Evolution of substrate specificity in the Nucleobase-Ascorbate Transporter (NAT) protein family

L-ascorbic acid (vitamin C) is an essential metabolite in animals and plants due to its role as an enzyme co-factor and antioxidant activity. In most eukaryotic organisms, L-ascorbate is biosynthesized enzymatically, but in several major groups, including the primate suborder Haplorhini, this ability is lost due to gene truncations in the gene coding for L-gulonolactone oxidase. Specific ascorbate transporters (SVCTs) have been characterized only in mammals and shown to be essential for life. These belong to an extensively studied transporter family, called Nucleobase-Ascorbate Transporters (NAT). The prototypic member of this family, and one of the most extensively studied eukaryotic transporters, is UapA, a uric acid-xanthine/H⁺ symporter in the fungus Aspergillus nidulans. Here, we investigate molecular aspects of NAT substrate specificity and address the evolution of ascorbate transporters apparently from ancestral nucleobase transporters. We present a phylogenetic analysis, identifying a distinct NAT clade that includes all known L-ascorbate transporters. This clade includes homologues only from vertebrates, and has no members in non-vertebrate or microbial eukaryotes, plants or prokaryotes. Additionally, we identify within the substrate-binding site of NATs a differentially conserved motif, which we propose is critical for nucleobase versus ascorbate recognition. This conclusion is supported by the amino acid composition of this motif in distinct phylogenetic clades and mutational analysis in the UapA transporter. Together with evidence obtained herein that UapA can recognize with extremely low affinity L-ascorbate, our results support that ascorbate-specific NATs evolved by optimization of a sub-function of ancestral nucleobase transporters.