Flavin binding to the high affinity riboflavin transporter RibU
Flavin binding to the high affinity riboflavin transporter RibU
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DOI：
10.1074/jbc.m608583200
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发表时间：
2007-04-06
影响因子：
4.8
通讯作者：
Slotboom, Dirk Jan
中科院分区：
文献类型：
--
作者：
Duurkens, Ria H.;Tol, Menno B.;Slotboom, Dirk Jan
首次提出了一种纯化的核黄素(维生素B-2)膜转运体的生化和光谱表征。对乳酸乳球菌核黄素转运蛋白Ribu进行了高效表达、溶解和纯化。纯化后的转运蛋白在富营养液中培养时呈亮黄色。我们使用与洗涤剂兼容的基质辅助激光解吸电离飞行时间质谱法(Cadene，M.和Chait，B.T.(2000)anal)。化学。72,5655-5658)，以表明黄色的来源是与转运体共纯化的核黄素。该方法对纯化的膜蛋白底物鉴定具有普遍的适用性。无底物核糖核酸是通过在培养的细胞中表达该蛋白而在化学定义的介质中产生的。核黄素、FMN和玫瑰黄素以高亲和力和1：1的化学计量比与核黄素结合(核黄素的K-d为0.6 nm)，但Fad不与转运蛋白结合。核黄素的吸收光谱在底物与核黄素结合后发生显著变化。在441、464和486 nm处出现了分辨良好的谱带，表明存在疏水结合口袋。核黄素与核黄素结合后荧光几乎完全猝灭，转运蛋白的色氨酸荧光也被猝灭。结果表明，核黄素与一个或多个色氨酸残基堆积在Ribu的结合口袋中。诱变实验表明，Trp-68直接参与了核黄素的结合。结合已知结构的可溶性核黄素结合蛋白，讨论了核黄素结合位点的结构性质和核黄素与其底物的高亲和力的机理。
The first biochemical and spectroscopic characterization of a purified membrane transporter for riboflavin ( vitamin B-2) is presented. The riboflavin transporter RibU from the bacterium Lactococcus lactis was overexpressed, solubilized, and purified. The purified transporter was bright yellow when the cells had been cultured in rich medium. We used a detergent- compatible matrix- assisted laser desorption ionization time-of-flight mass spectrometry method ( Cadene, M., and Chait, B. T. ( 2000) Anal. Chem. 72, 5655 - 5658) to show that the source of the yellow color was riboflavin that had been co-purified with the transporter. The method appears generally applicable for substrate identification of purified membrane proteins. Substrate- free RibU was produced by expressing the protein in cells cultured in chemically defined medium. Riboflavin, FMN, and roseoflavin bound to RibU with high affinity and 1: 1 stoichiometry ( K-d for riboflavin is 0.6 nM), but FAD did not bind to the transporter. The absorption spectrum of riboflavin changed dramatically when the substrate bound to RibU. Well resolved bands appeared at 441, 464, and 486 nm, indicating a hydrophobic binding pocket. The fluorescence of riboflavin was almost completely quenched upon binding to RibU, and also the tryptophan fluorescence of the transporter was quenched when flavins bound. The results indicate that riboflavin is stacked with one or more tryptophan residues in the binding pocket of RibU. Mutagenesis experiments showed that Trp-68 was involved directly in the riboflavin binding. The structural properties of the binding site and mechanistic consequences of the exceptionally high affinity of RibU for its substrate are discussed in relation to soluble riboflavin- binding proteins of known structure.