Departamento de Ingeniería Celular y Biocatálisis, Instituto de Biotecnología, Universidad Nacional Autónoma de México, Cuernavaca, Morelos, Mexico.
FEBS Lett. 2012 Feb 17;586(4):466-71. doi: 10.1016/j.febslet.2012.01.033. Epub 2012 Jan 27.
T-protein is composed of chorismate mutase (AroQ(T)) fused to the N-terminus of prephenate dehydrogenase (TyrA). Here, we report the replacement of AroQ(T) with the β1-domain of protein G (Gβ1). The TyrA domain shows a strong dehydrogenase activity within the context of this fusion, and our data indicate that Gβ1-TyrA folds into a dimeric conformation. Amino acid substitutions in the Gβ1 domain of Gβ1-TyrA identified residues involved in stabilizing the TyrA dimeric conformation. Gβ1 substitutions in the N-terminal β-hairpin eliminated Gβ1-TyrA expression, whereas Gβ1-TyrA tolerated Gβ1 substitutions in the C-terminal β-hairpin and in the α-helix. All of the characterized variants folded into a dimeric conformation. The importance of the β2-strand in forming a Gβ1 homo-dimerization interface explains the relevance of the first-β-hairpin in stabilizing the dimeric TyrA protein.
T 蛋白由侧翼氨酸合酶（AroQ（T））与预苯酸脱氢酶（TyrA）的 N 端融合而成。在这里，我们报告用蛋白 G 的β1 结构域（Gβ1）取代 AroQ（T）。在这种融合的背景下，TyrA 结构域表现出很强的脱氢酶活性，我们的数据表明 Gβ1-TyrA 折叠成二聚体构象。在 Gβ1-TyrA 的 Gβ1 结构域中的氨基酸取代鉴定出参与稳定 TyrA 二聚体构象的残基。Gβ1-TyrA 中 N 端β-发夹中的 Gβ1 取代消除了 Gβ1-TyrA 的表达，而 Gβ1-TyrA 可耐受 C 端β-发夹和α-螺旋中的 Gβ1 取代。所有表征的变体均折叠成二聚体构象。β2-链在形成 Gβ1 同源二聚化界面中的重要性解释了第一个β-发夹在稳定二聚体 TyrA 蛋白中的相关性。
