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

Anezia Kourkoulou,Alexandros A. Pittis,George Diallinas

doi:10.15698/mic2018.06.636

Abstract

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.

Highlights

Gene duplication and divergence is a major mechanism of evolutionary novelty, providing protein families with an expanded repertoire of activities and novel specificities
Together with evidence obtained that UapA can recognize with extremely low affinity L-ascorbate, our results support that ascorbate-specific Nucleobase-Ascorbate Transporters (NAT) evolved by optimization of a sub-function of ancestral nucleobase transporters
NATs are functionally classified into two distinct specificity groups: nucleobase versus L-ascorbate transporters More than 24 members of the NAT family have been functionally characterized via direct assessment of their transport activities or indirectly via genetic and physiological studies [19,20,21,22,23] (Table 1)

Summary

Introduction

Gene duplication and divergence is a major mechanism of evolutionary novelty, providing protein families with an expanded repertoire of activities and novel specificities. Novelty might evolve via promiscuous side-activities or sub-functions of proteins, which are optimized in the course of evolution and under specific selective pressures. This model puts emphasis on the multifunctional pre-duplication state of the evolving genes and introduces the possibility that positive selection pressure might drive evolution after gene duplication event. One way to approach these questions is to reproduce protein evolution in the laboratory [12] This can be achieved by systematic rational or random mutagenesis and DNA shuffling of specific genes, given a functional assay is designed to follow functional modification, or more efficiently, by direct genetic selection or genetic screens using model microorganisms. What is emerging from these approaches of directed evolution is that functional evolution necessitates conservation of sufficient protein stability, as conformational epistasis might suppress several routes of neofunctionalization [13]

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