Abstract
Ypr118w is a non-essential, low copy number gene product from Saccharomyces cerevisiae. It belongs to the PFAM family PF01008, which contains the alpha-, beta-, and delta-subunits of eukaryotic translation initiation factor eIF2B, as well as proteins of unknown function from all three kingdoms. Recently, one of those latter proteins from Bacillus subtilis has been characterized as a 5-methylthioribose-1-phosphate isomerase, an enzyme of the methionine salvage pathway. We report here the crystal structure of Ypr118w, which reveals a dimeric protein with two domains and a putative active site cleft. The C-terminal domain resembles ribose-5-phosphate isomerase from Escherichia coli with a similar location of the active site. In vivo, Ypr118w protein is required for yeast cells to grow on methylthioadenosine in the absence of methionine, showing that Ypr118w is involved in the methionine salvage pathway. The crystal structure of Ypr118w reveals for the first time the fold of a PF01008 member and allows a deeper discussion of an enzyme of the methionine salvage pathway, which has in the past attracted interest due to tumor suppression and as a target of aniprotozoal drugs.
Highlights
Ypr118w is a non-essential, low copy number gene product from Saccharomyces cerevisiae. It belongs to the PFAM family PF01008, which contains the ␣, , and ␦-subunits of eukaryotic translation initiation factor eIF2B, as well as proteins of unknown function from all three kingdoms. One of those latter proteins from Bacillus subtilis has been characterized as a 5-methylthioribose-1-phosphate isomerase, an enzyme of the methionine salvage pathway
Ypr118w Is a 5-Methylthioribose-1-phosphate Isomerase— The sequence similarity between Ypr118w and 5-methylthioribose-1-phosphate isomerase from B. subtilis led us to test whether Ypr118w is involved in the methionine salvage pathway
The first step is the conversion of MTA to 5-methylthioribose-1-phosphate (MTR) by MTA phosphorylase followed by its isomerization to methylthioribulose-1-P (Fig. 2A)
Summary
Yeast Strain—Yeast BY4741 wild type and derivative strain ⌬ypr118::kanXR (Euroscarf collection) were used. Batch purification of GST-Ypr118w was performed by incubating the extract with glutathione-Sepharose 4B beads (Amersham Biosciences) as described by the manufacturer followed by three washes with Tris-buffered saline. The protein was purified by gel filtration using a Superdex 75 (Amersham Biosciences) column equilibrated in 100 mM NaCl, 20 mM Tris-HCl, pH 7.5, 1 mM EDTA, and 1 mM dithiothreitol. The protein fraction was pooled, reincubated with glutathioneSepharose 4B beads, and loaded onto a 1-ml Resource QTM column (Amersham Biosciences), equilibrated with 20 mM Tris-HCl, pH 8.0. The Hamburg as well as the Grenoble MAD data sets were sufficient to solve the structure, both yielding clean electron density maps. The Grenoble data set extended to higher resolution due to the brighter beam and larger detector and was later used to obtain an experimental density map suitable for automated model building. Structure figures were created using the program PYMOL (www.pymol.org)
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