title: Enzymatic improvement of mitochondrial thiol oxidase Erv1 for oxidized glutathione fermentation by Saccharomyces cerevisiae.
abstract: BACKGROUND
  Oxidized glutathione (GSSG) is the preferred form for industrial mass production of glutathione due to its high stability compared with reduced glutathione (GSH). In our previous study, over-expression of the mitochondrial thiol oxidase ERV1 gene was the most effective for high GSSG production in Saccharomyces cerevisiae cells among three types of different thiol oxidase genes.
  
  
  RESULTS
  We improved Erv1 enzyme activity for oxidation of GSH and revealed that S32 and N34 residues are critical for the oxidation. Five engineered Erv1 variant proteins containing S32 and/or N34 replacements exhibited 1.7- to 2.4-fold higher in vitro GSH oxidation activity than that of parental Erv1, whereas the oxidation activities of these variants for γ-glutamylcysteine were comparable. According to three-dimensional structures of Erv1 and protein stability assays, S32 and N34 residues interact with nearby residues through hydrogen bonding and greatly contribute to protein stability. These results suggest that increased flexibility by amino acid replacements around the active center decrease inhibitory effects on GSH oxidation. Over-expressions of mutant genes coding these Erv1 variants also increased GSSG and consequently total glutathione production in S. cerevisiae cells. Over-expression of the ERV1S32Agene was the most effective for GSSG production in S. cerevisiae cells among the parent and other mutant genes, and it increased GSSG production about 1.5-fold compared to that of the parental ERV1 gene.
  
  
  CONCLUSIONS
  This is the first study demonstrating the pivotal effects of S32 and N34 residues to high GSH oxidation activity of Erv1. Furthermore, in vivo validity of Erv1 variants containing these S32 and N34 replacements were also demonstrated. This study indicates potentials of Erv1 for high GSSG production.
authors.affiliation: Graduate School of Science, Technology and Innovation, Kobe University, 1-1 Rokkodaicho, Nada-ku, Kobe, Hyogo, 657-8501, Japan.
authors.forename: Jyumpei
authors.initials: J
authors.lastname: Kobayashi
authors.affiliation: Graduate School of Science, Technology and Innovation, Kobe University, 1-1 Rokkodaicho, Nada-ku, Kobe, Hyogo, 657-8501, Japan.
authors.forename: Daisuke
authors.initials: D
authors.lastname: Sasaki
authors.affiliation: Graduate School of Nutritional and Environmental Sciences, University of Shizuoka, 52-1 Yada, Suruga-ku, Shizuoka, 422-8526, Japan.
authors.forename: Kiyotaka Y
authors.initials: KY
authors.lastname: Hara
authors.affiliation: Graduate School of Science, Technology and Innovation, Kobe University, 1-1 Rokkodaicho, Nada-ku, Kobe, Hyogo, 657-8501, Japan.
authors.forename: Tomohisa
authors.initials: T
authors.lastname: Hasunuma
authors.affiliation: Graduate School of Science, Technology and Innovation, Kobe University, 1-1 Rokkodaicho, Nada-ku, Kobe, Hyogo, 657-8501, Japan. akondo@kobe-u.ac.jp.|Department of Chemical Science and Engineering, Graduate School of Engineering, Kobe University, 1-1 Rokkodaicho, Nada-ku, Kobe, Hyogo, 657-8501, Japan. akondo@kobe-u.ac.jp.|RIKEN Center for Sustainable Resource Science, 1-7-22 Suehiro-cho, Tsurumi-ku, Yokohama, Kanagawa, 230-0045, Japan. akondo@kobe-u.ac.jp.
authors.forename: Akihiko
authors.identifier: http://orcid.org/0000-0002-2740-0384
authors.initials: A
authors.lastname: Kondo
mesh_terms: D004151:Dipeptides / Q000378:metabolism; D005285:Fermentation; D005978:Glutathione / Q000378:metabolism*; D060847:Metabolic Engineering / Q000379:methods; D008928:Mitochondria / Q000201:enzymology*; D024101:Mitochondrial Proteins / Q000235:genetics* / Q000378:metabolism*; D008958:Models, Molecular; D009154:Mutation; D010084:Oxidation-Reduction; D050862:Oxidoreductases Acting on Sulfur Group Donors / Q000235:genetics* / Q000378:metabolism*; D012441:Saccharomyces cerevisiae / Q000201:enzymology / Q000235:genetics / Q000378:metabolism*; D029701:Saccharomyces cerevisiae Proteins / Q000235:genetics* / Q000378:metabolism*
chemical_list: D004151:Dipeptides; D024101:Mitochondrial Proteins; D029701:Saccharomyces cerevisiae Proteins; D050862:Oxidoreductases Acting on Sulfur Group Donors; C073790:ERV1 protein, S cerevisiae; D005978:Glutathione; C017341:gamma-glutamylcysteine
keywords: Erv1; Glutathione; Mia40; Saccharomyces cerevisiae; Thiol oxidase