Please use this identifier to cite or link to this item:
Scopus Web of ScienceĀ® Altmetric
Type: Journal article
Title: Determination of the essentiality of the eight cysteine residues of the NrtA protein for high-affinity nitrate transport and the generation of a functional cysteine-less transporter
Author: Unkles, S.
Rouch, D.
Wang, Y.
Siddiqi, M.
Okamoto, M.
Stephenson, R.
Kinghorn, J.
Glass, A.
Citation: Biochemistry, 2005; 44(14):5471-5477
Publisher: Amer Chemical Soc
Issue Date: 2005
ISSN: 0006-2960
Statement of
Shiela E. Unkles, Duncan A. Rouch, Ye Wang, M. Yaeesh Siddiqi, Mamoru Okamoto, Raymond M. Stephenson, James R. Kinghorn, and Anthony D. M. Glass
Abstract: All eight cysteine residues, C90, C94, C143, C147, C219, C325, C367, and C431, present in transmembrane domains of the Aspergillus nidulans NrtA nitrate transporter protein were altered individually by site-specific mutagenesis. The results indicate that six residues, C90, C147, C219, C325, C367, and C431, are not required for nitrate transport. Although alterations of C94 and C143 are less well tolerated, these residues are not mandatory and their possible role is discussed. A series of constructs, all completely devoid of cysteine residues, was generated to permit future cysteine-scanning mutagenesis. The optimum cysteine-less combination was identified as C90A, C94A, C143A, C147T, C219S, C325S, C367S, and C431S. This mutant combination yielded transformant strains with up to 40% of wild-type nitrate transport activity. Furthermore, the K(m) value and the level of protein expression were found to be similar to those of the wild-type. This cysteine-less vector should allow us to investigate in detail potentially interesting NrtA amino acids (e.g. identified from homology comparisons) which may be involved in transport, by altering these singly to cysteine and studying such residues by thiol chemistry.
Keywords: Aspergillus nidulans
Carrier Proteins
Anion Transport Proteins
Fungal Proteins
Mutagenesis, Site-Directed
Biological Transport
DOI: 10.1021/bi047732e
Published version:
Appears in Collections:Agriculture, Food and Wine publications
Aurora harvest 5

Files in This Item:
There are no files associated with this item.

Items in DSpace are protected by copyright, with all rights reserved, unless otherwise indicated.