Abstract
The evolutionarily broad family nucleobase-cation symporter-2 (NCS2) encompasses transporters that are conserved in binding site architecture but diverse in substrate selectivity. Putative purine transporters of this family fall into one of two homology clusters: COG2233, represented by well studied xanthine and/or uric acid permeases, and COG2252, consisting of transporters for adenine, guanine, and/or hypoxanthine that remain unknown with respect to structure-function relationships. We analyzed the COG2252 genes of Escherichia coli K-12 with homology modeling, functional overexpression, and mutagenesis and showed that they encode high affinity permeases for the uptake of adenine (PurP and YicO) or guanine and hypoxanthine (YjcD and YgfQ). The two pairs of paralogs differ clearly in their substrate and ligand preferences. Of 25 putative inhibitors tested, PurP and YicO recognize with low micromolar affinity N(6)-benzoyladenine, 2,6-diaminopurine, and purine, whereas YjcD and YgfQ recognize 1-methylguanine, 8-azaguanine, 6-thioguanine, and 6-mercaptopurine and do not recognize any of the PurP ligands. Furthermore, the permeases PurP and YjcD were subjected to site-directed mutagenesis at highly conserved sites of transmembrane segments 1, 3, 8, 9, and 10, which have been studied also in COG2233 homologs. Residues irreplaceable for uptake activity or crucial for substrate selectivity were found at positions occupied by similar role amino acids in the Escherichia coli xanthine- and uric acid-transporting homologs (XanQ and UacT, respectively) and predicted to be at or around the binding site. Our results support the contention that the distantly related transporters of COG2233 and COG2252 use topologically similar side chain determinants to dictate their function and the distinct purine selectivity profiles.
Highlights
Four putative purine transporter genes represent a distinct homology cluster within family nucleobase-cation symporter-2 (NCS2) in Escherichia coli
We provide a first insight on the structure-function relationships of COG2252 members of family NCS2 using the homologs of E. coli K-12 as study paradigms
Several K-12 strains were used as E. coli host in these experiments to establish optimal conditions for assaying the uptake of each purine nucleobase
Summary
Four putative purine transporter genes represent a distinct homology cluster within family NCS2 in Escherichia coli. Putative purine transporters of this family fall into one of two homology clusters: COG2233, represented by well studied xanthine and/or uric acid permeases, and COG2252, consisting of transporters for adenine, guanine, and/or hypoxanthine that remain unknown with respect to structure-function relationships. We analyzed the COG2252 genes of Escherichia coli K-12 with homology modeling, functional overexpression, and mutagenesis and showed that they encode high affinity permeases for the uptake of adenine (PurP and YicO) or guanine and hypoxanthine (YjcD and YgfQ). The purine transporters of this family fall into one of two homology clusters: COG2233, represented by well studied permeases for xanthine and/or uric acid (2-oxy purines), and COG2252, consisting of transporters for adenine, guanine, and/or hypoxanthine (2-non-oxy purines) that remain largely unknown with respect to structure-function relationships (Fig. 1). Our data provide support to the contention that the distantly related purine transporters of the two homology clusters use distinct but topologically equivalent side chains to dictate the binding site function and selectivity
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