作用。无法达到天然构象也会阻止FAD的附着，这表明蛋白质的特定构象是FAD-组氨酰键形成的先决条件。从含有458个氨基酸残基的酶中删除Phe448和Arg449会导致对蛋白酶完全敏感，这表明黄素化是在翻译后发生的。
