Saccharomyces cerevisiae Forms D-2-Hydroxyglutarate and Couples Its Degradation to D-Lactate Formation via a Cytosolic Transhydrogenase

Abstract

The D or L form of 2-hydroxyglutarate (2HG) accumulates in certain rare neurometabolic disorders, and high D-2-hydroxyglutarate (D-2HG) levels are also found in several types of cancer. Although 2HG has been detected in Saccharomyces cerevisiae, its metabolism in yeast has remained largely unexplored. Here, we show that S. cerevisiae actively forms the D enantiomer of 2HG. Accordingly, the S. cerevisiae genome encodes two homologs of the human D-2HG dehydrogenase: Dld2, which, as its human homolog, is a mitochondrial protein, and the cytosolic protein Dld3. Intriguingly, we found that a dld3Δ knock-out strain accumulates millimolar levels of D-2HG, whereas a dld2Δ knock-out strain displayed only very moderate increases in D-2HG. Recombinant Dld2 and Dld3, both currently annotated as D-lactate dehydrogenases, efficiently oxidized D-2HG to α-ketoglutarate. Depletion of D-lactate levels in the dld3Δ, but not in the dld2Δ mutant, led to the discovery of a new type of enzymatic activity, carried by Dld3, to convert D-2HG to α-ketoglutarate, namely an FAD-dependent transhydrogenase activity using pyruvate as a hydrogen acceptor. We also provide evidence that Ser3 and Ser33, which are primarily known for oxidizing 3-phosphoglycerate in the main serine biosynthesis pathway, in addition reduce α-ketoglutarate to D-2HG using NADH and represent major intracellular sources of D-2HG in yeast. Based on our observations, we propose that D-2HG is mainly formed and degraded in the cytosol of S. cerevisiae cells in a process that couples D-2HG metabolism to the shuttling of reducing equivalents from cytosolic NADH to the mitochondrial respiratory chain via the D-lactate dehydrogenase Dld1.

Keywords: 2-hydroxyglutarate; dehydrogenase; enzyme kinetics; flavoprotein; inborn error of metabolism; transhydrogenase; yeast metabolism.

FIGURE 1.

Sequence evolution of yeast d-2-hydroxyglutarate dehydrogenase candidate proteins. Phylogenetic tree obtained by Neighbor-Joining analysis of Dld2- and Dld3-like protein sequences of Hemiascomycetes yeasts and the fission yeast Schizosaccharomyces pombe (green background). Numbers represent the bootstrap values from 1000 replicates. The protein identifiers of the sequences used for generating the phylogenetic tree are indicated in parentheses. The protein sequences were all extracted from the NCBI protein database, except for those indicated by an asterisk (Saccharomyces paradoxus, Saccharomyces mikatae, and Saccharomyces uvarum), which were retrieved from the Saccharomyces Genome Database. The three main groups that are commonly distinguished within the Hemiascomycetes species are highlighted by colored backgrounds (red, Saccharomycetaceae; orange, CTG group; blue, Dipodascaceae). Within the Saccharomycetaceae group, two subgroups are further distinguished here as follows: the pre-duplication or protoploid species (light red background) and the post-duplication species (dark red background). The Whole Genome Duplication occurrence is indicated by a red star.

FIGURE 2.

2-Hydroxyglutarate levels in wild-type, dld1Δ, dld2Δ, and dld3Δ deletion strains. The indicated yeast strains were cultivated in minimal defined medium with 1% glucose, and intracellular metabolites were extracted at different stages of growth. A, left panel, 2HG was measured by an MRM-based LC-MS/MS method allowing for differential quantification of l-2HG and d-2HG. Only d-2HG concentrations are shown as l-2HG levels were below the quantification limit in all the samples measured. Right panel, total 2HG was quantified using an HRAM LC-MS method in which d-2HG and l-2HG cannot be distinguished. B, the latter method was also used to measure 2HG in wild-type or dld3Δ mutant strains transformed with an “empty vector” (P_GPD-ccdB) or a plasmid expressing DLD3 from a strong constitutive promoter (P_GPD-DLD3). For this rescue experiment, uracil was omitted from our standard cultivation medium for plasmid maintenance. Means and standard deviations of three biological replicates are shown. exp., exponential; stat., stationary.

FIGURE 3.

