Substrate Selection in Homologous Bacterial Transporters

同源细菌转运蛋白的底物选择

• 批准号: 6753938
• 负责人: MANUEL F VARELA
• 金额: $ 20.91万
• 依托单位: EASTERN NEW MEXICO UNIVERSITY PORTALES
• 依托单位国家: 美国
• 财政年份: 2004
• 资助国家: 美国
• 起止时间: 2004-07-01 至 2008-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6753938
• 关键词: Escherichia coli; aminoacid; binding sites; carbohydrate sequence; carbohydrate transport; disaccharides; enzyme activity; fermentation; growth media; lactose; maltose; mutant; nucleic acid sequence; oligosaccharides; permease; site directed mutagenesis; sucrose

DESCRIPTION (provided by applicant): Solute transporters are defective in genetic diseases and confer bacterial and cancer drug resistance. Transporters have substrate specificity, which is poorly understood, thus hindering our understanding of transport. It is unclear whether amino acids that confer sugar specificity in the lactose permease of E. coli (LacY) also confer sugar selection in homologous transporters for melibiose (MelY), raffinose (RafB) and sucrose (CseB). Until solute specificity is understood in homologous transporters, it will be difficult to know how transport is accomplished. The long range goal is to understand the molecular basis of substrate recognition during transport. The specific objective of this research is to determine whether (the molecular properties) the solute binding sites in homologous solute transporters differ in their amino acids that determine specificities. The central hypothesis is that functional differences between transporters (i.e., solute specificities) are dictated by subtle differences in sequence (and thus, structure), since the seemingly diverse transporters share related amino acid sequences. The rationale for the proposed research is that once it is known whether homologous sugar transporters mediate substrate specificity by similar or distinct binding and transport properties, we will know whether a common molecular basis exists for transport in seemingly diverse transporters, and the potential will exist for engineering transporters to translocate highly desirable solutes for therapeutic purposes. The solute specificity profiles for MelY RafB and CscB homologues are distinct and thus make our experimental system unique for a comparative study of substrate specificity. The specific aims are independent of the completion of one another. Specific Aim 1: Identify amino acid residues that determine sucrose transport in MelY. We propose to isolate and characterize mutants containing MelY that transport sucrose. In preliminary work, we have found several sucrose fermentation positive mutants. We have published data showing mutations in helices 3 and 6 of MelY that transport maltose. Specific Aim 2: Identify amino acids that determine sucrose and maltose transport in RafB. We propose to isolate and characterize RafB mutants that transport sucrose. We isolated maltose positive mutants; we will study maltose uptake and accumulation in these mutants using transport assays. We have preliminary data showing that RafB has distinct binding sites for raffinose and lactose. Specific Aim 3: Identify residues that determine melibiose and maltose transport in CscB. We will isolate cells with mutated CscB that transport melibiose or maltose, and then study transport. We have found maltose positive mutants. We have preliminary data showing that CscB transports lactose. The comparative nature of the study of solute specificity in homologous transporters is innovative, as most other studies focus on solute binding and transport of naturally occurring substrates. Data generated by the proposed work will be innovative because MelY, RafB and CscB are homologous to many transporters, and knowledge of solute specificity mechanisms gained here will be applicable to transporters involved in genetic diseases and to infectious disease-causing bacteria and their drug resistances.

描述（由申请人提供）：溶质转运体在遗传病中存在缺陷，并赋予细菌和癌症耐药性。转运蛋白具有底物特异性，这一点我们了解得很少，因此阻碍了我们对转运的理解。目前尚不清楚，在大肠杆菌的乳糖渗透酶（LacY）中赋予糖特异性的氨基酸，是否也赋予了同源转运蛋白对糖的选择，如糖二糖（MelY）、棉子糖（RafB）和蔗糖（CseB）。在了解同源转运蛋白的溶质特异性之前，很难知道转运是如何完成的。长期目标是了解转运过程中底物识别的分子基础。本研究的具体目的是确定同源溶质转运体中的溶质结合位点（分子性质）在决定特异性的氨基酸上是否不同。核心假设是，转运蛋白之间的功能差异（即溶质特异性）是由序列（以及结构）的细微差异决定的，因为表面上不同的转运蛋白共享相关的氨基酸序列。提出这项研究的基本原理是，一旦知道同源糖转运蛋白是否通过相似或不同的结合和运输特性介导底物特异性，我们就会知道在看似不同的转运蛋白中是否存在一个共同的分子基础，并且工程转运蛋白转运高度理想的溶质用于治疗目的的潜力将存在。MelY RafB和CscB同源物的溶质特异性特征是不同的，因此我们的实验系统对于底物特异性的比较研究是独一无二的。具体的目标是独立于彼此的完成。特异性目标1：鉴定在MelY中决定蔗糖转运的氨基酸残基。我们建议分离和鉴定含有转运蔗糖的MelY突变体。在前期工作中，我们发现了几个蔗糖发酵阳性突变体。我们已经发表的数据显示，MelY的螺旋3和6突变转运麦芽糖。特异性目标2：鉴定决定蔗糖和麦芽糖转运的氨基酸。我们建议分离和鉴定转运蔗糖的RafB突变体。我们分离出麦芽糖阳性突变体；我们将研究麦芽糖的吸收和积累在这些突变体使用运输试验。我们有初步的数据表明，RafB对棉子糖和乳糖有不同的结合位点。特异性目标3：鉴定在CscB中决定美利二糖和麦芽糖运输的残基。我们将分离携带转运美利二糖或麦芽糖的突变CscB细胞，然后研究转运。我们发现了麦芽糖阳性突变体。我们有初步数据显示CscB运输乳糖。同源转运体中溶质特异性研究的比较性质是创新的，因为大多数其他研究都集中在天然底物的溶质结合和运输上。由于MelY， RafB和CscB与许多转运蛋白同源，因此拟议工作产生的数据将具有创新性，并且在此获得的溶质特异性机制的知识将适用于涉及遗传疾病的转运蛋白和感染性致病细菌及其耐药性。