4 research outputs found
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
Context-dependent Cryptic Roles of Specific Residues in Substrate Selectivity of the UapA Purine Transporter
Members of the ubiquitous Nucleobase Ascorbate Transporter (NAT) family
are H+ or Na+ symporters specific for the cellular uptake of either
purines and pyrimidines or L-ascorbic acid. Despite the fact that
several bacterial and fungal members have been extensively characterised
at a genetic, biochemical or cellular level, and crystal structures of
NAT members from Escherichia coli and Aspergillus nidulans have been
determined pointing to a mechanism of transport, we have little insight
on how substrate selectivity is determined. Here, we present systematic
mutational analyses, rational combination of mutations, and novel
genetic screens that reveal cryptic context-dependent roles of partially
conserved residues in the so-called NAT signature motif in determining
the specificity of the UapA transporter of A. nidulans. We show that
specific NAT signature motif substitutions, alone and in combinations
with each other or with distant mutations in residues known to affect
substrate selectivity, lead to novel UapA versions possessing variable
transport capacities and specificities for nucleobases. In particular,
we show that a UapA version including the quadruple mutation
T405S/F406Y/A407S/Q408E in the NAT signature motif (UapA-SYSE) becomes
incapable of purine transport, but gains a novel pyrimidine-related
profile, which can be further altered to a more promiscuous
purine/pyrimidine profile when combined with replacements at distantly
located residues, especially at F528. Our results reveal that UapA
specificity is genetically highly modifiable and allow us to speculate
on how the elevator-type mechanism of transport might account for this
flexibility. (C) 2021 Elsevier Ltd. All rights reserved