Growth and metabolic characterization of the wild-type and dld mutant strains. Cell concentration (A), extracellular glucose concentration (B), intra- and extracellular 2HG concentrations (C and D), and intra- and extracellular lactate concentrations (E and F) were measured at the indicated times during batch cultivations in minimal defined medium containing 1% glucose. For cell concentration and intracellular metabolite concentrations, means and standard deviations of three biological replicates are shown. Extracellular metabolite concentrations represent single measurements.

FIGURE 4.

SDS-PAGE and Western blot analyses of purified recombinant Dld2, Dld3, Ser3, and Ser33. Purified preparations of recombinant Dld2 (1.2 μg of protein), Dld3 (1.2 μg of protein), Ser3 (1.0 μg of protein), and Ser33 (0.7 μg of protein) used in this study were analyzed by SDS-PAGE using Coomassie Blue staining (A) and Western blotting using an antibody directed against the N-terminal polyhistidine tag fused to each of the recombinant proteins (B). Commercial BSA (Sigma), which had been added to the Dld3, Ser3, and Ser33 preparations for improved protein preservation during storage, was also loaded alone (1.2 μg of protein) in a control lane. BSA (66.43 kDa) is therefore also present in the Dld3 lane (0.12 μg) and in the Ser3 and Ser33 lanes (1.2 μg). The expected molecular weights of the recombinant proteins are 57.64 kDa for Dld2 (truncated MTS), 58.80 kDa for Dld3, and 54.77 kDa for Ser3 and Ser33. Only the upper band of the double band revealed by Coomassie staining for the Ser3 and Ser33 preparations was detected by the anti-His antibody, indicating that the lower band corresponds to a proteolytic cleavage product lacking the N-terminal His tag.

FIGURE 5.

Substrate specificity of the dehydrogenase activities of Dld2 and Dld3. The dehydrogenase activity of Dld2 (A) and Dld3 (B) was assayed in the presence of the artificial electron acceptor DCIP and of the indicated 2-hydroxyacids at a final concentration of 0.5 mm (black bars) or 5 mm (white bars). Control reactions without substrate were run for background correction. Dehydrogenase activities are represented relative to the activities measured in the presence of 0.5 or 5 mm d-2HG. For Dld2, dehydrogenase activities of 0.186 ± 0.003 and 0.20 ± 0.02 μmol·min⁻¹·mg protein⁻¹ were measured in the presence of 0.5 and 5 mm d-2HG, respectively. For Dld3, dehydrogenase activities of 4.0 ± 0.8 and 4.2 ± 0.5 μmol·min⁻¹·mg protein⁻¹ were measured in the presence of 0.5 and 5 mm d-2HG, respectively. The values shown are means ± S.D. of three independent replicates. The structures of the 2-hydroxyacids tested as substrates are shown in C.

FIGURE 6.

Spectral analysis of purified recombinant Dld2 and Dld3. A 1-ml mixture containing 20 mm Tris-HCl, pH 8.0, 5 μm ZnCl₂, and purified Dld2 or Dld3 (0.7 mg) was flushed for 5 min with N₂ in a quartz glass cuvette. The absorbance spectra were recorded at 30 °C for both Dld2 (A) and Dld3 (B). After addition of d-2HG at a final concentration of 200 μm, the change of absorbance at 378 and 450 nm was followed over time for Dld2 (C) and Dld3 (D) until no further decrease was observed.

FIGURE 7.

Effect of bivalent metal ions on the d-2HG dehydrogenase activity of Dld2 and Dld3. The d-2HG dehydrogenase activities of recombinant purified Dld2 (A) and Dld3 (B) were assayed spectrophotometrically in the absence or presence of 5 or 50 μm of the chloride salts of the indicated metal ions and with 500 μm d-2HG as substrate. The effect of EDTA at a final concentration of 1 mm was also tested. Control reactions without substrate were run for background correction. The values shown correspond to means ± S.D. from three independent replicates.

FIGURE 8.

Dld2 and Dld3 convert d-2HG into α-ketoglutarate in the presence of DCIP. A reaction mixture containing 50 mm Tris-HCl, pH 8.0, 120 μm DCIP, 100 μg/ml BSA, 5 μm ZnCl₂, and 50 μm d-2HG was incubated at 30 °C in the absence or presence of recombinant purified Dld2 (6.6 μg/ml) or Dld3 (0.25 μg/ml). Aliquots were heat-inactivated (3 min at 95 °C) after a reaction time of 0, 20, 40, or 60 min and analyzed with a targeted quantitative HRAM LC-MS method. Substrate and product of the enzymatic reaction were identified by comparison of the retention time (A) and the mass spectrum (B) with a mixed standard containing 50 μm d-2HG and 50 μm α-ketoglutarate. Exemplary extracted ion chromatograms and mass spectra are shown after a reaction time of 60 min for both proteins. Considering theoretical masses (82) of 145.01370 for α-ketoglutarate and 147.02935 for d-2HG, the error on the detected masses in the standard as well as in the experimental samples corresponded to 6–7 ppm. Presumably due to ion suppression effects induced by the artificial electron acceptor DCIP, absolute quantifications of d-2HG and α-ketoglutarate were not possible in these experiments. For this reason, peak areas (C) are shown instead of concentrations. The values represented correspond to single point measurements. α-KG, α-ketoglutarate; cps, counts/s; Enz, enzyme; RT, retention time; t, time.

