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 ERV1 ^S32A gene 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.

Keywords: Erv1; Glutathione; Mia40; Saccharomyces cerevisiae; Thiol oxidase.

Fig. 1

Relative activities of Erv1 and its variants. a GSH was used as a substrate; b γ-GC was used as a substrate. Erv1 and variants were assayed for 10 min at 30 °C and pH 7.0 (potassium phosphate buffer) using 10 mM substrates. The 100% relative activities of Erv1 using GSH and γ-GC as substrates were 1.62 and 1.59 mU mg⁻¹, respectively. All assays were separately performed three times. Data are presented as the mean ± standard deviation (n = 3)

Fig. 2

Structural models of the area surrounding catalytic cysteine residues.  a Catalytic cysteine and its surrounding residues; b lateral view of the catalytic site; c, d surface around the active site. The structures were constructed from Erv1 variant protein of S. cerevisiae (PDB ID: 4E0I) by PyMOL software. Each subunit was separately rendered in green and pink. Oxygen, nitrogen, and sulfur atoms are rendered in red, blue, and yellow, respectively. Hydrogen bonds are shown as black dashed lines

Fig. 3

Temperature effect on enzyme activity. a Specific activities of Erv1, Erv1^S32A and Erv1^S32T; b specific activities of Erv1^N34A, Erv1^S32A/N34A and Erv1^S32T/N34A. Data are presented as the mean ± standard deviation (n = 3)

Fig. 4

Glutathione production by S. cerevisiae GCIΔGLR1 strains over-expressing ERV1 and its mutant genes. a Intracellular GSH content; b intracellular GSSG content; c intracellular GSH and GSSG content; d intracellular GSSG ratio to GSH and GSSG. All fermentation for glutathione production were separately performed three times. Data are presented as the mean ± standard deviation (n = 3). Asterisks indicate statistical significances determined by Student’s t test. One asterisk indicates a p value smaller than 0.05 (p < 0.05). Two asterisks indicate a p value smaller than 0.01 (p < 0.01)

Fig. 5

Lateral view of the area surrounding catalytic cysteine residues of Erv1 and its variants. a Erv1; b Erv1^S32A; c Erv1^S32T; d different rendering of Erv1 with solvents. The structures were constructed from Erv1 mutant protein of S. cerevisiae (PDB ID: 4E0I) (a–c) and the Erv1 core of S. cerevisiae (PDB ID: 3W4Y) (d) by PyMOL software. Each subunit was separately rendered in green and pink. Oxygen, nitrogen, and sulfur atoms are rendered in red, blue, and yellow, respectively. The hydrogen bond and water molecule are shown as a black dashed line and cyan sphere, respectively