Abstract
We present the first account of the structure–function relationships of a protein of the subfamily of urea/H+ membrane transporters of fungi and plants, using Aspergillus nidulans UreA as a study model. Based on the crystal structures of the Vibrio parahaemolyticus sodium/galactose symporter (vSGLT) and of the Nucleobase-Cation-Symport-1 benzylhydantoin transporter from Microbacterium liquefaciens (Mhp1), we constructed a three-dimensional model of UreA which, combined with site-directed and classical random mutagenesis, led to the identification of amino acids important for UreA function. Our approach allowed us to suggest roles for these residues in the binding, recognition and translocation of urea, and in the sorting of UreA to the membrane. Residues W82, Y106, A110, T133, N275, D286, Y388, Y437 and S446, located in transmembrane helixes 2, 3, 7 and 11, were found to be involved in the binding, recognition and/or translocation of urea and the sorting of UreA to the membrane. Y106, A110, T133 and Y437 seem to play a role in substrate selectivity, while S446 is necessary for proper sorting of UreA to the membrane. Other amino acids identified by random classical mutagenesis (G99, R141, A163, G168 and P639) may be important for the basic transporter's structure, its proper folding or its correct traffic to the membrane.
Highlights
Urea is a readily available nitrogen source for bacteria, fungi and plants, occurring in nature as a product of animal, plant and microbial metabolism of nitrogenous compounds [1,2,3,4,5,6]
Which constitutes a subfamily of Hþ-symporters within the sodium : solute symporter (SSS) superfamily. The latter is composed by hundreds of members, present in both eukaryotic and prokaryotic organisms, which usually function as Naþcotransporters of a variety of substrates, such as sugars, vitamins, amino acids, inorganic ions, etc
Results obtained for strains bearing mutations Y106F and W82F show that there is a reduction in the initial transport rate which does not correlate with Km and Ki changes
Summary
Urea is a readily available nitrogen source for bacteria, fungi and plants, occurring in nature as a product of animal, plant and microbial metabolism of nitrogenous compounds [1,2,3,4,5,6]. Together with ammonium nitrate it is one of the most widely used nitrogen-based fertilizers (http://espere.mpch-mainz.mpg.de/documents/ACCENT/Edition08/texts%20material/english/fertilizer_usage_2001.pdf [7]). The internalization of this highly polar molecule necessitates specialized cellular transport systems. License http://creativecommons.org/licenses/by/3.0/, which permits unrestricted use, provided the original author and source are credited. Which constitutes a subfamily of Hþ-symporters within the sodium : solute symporter (SSS) superfamily. The latter is composed by hundreds of members, present in both eukaryotic and prokaryotic organisms, which usually function as Naþcotransporters of a variety of substrates, such as sugars, vitamins, amino acids, inorganic ions, etc. The latter is composed by hundreds of members, present in both eukaryotic and prokaryotic organisms, which usually function as Naþcotransporters of a variety of substrates, such as sugars, vitamins, amino acids, inorganic ions, etc. (http://www.tcdb.org [8])
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