Poolman B, Modderman R, Reizer J
Department of Microbiology, State University of Groningen, The Netherlands.
J Biol Chem. 1992 May 5;267(13):9150-7.
The lactose transport protein (LacS) of Streptococcus thermophilus is a chimeric protein consisting of an amino-terminal carrier domain and a carboxyl-terminal phosphoenolpyruvate:sugar phosphotransferase system (PTS) IIA protein domain. The histidine residues of LacS were changed individually into glutamine or arginine residues. Of the 11 histidine residues present in LacS, only the His-376 substitution in the carrier domain significantly affected sugar transport. The region around His-376 was found to exhibit sequence similarity to the region around His-322 of the lactose transport protein (LacY) of Escherichia coli, which has been implicated in sugar binding and in coupling of sugar and H+ transport. The H376Q mutation resulted in a reduced rate of uptake and altered affinity for lactose (beta-galactoside), melibiose (alpha-galactoside), and the lactose analog methyl-beta-D-thiogalactopyranoside. Similarly, the extent of accumulation of the galactosides by cells expressing LacS(H376Q) was highly reduced in comparison to cells bearing the wild-type protein. Nonequilibrium exchange of lactose and methyl-beta-D-thiogalactopyranoside by the H376Q mutant was approximately 2-fold reduced in comparison to the activity of the wild-type transport protein. The data indicate that His-376 is involved in sugar recognition and is important, but not essential, for the cotransport of protons and galactosides. The carboxyl-terminal domain of LacS contains 2 histidine residues (His-537 and His-552) that are conserved in seven homologous IIA protein(s) (domains) of PTSs. P-enolpyruvate-dependent phosphorylation of wild-type LacS, but not of the mutant H552Q, was demonstrated using purified Enzyme I and HPr, the general energy coupling proteins of the PTS, and inside-out membrane vesicles isolated from E. coli in which the lactose transport gene was expressed. The His-537 and His-552 mutations did not affect transport activity when the corresponding genes were expressed in E. coli.
嗜热链球菌的乳糖转运蛋白（LacS）是一种嵌合蛋白，由一个氨基末端载体结构域和一个羧基末端磷酸烯醇丙酮酸：糖磷酸转移酶系统（PTS）IIA蛋白结构域组成。LacS的组氨酸残基被逐个替换为谷氨酰胺或精氨酸残基。在LacS中存在的11个组氨酸残基中，只有载体结构域中的His-376替换显著影响糖转运。发现His-376周围的区域与大肠杆菌乳糖转运蛋白（LacY）的His-322周围区域具有序列相似性，后者与糖结合以及糖和H⁺转运的偶联有关。H376Q突变导致摄取速率降低，并改变了对乳糖（β-半乳糖苷）、蜜二糖（α-半乳糖苷）和乳糖类似物甲基-β-D-硫代半乳糖吡喃糖苷的亲和力。同样，与携带野生型蛋白的细胞相比，表达LacS(H376Q)的细胞中半乳糖苷的积累程度大大降低。与野生型转运蛋白的活性相比，H376Q突变体对乳糖和甲基-β-D-硫代半乳糖吡喃糖苷的非平衡交换减少了约2倍。数据表明His-376参与糖识别，对于质子和半乳糖苷的共转运很重要，但不是必需的。LacS的羧基末端结构域包含2个组氨酸残基（His-537和His-552），它们在PTS的7个同源IIA蛋白（结构域）中是保守的。使用纯化的酶I和HPr（PTS的一般能量偶联蛋白）以及从表达乳糖转运基因的大肠杆菌中分离的内翻膜囊泡，证明了野生型LacS的磷酸烯醇丙酮酸依赖性磷酸化，但突变体H552Q没有。当相应基因在大肠杆菌中表达时，His-537和His-552突变不影响转运活性。
