Methionine Metabolism Alters Oxidative Stress Resistance via the Pentose Phosphate Pathway

Abstract

Nutrient uptake and metabolism have a significant impact on the way cells respond to stress. The amino acid methionine is, in particular, a key player in the oxidative stress response, and acting as a reactive oxygen species scavenger, methionine is implicated in caloric restriction phenotypes and aging. We here provide evidence that some effects of methionine in stress situations are indirect and caused by altered activity of the nicotinamide adenine dinucleotide phosphate (NADPH) producing oxidative part of the pentose phosphate pathway (PPP). In Saccharomyces cerevisiae, both methionine prototrophic (MET15) and auxotrophic (met15Δ) cells supplemented with methionine showed an increase in PPP metabolite concentrations downstream of the NADPH producing enzyme, 6-phosphogluconate dehydrogenase. Proteomics revealed this enzyme to also increase in expression compared to methionine self-synthesizing cells. Oxidant tolerance was increased in cells preincubated with methionine; however, this effect was abolished when flux through the oxidative PPP was prevented by deletion of its rate limiting enzyme, ZWF1. Stress resistance phenotypes that follow methionine supplementation hence involve the oxidative PPP. Effects of methionine on oxidative metabolism, stress signaling, and aging have thus to be seen in the context of an altered activity of this NADP reducing pathway.

FIG. 1.

Methionine supplementation alters intracellular PPP activity. (A) Methionine exposed wild-type (MET15) and met15Δ cells have increased concentrations of PPP metabolites (n = 3). Absolute metabolite concentrations are shown in Table 1. Glycolysis: G6P, glucose 6-phosphate; F6P, fructose 6-phosphate; F1,6BP, fructose 1,6-bisphosphate; DHAP, dihydroxyacetone phosphate; G3P, glyceraldehyde 3-phosphate; 3PG, 3-phosphoglycerate; 2PG, 2-phosphoglycerate; PEP, phosphoenolpyruvate; Pyr, pyruvate. Pentose phosphate pathway: 6PG, 6-phosphogluconate; Rl5P, ribulose 5-phosphate; R5P, ribose 5-phosphate; X5P, xylulose 5-phosphate; S7P, sedoheptulose 7-phosphate; E4P, erythrose 4-phosphate. (B) Top: Reaction scheme of 6-phosphogluconate (6PG) to ribulose 5-phosphate (Rl5P) catalyzed by 6-phosphogluconate dehydrogenase (6PGDH), yielding NADPH. 6PG and Rl5P are highlighted in green and 6PGDH in brown, here and in (A). Bottom: The expression level of 6PGDH in wild-type (MET15) as well as in methionine supplemented wild-type and met15Δ cells, as determined by SWATH-MS. Bottom left: Reconstruction of SWATH-MS chromatographic spectra in Skyline; shown are two transitions of the representative 6PGDH peptide DYFGAHTFR. Bottom center: Expression level (fold change to wild type) of 6PGDH. c.p.s., counts per second. n = 3, error bars, ± SD. (C) Increased resistance of wild-type cells (ZWF1) to diamide upon methionine supplementation. This phenotype is lost in cells deleted for the rate-limiting oxidative PPP enzyme G6PDH (zwf1Δ). PPP, pentose phosphate pathway. G6PDH, glucose 6-phosphate dehydrogenase. To see this illustration in color, the reader is referred to the web version of this article at www.liebertpub.com/ars