NAT/NCS2-hound: A Webserver for the detection and evolutionary classification of prokaryotic and eukaryotic nucleobase–cation symporters of the NAT/NCS2 family

Abstract

Nucleobase transporters are important for supplying the cell with purines and/or pyrimidines, for controlling the intracellular pool of nucleotides and for obtaining exogenous nitrogen/carbon sources for the metabolism. Nucleobase transporters are also evaluated as potential targets for antimicrobial therapies, since several pathogenic microorganisms rely on purine/pyrimidine salvage from their hosts. The majority of known nucleobase transporters belong to the evolutionarily conserved and ubiquitous NAT/NCS2 protein family. Based on a large-scale phylogenetic analysis that we performed on thousands of prokaryotic proteomes, we have developed a webserver that can detect and distinguish this family of transporters from other homologous families that recognize different substrates. We can further categorize these transporters to certain evolutionary groups with distinct substrate preferences. The webserver scans whole proteomes and graphically displays which proteins are identified as NAT/NCS2, to which evolutionary groups and subgroups they belong to and which conserved motifs they have.For key subgroups and motifs, the server displays annotated information from published crystal-structures and mutational studies pointing to key functional amino acids that may help experts assess the transport capability of the target sequences. The server is 100% accurate in detecting NAT/NCS2 family members. We also used the server to analyze 9109 prokaryotic proteomes and identified Clostridia, Bacilli, β-and γ-Proteobacteria, Actinobacteria and Fusobacteria as the taxa with the largest number of NAT/NCS2 transporters per proteome. An analysis of 120 representative eukaryotic proteomes also demonstrates the server's capability of correctly analyzing this major lineage, with plants emerging as the group with the highest number of NAT/NCS2 members per proteome.

Specificity profile of NAT/NCS2 purine transporters inSinorhizobium(Ensifer)meliloti

“…The results show lack of NAT/NCS2 homologs in most of the Rhizobiales except in the genus Sinorhizobium ( Ensifer ), in particular all strains of S. meliloti or S. medicae which contain five or six NAT/NCS2 homologs per strain, and sporadic presence in Bradyrhizobium (containing 0–3 homologs per strain), Rhizobium ( R. tropici ), and Mesorhizobium ( M. australicum ). A phylogenetic tree of these rhizobial NAT/NCS2 homologs reveals that four Sinorhizobium homologs classify in NAT/COG2233 (in Cluster C1_Xanthine‐Uric Acid; see Chaliotis et al , 2018) and two in AzgA‐like/COG2252, whereas the Bradyrhizobium homologs classify in different clusters of these subfamilies (Figure 1b). Of the six Sinorhizobium homologs, only SmVC3 (AzgA‐like/COG2252) is consistently present throughout the genus, whereas SmX28, SmLL8, SmLL9, SmWQ0 (NAT/COG2233), and SmYE1 (AzgA‐like/COG2252) are confined in S. meliloti and S. medicae .…”

“…Asp266 (TM8) is replaceable only by E or N and irreplaceable by C, H, M, or V (Figure 4b), in close resemblance with the properties of Asp276 in XanQ (Mermelekas et al , 2010); a difference is that D276E in XanQ displays high activity, in contrast to D266E in SmLL9 which transports at a rate of 20% relative to wild type (Figure 4b) pointing to a more crucial role of this carboxyl group in SmLL9. In the uric acid transporter UacT which also belongs to the NAT/COG2233 Cluster C1_Xanthine‐Uric Acid (Chaliotis et al , 2018) and is closely related with SmLL8 (Figure 1b), all residues corresponding to the above six crucial residues of SmLL9 were found to be irreplaceable (Papakostas and Frillingos, 2012). Of the corresponding residues in the fungal UapA, the two substrate‐binding residues Glu356 (TM8) (Papageorgiou et al , 2008) and Gln408 (TM10) (Koukaki et al , 2005) are also essential and Asp388 (TM9) is also functionally irreplaceable (Papageorgiou et al , 2008), but the other residues are less stringently related with activity: Asn409 (TM10) is replaceable by Ala, Ser or Gln with retention of wild‐type properties, and only N409D results in loss of function (Koukaki et al , 2005), His86 (TM1) is linked with defects in folding and targeting to the plasma membrane as shown with the low‐activity mutants H86A, H86K, or H86D, while H86N is active and indistinguishable in function from wild type (Pantazopoulou and Diallinas, 2006), and Asp360 (TM8) is replaceable by Ala retaining wild‐type properties (Kosti et al , 2012).…”

“…Cluster 1 (C1_Xanthine-Uric Acid) (Chaliotis et al, 2018) which also includes two very well studied homologs, the xanthine-specific…”

“…The percentage (given as a decimal) of trees in which the associated taxa clustered together is shown next to the branches. Branches with a bootstrap value < 0.7 have been deleted and bootstrap values < 0.9 are not shown in these presentations [Colour figure can be viewed at wileyonlinelibrary.com] from purine or pyrimidine permeases in bacteria, fungi, plants, and animals to Na + -dependent L-ascorbic acid transporters in human and other mammals Yamamoto et al, 2010;Frillingos, 2012;Bürzle et al, 2013;Girke et al, 2014;Botou et al, 2018;Chaliotis et al, 2018). Phylogenetically, all homologs of S. meliloti cluster together with known purine nucleobase transporters from Escherichia coli or other proteobacteria (Frillingos, 2012;Papakostas and Frillingos, 2012;Papakostas et al, 2013;Karena et al, 2015).…”

The guanine–hypoxanthine permease GhxP of Erwinia amylovora facilitates the influx of the toxic guanine derivative 6‐thioguanine

“…In addition, analysis of the EaGhxP amino acid sequence by the web‐based NAT/NCS2‐Hound, a web‐based program designed for identifying and classifying nucleobase cation symporter 2, clearly places this protein among GhxP cluster (Chaliotis et al . 2018).…”