期刊
JOURNAL OF MOLECULAR BIOLOGY
卷 433, 期 16, 页码 -出版社
ACADEMIC PRESS LTD- ELSEVIER SCIENCE LTD
DOI: 10.1016/j.jmb.2021.166814
关键词
Aspergillus nidulans; Fungi; UapA; Genetics; Nucleobase
资金
- Stavros S. Niarchos Foundation
- Greek Research & Technology Network (GRNET) in the National HPC facility -ARIS- under project NCS1_Mechanism [pr006040]
Members of the NAT family are symporters specific for the uptake of purines, pyrimidines, or L-ascorbic acid. This study reveals the roles of partially conserved residues in determining the specificity of the UapA transporter. Results show that UapA specificity is genetically modifiable, allowing for speculation on the flexible mechanism of transport.
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.
作者
我是这篇论文的作者
点击您的名字以认领此论文并将其添加到您的个人资料中。
推荐
暂无数据