Cloning and functional characterization of two bacterial members of the NAT/NCS2 family in Escherichia coli.
The coding potential of the genome of E. coli K-12 includes YgfO and YicE, two members of the evolutionarily conserved NAT/NCS2 transporter family that are highly homologous to each other (45% residue identity) and closely related to UapA of Aspergillus nidulans, a most extensively studied microbial member of this family. YgfO and yicE were cloned from the genome, over-expressed extrachromosomally and assayed for uptake of [(3)H]xanthine and other nucleobases, in E. coli K-12, under conditions of negligible activity of the corresponding endogenous systems. Alternative, essentially equivalent functional versions of YgfO and YicE were engineered by C-terminal tagging with an epitope from the E. coli lactose permease and a biotin-acceptor domain from Klebsiella pneumoniae. Both YgfO and YicE were shown to be present in the plasma membrane of E. coli and function as specific, high-affinity transporters for xanthine (K(m) 4.2-4.6 microM for YgfO, or 2.9-3.8 microM for YicE), in a proton motive force-dependent manner; they display no detectable transport of uracil, hypoxanthine, or uric acid at external concentrations of up to 0.1 mM. Both YgfO and YicE are inefficient in recognizing uric acid or xanthine analogues modified at position 8 of the purine ring (8-methylxanthine, 8-azaxanthine, oxypurinol, allopurinol), which distinguishes them from their fungal homologues UapA and Xut1.
Karatza
Panayiota
P
Laboratory of Biological Chemistry, University of Ioannina Medical School, Ioannina 45110, Greece.
Frillingos
Stathis
S
D000818:Animals; D001483:Base Sequence; D001692:Biological Transport; D003001:Cloning, Molecular; D000939:Epitopes; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism*; D029968:Escherichia coli Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D033702:Nucleobase Transport Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D010957:Plasmids; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D013329:Structure-Activity Relationship; D013379:Substrate Specificity; D014498:Uracil / Q000378:metabolism; D014527:Uric Acid / Q000378:metabolism; D019820:Xanthine / Q000378:metabolism
D000939:Epitopes; D029968:Escherichia coli Proteins; D033702:Nucleobase Transport Proteins; D019820:Xanthine; D014527:Uric Acid; D014498:Uracil