Molecular Microbiology
Molecular Microbiology
5 years ago

Insight on specificity of uracil permeases of the NAT/NCS2 family from analysis of the transporter encoded in the pyrimidine utilization operon of Escherichia coli

Insight on specificity of uracil permeases of the NAT/NCS2 family from analysis of the transporter encoded in the pyrimidine utilization operon of Escherichia coli
Panayiota Lazou, Thomas Evangelidis, Emmanuel Mikros, George Lambrinidis, Maria Botou, Konstantinos Papakostas, Stathis Frillingos
The uracil permease UraA of Escherichia coli is a structurally known prototype for the ubiquitous Nucleobase-Ascorbate Transporter (NAT) or Nucleobase-Cation Symporter-2 (NCS2) family and represents a well defined subgroup of bacterial homologs that remain functionally unstudied. Here, we analyze four of these homologs, including RutG of E. coli which shares 35% identity with UraA and is encoded in the catabolic rut (pyrimidine utilization) operon. Using amplified expression in E. coli K-12, we show that RutG is a high-affinity permease for uracil, thymine and, at low efficiency, xanthine, and recognizes also 5-fluorouracil and oxypurinol. In contrast, UraA and the homologs from Acinetobacter calcoaceticus and Aeromonas veronii are permeases specific for uracil and 5-fluorouracil. Molecular docking indicates that thymine is hindered from binding to UraA by a highly conserved Phe residue which is absent in RutG. Site-directed replacement of this Phe with Ala in the three uracil-specific homologs allows high-affinity recognition and/or transport of thymine, emulating the RutG profile. Furthermore, all RutG orthologs from enterobacteria retain an Ala at this position, implying that they can use both uracil and thymine and, possibly, xanthine as substrates, and provide the bacterial cell with a range of catabolizable nucleobases. This article is protected by copyright. All rights reserved. RutG is a broad-specificity uracil/thymine permease encoded in the pyrimidine utilization operon of enterobacteria. An essential difference from the closely related UraA and other uracil-specific permeases is the absence of a crucial Phe that is predicted to hinder coordination of thymine at the binding site. Replacing this Phe with Ala allows high-affinity binding and/or transport of thymine in addition to uracil.

Publisher URL: http://onlinelibrary.wiley.com/resolve/doi

DOI: 10.1111/mmi.13931

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