Functional mapping and implications of substrate specificity of the yeast high-affinity leucine permease Bap2
Functional mapping and implications of substrate specificity of the yeast high-affinity leucine permease Bap2
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DOI：
10.1016/j.bbamem.2014.03.018
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发表时间：
2014-07-01
影响因子：
3.4
通讯作者：
Abe, Fumiyoshi
中科院分区：
文献类型：
--
作者：
Usami, Yuki;Uemura, Satsohi;Abe, Fumiyoshi
亮氨酸是营养物质和蛋白质中的主要氨基酸，也是酿造过程中高级醇的重要前体。在酿酒酵母中，亮氨酸摄取由多种氨基酸通透酶介导，包括高亲和力亮氨酸通透酶 Bap2。尽管 BAP2 转录已被广泛分析，但底物被识别并通过通透酶移动的机制仍然未知。最近，我们确定了 Tat2 介导的色氨酸导入所需的 15 个氨基酸残基。在这里，我们向 Bap2 氨基酸残基引入同源突变，并显示 7 个残基在亮氨酸输入中发挥作用。根据结构同源的大肠杆菌精氨酸/胍丁胺逆向转运蛋白 AdiC，残基 1109/G110/T111 和 E305 分别位于 TMD1 和 TMD6 中推定的 a 螺旋断裂内。亮氨酸结合后，这些 α 螺旋断裂被认为介导 Bap2 中从向外开放状态到底物结合封闭状态的构象转变。残基Y336 (TMD7)和Y181 (TMD3)分别位于1109和E305附近。 Bap2介导的亮氨酸输入被一些氨基酸抑制，按以下严重程度顺序：苯丙氨酸、亮氨酸>异亮氨酸>蛋氨酸、酪氨酸>缬氨酸>色氨酸；组氨酸和天冬酰胺没有作用。此外，这种严重性顺序显然与除色氨酸之外的所有氨基酸的 logP 值（辛醇-水分配系数）一致。该结果表明，埋入 Bap2 结合袋的底物分配效率是底物特异性的主要决定因素，而不是结构氨基酸侧链识别。 (C) 2014 Elsevier B.V. 保留所有权利。
Leucine is a major amino acid in nutrients and proteins and is also an important precursor of higher alcohols during brewing. In Saccharomyces cerevisiae, leucine uptake is mediated by multiple amino acid permeases, including the high-affinity leucine permease Bap2. Although BAP2 transcription has been extensively analyzed, the mechanisms by which a substrate is recognized and moves through the permease remain unknown. Recently, we determined 15 amino acid residues required for Tat2-mediated tryptophan import. Here we introduced homologous mutations into Bap2 amino acid residues and showed that 7 residues played a role in leucine import Residues 1109/G110/T111 and E305 were located within the putative a-helix break in TMD1 and TMD6, respectively, according to the structurally homologous Escherichia coli arginine/agmatine antiporter AdiC. Upon leucine binding, these a-helix breaks were assumed to mediate a conformational transition in Bap2 from an outward-open to a substratebinding occluded state. Residues Y336 (TMD7) and Y181 (TMD3) were located near 1109 and E305, respectively. Bap2-mediated leucine import was inhibited by some amino acids according to the following order of severity: phenylalanine, leucine > isoleucine > methionine, tyrosine > valine > tryptophan; histidine and asparagine had no effect. Moreover, this order of severity clearly coincided with the logP values (octanol-water partition coefficients) of all amino acids except tryptophan. This result suggests that the substrate partition efficiency to the buried Bap2 binding pocket is the primary determinant of substrate specificity rather than structural amino acid side chain recognition. (C) 2014 Elsevier B.V. All rights reserved.