Laboratory of Applied Stress MicrobiologyLaboratory of Applied Stress Microbiology

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Research Projects

1. Novel stress-tolerant mechanisms of yeast cells and their applications for breeding of industrial yeast

2) N-Acetyltransferase Mpr1: Antioxidative mechanism, structural and functional analysis

The Mpr1 gene found in S. cerevisiaeS background strain encodes a novel N-acetyltransferase that detoxifies the Pro analogue azetidine-2-carboxylate (AZC). Many yeasts and fungi contain its homologous genes, suggesting that Mpr1 is widely present in eukaryotic microorganisms. Previously, we found that Mpr1 protects yeast cells by regulating ROS levels under oxidative stress conditions. So, we proposed that Mpr1 is a novel antioxidant enzyme that acetylates the unknown substrate involved in ROS generation. Mpr1 converts the Pro catabolism intermediate L-Δ1-pyrroline-5-carboxylic acid (P5C)/L-glutamate-γ-semialdehyde (GSA) into N-acetyl-GSA for arginine (Arg) synthesis, and more importantly, regulates ROS levels by acetylating P5C/GSA involved in ROS generation. Mpr1 was also found to convert the Pro analogue cis-4-hydroxy-L-proline (CHOP) into N-acetyl CHOP, which might be a new anticancer prodrug, instead of toxic CHOP. We recently determined the 3D structure to understand the catalytic reaction mechanism of this unique enzyme. Mpr1 protects yeast cells from oxidative stresses possibly by activating a novel L-arginine (Arg) biosynthesis. We currently try to identify the cellular substrate in the Arg synthetic pathway. We also engineer Mpr1 to enhance functions by the structure-based design. For example, the stable Mpr1 variant (Asn203Lys) was suggested to contributes to the construction of new industrial yeast strains with improved fermentation ability and diversity of taste and flavor.

