Substrate Selection in Homologous Bacterial Transporters

Eastern New Mexico University Portales, Portales NM

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Abstract

[unreadable] 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. [unreadable] [unreadable]