Modelling, substrate docking and mutational analysis identify residues essential for the function and specificity of the purine-cytosine transporter FcyB.

Abstract

The Nucleobase Cation Symporter-1 (NCS1) family includes bacterial and fungal transmembrane transporters specific for the uptake of purines-pyrimidines as well as other structurally similar metabolites (e.g. allantoin, hydantoin, pyridoxine, thiamine). The recent elucidation of crystal structures of a bacterial member of the NCS1 family, the Mhp1 benzyl-hydantoin permease from Microbacterium liquefaciens, allowed us to construct and validate a three-dimensional model of the Aspergillus nidulans purine-cytosine/H+ FcyB symporter. Based on primary sequence alignment, three-dimensional topology, and substrate docking, we identified residues as potentially essential for substrate binding in FcyB. To validate the role of these and other putatively critical for transport residues, we performed a systematic functional analysis of relevant mutants. The characterization of the mutated forms of FcyB was performed by growth tests, epifluorescence microscopic analysis, detailed kinetic studies and western blot analysis. Among the residues mutated we identified critical ones for the substrate binding affinity and/or the specificity, providing information concerning the molecular mechanism and evolution of the transporter specificity.