A Novel Acetyltransferase Found in Saccharomyces cerevisiae Σ1278b That Detoxifies a Proline Analogue, Azetidine-2-carboxylic Acid

Abstract

L-Azetidine-2-carboxylic acid (AZC), a toxic four-membered ring analogue of L-proline, is transported into the cells via proline transporters. It causes misfolding of the proteins into which it is incorporated competitively with L-proline and thereby inhibits the growth of the cells. We recently have discovered, on the chromosome of Saccharomyces cerevisiae ⌺1278b, a novel gene MPR1 required for the resistance of ⌺1278 background strains to toxic AZC. This gene was missing in the particular yeast strain used for the genomic sequence determination. Although the protein sequence was homologous to that of the S. cerevisiae transcriptional regulator, Mpr1p did not affect the expression of genes involved in proline uptake. However, gene expression in Escherichia coli and enzymatic analysis showed that the MPR1 gene encodes a novel AZC acetyltransferase, by which L-proline itself and other L-proline analogues are not acetylated. Mpr1p was considered to be a member of the N-acetyltransferase superfamily based on the results of an Alascan mutagenesis through the highly conserved region involved in binding acetyl-CoA in members of the superfamily. Our findings suggest that Mpr1p detoxifies AZC by acetylating it in the cytoplasm. This enzyme might be utilized as a selective marker in a wide variety of organisms, because the cells expressing the MPR1 gene acquire the AZC-resistant phenotype.Some amino acid analogues are known to induce in cells a transient stress response comparable with that of heat shock stress (1-3). The addition of certain amino acid analogues or mild heat shock causes a rapid increase in the synthesis of heat shock proteins in cells. The elevated levels of synthesis for this set of proteins begins to decrease shortly after restoration of the normal amino acids or normal temperature. Therefore, amino acid analogues have proved to be valuable reagents for studying cellular metabolism and the regulation of synthesis of macromolecules in both prokaryotic and eukaryotic cells (1-3). A toxic four-membered ring analogue of L-proline, L-azetidine-2-carboxylic acid (AZC) 1 , is incorporated into proteins competitively with L-proline and causes the synthesis of abnormal misfolded proteins, thereby inhibiting cell growth in both bacterial and animal cells (4 -7). In bacteria such as Escherichia coli and Serratia marcescens, L-proline biosynthesis from Lglutamate is regulated mainly through feedback inhibition by L-proline of ␥-glutamyl kinase, a key enzyme in the pathway (8,9). Because this enzyme is also feedback-inhibited by L-proline analogues such as AZC, L-proline-overproducing strains have been successfully isolated from L-proline-analogue-resistant mutants, which have a mutation in the proB gene coding for ␥-glutamyl kinase that causes desensitization to feedback inhibition (10, 11).To investigate the cryoprotective effect of L-proline on the freezing stress of yeast, we previously isolated AZC-resistant mutants from an L-proline-nonutilizing strain of Saccharomyces cerevisiae (12). In the process of this...

Role of the yeast acetyltransferase Mpr1 in oxidative stress: Regulation of oxygen reactive species caused by a toxic proline catabolism intermediate

“…The yeast haploid strains used in this study are listed in Table 1. The S. cerevisiae strains with a ⌺1278b background were the WT strain L5685 supplied by G. Fink (Massachusetts Institute of Technology, Cambridge) and the mpr-disrupted strain LD1014 (2). Strains L5685Dput1 and L5685Dput2 were constructed from strain L5685.…”

“…Although one amino acid change at position 85 occurred between MPR1 and MPR2, both genes are expressed in S. cerevisiae and play similar roles in AZC resistance (1). Gene expression in Escherichia coli and enzymatic analysis showed that MPR1 encodes an AZC acetyltransferase, by which proline itself and other proline analogues are not acetylated (2). The MPR1-encoded protein (Mpr1) is a member of the N-acetyltransferase superfamily (3).…”

“…We believe that Mpr1 converts AZC into N-acetyl AZC (Fig. 1A), and consequently that N-acetyl AZC does not replace proline during the biosynthesis of protein (2).…”

Distribution ofL-Azetidine-2-carboxylateN-Acetyltransferase in Yeast

“…There are several possible explanations for the AZC resistance of the cells: (i) the cells do not incorporate AZC, suggesting that they possess a different type of amino acid transporter than S. cerevisiae; (ii) the cells accumulate large quantities of L-proline, which dilutes the effect of AZC; 7) and (iii) the cells degrade or modify AZC directly. The hitherto known AZC-resistant strains S. cerevisiae, S. paradoxus, and S. pombe use the third of these mechanisms, because modification of AZC by Mpr1 in these strains eventually converts AZC into Nacetyl-AZC for detoxification, 2,4,5) but, the mechanisms used by the newly identified strains (Table 1) are currently unknown.…”

“…1). 2) Homologous genes (Spa MPR1 and ppr1 þ ) that encode similar acetyltransferases have been identified in S. paradoxus, an S. cerevisiae-complex species, 4) and in the fission yeast Schizosaccharomyces pombe, 5) respectively. Moreover, genomic PCR analysis has revealed that most S. cerevisiae-complex species have sequences highly homologous to that of MPR1.…”