The nucleoside transport proteins, NupC and NupG, from Escherichia coli: specific structural motifs necessary for the binding of ligands

Abstract

A series of 46 natural nucleosides and analogues (mainly adenosine-based) were tested as inhibitors of [U-(14)C]uridine uptake by the concentrative, H(+)-linked nucleoside transport proteins NupC and NupG from Escherichia coli. The two evolutionarily unrelated transporters showed similar but distinct patterns of inhibition, revealing differing selectivities for the different nucleosides and their analogues. Binding of nucleosides to NupG required the presence of hydroxyl groups at each of the C-3' and C-5' positions of ribose, while binding to NupC required only the C-3' hydroxyl substituent. The greater importance of the ribose moiety for binding to NupG is consistent with the evolutionary relationship between this protein and the oligosaccharide: H(+) symporter (OHS) subfamily of the major facilitator superfamily (MFS) of transporters. For both proteins the natural alpha-configuration at C-3' and the natural beta-configuration at C-1' was mandatory for ligand binding. N-7 in the imidazole ring of adenosine and the amino group at C-6 were found not to be important for binding and both transporters showed flexibility for substitution at C-6/N(6); one or both of N-1 and N-3 were important for adenosine analogue binding to NupC but significantly less so for binding to NupG. From the different effects of 8-bromoadenosine on the two transporters it appears that adenosine selectively binds to NupC in an anti- rather than a syn-conformation, whereas NupG is less prescriptive. The pattern of inhibition of NupC by differing nucleoside analogues confirmed the functional relationship of the bacterial transporter to members of the human concentrative nucleoside transporter (CNT) family and reaffirmed the use of the bacterial protein as an experimental model for these physiologically and clinically important mammalian proteins. The specificity data for NupG have been used to develop a homology model of the protein's binding site, based on the X-ray crystallographic structure of the disaccharide transporter LacY from E. coli. We have also developed an efficient general protocol for the synthesis of adenosine and three of its analogues, which is illustrated by the synthesis of [1'-(13)C]adenosine.

Influence of dTMP on the Phenotypic Appearance and Intracellular Persistence of Staphylococcus aureus

“…The influence of uridine, an inhibitor of nucleoside transport (18,33), on the phenotype of S. aureus is shown in Fig. 1E and 1F.…”

“…In Escherichia coli there are at least two nucleoside uptake enzymes, NupC and NupG, that are efficient in pyrimidine nucleoside transport (6), whereas in Bacillus subtilis and S. aureus only genes belonging to the nupC family have been reported (20, 24; www.ncbi.nlm.nih.gov). Thymidine transport can be inhibited by uridine in eukaryotic mouse cells (33) and in prokaryotic E. coli cells (18). Hence, it is tempting to speculate that uridine impairs the transport of thymidine, probably mediated by the NupC transport system in both clinical and 5-fluorouracil/SXT-induced S. aureus SCVs.…”

Sequence Alignment and Homology Threading Reveals Prokaryotic and Eukaryotic Proteins Similar to Lactose Permease

“…[11][12][13][14][15]54,55 Nevertheless, significant sequence homology with LacY has been found with only a few bacterial transporters grouped in the Oligosaccharide/H + Symport subfamily. 3 A BLAST search was performed using the translated genomic databases starting from the amino acid sequence of the maltose permease (MaltP) homolog from the deep sea alkalophile B. halodurans ,56 a homolog of LacY with conservation of the most essential amino acid residues (17% identity, see Table 1).…”

Role of Purine Biosynthesis in Bacillus anthracis Pathogenesis and Virulence

“…The salvage of purines can often be used to complement a loss of de novo purine biosynthesis depending on the availability of exogenous purines. While specific nucleoside transporters have not been well studied in B. anthracis, proteins homologous to those studied in model organisms, such as Escherichia coli and Bacillus subtilis (3,21,35,56), are present in B. anthracis. The homology between these proteins suggests that B. anthracis has a similar capacity for nucleoside transport and salvage.…”