  1. M. Ohashi, R. Nasuno, D. Watanabe and H, Takagi: Stable N-acetyltransferase Mpr1 improves ethanol productivity in the sake yeast Saccharomyces cerevisiae. J. Ind. Microbiol. Biotechnol., 46, 1039-1045 (2019).
  2. R. Nasuno, S. Hirase, S. Norifune, D. Watanabe, and H. Takagi: Structure-based molecular design for thermostabilization of N-acetyltransferase Mpr1 involved in a novel pathway of L-arginine synthesis in yeast. J. Biochem., 159, 271-277 (2016).
  3. R. Nasuno, Y. Hirano, T. Itoh, T. Hakoshima, T. Hibi and H. Takagi: Structural and functional analysis of the yeast N-acetyltransferase Mpr1 involved in oxidative stress tolerance via proline metabolism. Proc. Natl. Acad. Sci. USA, 110, 11821-11826 (2013).
  4. T. M. H. Bach and H. Takagi: Properties, metabolisms, and applications of L-proline analogues. Appl. Microbiol. Biotech., 97, 6623-6634 (2013).
  5. T. M. H. Bach, R. Hara, K. Kino, I. Ohtsu, N. Yoshida and H. Takagi: Microbial production of N-acetyl cis-4-hydroxy-L-proline by coexpression of the Rhizobium L-proline cis-4-hydroxylase and the yeast N-acetyltransferase Mpr1. Appl. Microbiol. Biotech., 97, 247-257 (2013).
  6. A. Nishimura, R. Nasuno and H. Takagi: The proline metabolism intermediate Δ1-pyrroline-5-carboxylate directly inhibits the mitochondrial respiration in budding yeast. FEBS Lett., 586, 2411-2416 (2012).
  7. T. M. H. Bach, T. Hibi, R. Nasuno, G. Matsuo, Y. Sasano and H. Takagi: Production of N-acetyl cis-4-hydroxy-L-proline by the yeast N-acetyltransferase Mpr1. J. Biosci. Bioeng., 114, 160-165 (2012).
  8. A. Nishimura, T. Kotani, Y. Sasano and H. Takagi: An antioxidative mechanism mediated by the yeast N-acetyltransferase Mpr1: Oxidative stress-induced arginine synthesis and its physiological role. FEMS Yeast Res., 10, 687-698 (2010).
  9. Y. Sasano*, S. Takahashi*, J. Shima and H. Takagi: Antioxidant N-acetyltransferase Mpr1/2 of industrial baker's yeast enhances fermentation ability after air-drying stress in bread dough. *These authors contributed equally to this work. Int. J. Food Microbiol., 138, 181-185 (2010).
  10. K. Ogawa-Mitsuhashi, K. Sagane, J. Kuromitsu, H. Takagi and K. Tsukahara: MPR1 as a novel selection marker in Saccharomyces cerevisiae. Yeast, 26, 587-593 (2009).
  11. T. Hibi, H. Yamamoto, G. Nakamura, and H. Takagi: Crystallization and preliminary crystallographic analysis on N-acetyltransferase Mpr1 from Saccharomyces cerevisiae. Acta Crystallogr., F65, 169-172 (2009).
  12. K. Iinoya, T. Kotani, Y. Sasano and H. Takagi: Engineering of the yeast antioxidant enzyme Mpr1 for enhanced activity and stability. Biotechnol. Bioeng., 103, 341-352 (2009).
  13. T. Kotani and H. Takagi: Identification of amino acid residues essential for the yeast N-acetyltransferase Mpr1 activity by site-directed mutagenesis. FEMS Yeast Res., 8, 607-614 (2008).
  14. M. Wada, K. Okabe, M. Kataoka, S. Shimizu, A. Yokota and H. Takagi: Distribution of L-azetidine-2-carboxylate N-acetyltransferase in yeast. Biosci. Biotech. Biochem., 72, 582-586 (2008).
  15. X. Du and H. Takagi: N-Acetyltransferase Mpr1 confers ethanol tolerance on Saccharomyces cerevisiae by reducing reactive oxygen species. Appl. Microbiol. Biotech., 75, 1343-1351 (2007).
  16. X. Du and H. Takagi: N-Acetyltransferase Mpr1 conferred freeze tolerance in Saccharomyces cerevisiae by reducing reactive oxygen species. J. Biochem., 138, 391-397 (2005).
  17. M. Nomura and H. Takagi: Role of the yeast acetyltransferase Mpr1 in oxidative stress: Regulation of oxygen reactive species caused by a toxic proline catabolism intermediate. Proc. Natl. Acad. Sci. USA, 101, 12616-12621 (2004).
  18. T. Iwaki, N. Tanaka, H. Takagi, Y. Giga-Hama and K. Takegawa: Characterization of end4+, a gene required for endocytosis in Schizosaccharomyces pombe. Yeast, 21, 867-881 (2004).
  19. X.-H. Zhang, H. Takagi and J. M. Widholm: Expression of a novel yeast gene that detoxifies the proline analog azetidine-2-carboxylate confers resistance during tobacco seed germination, callus and shoot formation. Plant Cell Rep., 22, 615-622 (2004).
  20. M. Nomura, S. Nakamori and H. Takagi: Characterization of novel acetyltransferases found in the budding and fission yeasts that detoxifies a proline analogue, azetidine-2-carboxylic acid. J. Biochem., 133, 67-74 (2003).
  21. Y. Kimura, S. Nakamori and H. Takagi: Polymorphism of the MPR1 gene required for toxic proline analogue resistance in the Saccharomyces cerevisiae complex species. Yeast, 19, 1437-1445 (2002).
  22. M. Shichiri, C. Hoshikawa, S. Nakamori and H. Takagi: A novel acetyltranferase found in Saccharomyces cerevisiae 1278b that detoxifies a proline analogue, azetidine-2-carboxylic acid. J. Biol. Chem., 276, 41998-42002 (2001).
  23. H. Takagi, M. Shichiri, M. Takemura, M. Mohri and S. Nakamori: Saccharomyces cerevisiae S1278b has novel gene of the N-acetyltransferase gene superfamily required for L-proline analogue resistance. J. Bacteriol., 182, 4249-4256 (2000).