Molecular mechanism of ethanol fermentation inhibition via protein tyrosine nitration of pyruvate decarboxylase by reactive nitrogen species in yeast

Abstract

Protein tyrosine nitration (PTN), in which tyrosine (Tyr) residues on proteins are converted into 3-nitrotyrosine (NT), is one of the post-translational modifications mediated by reactive nitrogen species (RNS). Many recent studies have reported that PTN contributed to signaling systems by altering the structures and/or functions of proteins. This study aimed to investigate connections between PTN and the inhibitory effect of nitrite-derived RNS on fermentation ability using the yeast Saccharomyces cerevisiae. The results indicated that RNS inhibited the ethanol production of yeast cells with increased intracellular pyruvate content. We also found that RNS decreased the activities of pyruvate decarboxylase (PDC) as a critical enzyme involved in ethanol production. Our proteomic analysis revealed that the main PDC isozyme Pdc1 underwent the PTN modification at Tyr38, Tyr157, and Tyr344. The biochemical analysis using the recombinant purified Pdc1 enzyme indicated that PTN at Tyr157 or Tyr344 significantly reduced the Pdc1 activity. Interestingly, the substitution of Tyr157 or Tyr344 to phenylalanine, which is no longer converted into NT, recovered the ethanol production under the RNS treatment conditions. These findings suggest that nitrite impairs the fermentation ability of yeast by inhibiting the Pdc1 activity via its PTN modification at Tyr157 and Tyr344 of Pdc1.

Figure 1

Effect of acidified nitrite on ethanol production and the related enzymatic activity. (A) Ethanol concentration in the culture medium, or (B) intracellular pyruvate content of yeast with or without acidified nitrite treatment was analyzed. Each content was normalized by OD₆₀₀ of the culture medium. (C) PDC, or (D) ADH activity in the cell-free extract from yeast exposed to acidified nitrite was measured. The values represent the averages and standard deviations from three independent experiments. **ρ < 0.01 by Student’s t-test.

Figure 2

PTN modification of Pdc1 in response to RNS. The PDC1-myc7His strain grown until the exponential phase was treated with acidified nitrite and then the extracted lysate was subjected to pull-down assay, followed by western blot analysis with anti-NT or anti-myc antibody. The PVDF membrane was incubated with sodium dithionite before the treatment with anti-NT antibody as a negative control for the PTN analysis.

Figure 3

Effect of the PTN modification of Pdc1 on its enzymatic activity. (A) The enzymatic activity of recombinant WT-Pdc1 treated with the indicated concentration of ONOO^- was measured. The values are the averages and standard deviations from three independent experiments. (B) Immunoblotting of WT-Pdc1 treated with various concentrations of ONOO^- using anti-NT antibody was shown. The membrane was stained with Ponceau S to confirm the unified protein loaded. (C) Incorporation of NT into the recombinant Pdc1 prepared the heterologous expression system in E. coli was examined by western blotting with anti-NT antibody. The staining with Ponceau S was used for a loading control. (D) The enzymatic activity of WT-, Tyr38NT-, Tyr157NT-, or Tyr344NT-Pdc1 was measured. The values represent the averages and standard deviations from three independent experiments. **ρ < 0.01 by Student’s t-test.

Figure 4

Ethanol production of yeast cells Pdc1 variants resistant to the PTN modification. (A) Ethanol contents in the culture medium of yeast producing Pdc1-myc7His with Tyr157Phe or Tyr344Phe substitutions in the presence or absence of acidified nitrite. The values represent the averages and standard deviations from three independent experiments. **ρ < 0.01 by Student’s t-test. (B) Protein extracts from the cells prepared same as (A) were analyzed by western blotting with anti-NT antibody. The pdc1Δ ura3Δ cells harboring an empty vector pRS416 was used as a negative control.

Figure 5

Three-dimensional structure and amino acid sequence conservation of Pdc1. (A, B) The crystal structures of Pdc1 from S. cerevisiae with the PDB ID code of 2VK1 were shown with the molecular surface of protein. Oxygen or nitrogen atom is colored by red or blue. The values nearby dotted lines indicate the distances between two atoms. (A) Local structure around Tyr157 was exhibited with Gly66, Tyr157, and Met187 in a stick model. Carbon atoms were colored by cyan, yellow, or green in Tyr157, Gly66 and Met187, or the other residues, respectively. The side chain of Tyr157 is located close to Gly66 and Met187. (B) The structure surrounding Tyr344 was shown with Asn213, Pro214, Phe240, Tyr344, and Val347 in a stick model. Carbon atoms were colored by cyan in Tyr344, yellow in Asn213, Pro214, Phe240, and Val347, or green in the other residues, respectively. (C) Amino acid sequences of Pdc1 from S. cerevisiae, Candida glabrata, Kluyveromyces lactis, Arabidopsis thaliana, and Oryza sativa were aligned using the sequence alignment tool Align in the Universal Protein Resource (UniProt) (https://www.uniprot. org/align/) and the part of alignment was shown. Tyr157 and Tyr344 in Pdc1 from S. cerevisiae and their corresponding residues in PDCs from the other species were highlighted by red boxes.