FIGURE 9.

Dld3 can use pyruvate and oxaloacetate as electron acceptors and acts as a transhydrogenase. A reaction mixture containing 50 mm Tris-HCl, pH 8.0, 100 μg/ml BSA, 5 μm ZnCl₂, and either 100 μm d-2HG and 100 μm pyruvate (A) or 50 μm d-2HG and 100 μm oxaloacetate (B) was incubated at 30 °C in the absence or presence of recombinant purified Dld3. Aliquots were heat-inactivated at the indicated times and analyzed with a targeted quantitative HRAM LC-MS method. α-KG, α-ketoglutarate; Enz, enzyme; LAC, lactate; MAL, malate; OAA, oxaloacetate; PYR, pyruvate.

FIGURE 10.

2HG and serine levels in yeast strains overexpressing SER3 or SER33. SER3 and SER33 were overexpressed in wild-type and dld3Δ mutant strains from a low copy number plasmid under the control of the GPD promoter (P_GPD-SER3 and P_GPD-SER33, respectively). The overexpression strains were cultivated along with control strains transformed with an “empty vector” (P_GPD-ccdB) in minimal medium with 1% glucose and without uracil. Metabolites were extracted from yeast cells at different stages of growth and 2HG (A) and serine (B) concentrations were measured by HRAM LC-MS. Intracellular concentrations normalized to the concentrations measured in the respective control strains are shown. Values are means ± S.D. of three biological replicates. Intracellular 2HG levels of the controls were, in the wild-type background, 64 ± 5 and 64 ± 3 μm at early and mid-exponential phase, respectively, and in the dld3Δ mutant background 392 ± 28 and 584 ± 18 μm at early and mid-exponential phase, respectively. Intracellular serine levels of the controls were, in the wild-type background, 1438 ± 202 and 1382 ± 73 μm at early and mid-exponential phase, respectively, and in the dld3Δ mutant background, 1001 ± 28 and 1191 ± 61 μm at early and mid-exponential phase, respectively. exp., exponential.

FIGURE 11.

2HG and serine levels in yeast deletion strains of SER3 and/or SER33. Single knock-out mutants (ser3Δ and ser33Δ) and the wild-type control strain were cultivated in minimal defined medium with 1% glucose, while for the double knock-out strain (ser3Δser33Δ) and the wild-type control strain this medium was supplemented with serine and lysine at a final concentration of 240 mg/liter each. All the strains were isogenic to BY4741, except for the double knock-out strain, which was isogenic to BY4742. Metabolites were extracted at the indicated growth stages, and 2HG (A) and serine (B) concentrations were measured by targeted HRAM LC-MS. Intracellular concentrations normalized to the concentrations measured in the control wild-type strain are shown. Values are means ± S.D. of three biological replicates. The intracellular wild-type 2HG concentrations corresponded to 114 ± 10 μm (mid exp. phase) and 119 ± 3 μm (early stat. phase) without serine and lysine supplementation (A, left panel) and to 15 ± 2 μm (mid exp. phase) and 24 ± 4 μm (early stat. phase) with serine and lysine supplementation (A, right panel). The intracellular wild-type serine concentrations were 1191 ± 55 μm (mid exp. phase) and 1217 ± 73 μm (early stat. phase) without serine and lysine supplementation (B). exp., exponential; stat., stationary.

FIGURE 12.

Ser3 and Ser33 reduce α-ketoglutarate to 2HG. A reaction mixture containing 45 mm Hepes, pH 7.4, 1 mm DTT, 0.25 mm NADH, and 50 μm α-ketoglutarate was incubated at 30 °C in the absence or presence of recombinant purified Ser3 (A) or Ser33 (B). Aliquots were heat-inactivated at the indicated times and analyzed with a targeted quantitative HRAM LC-MS method. α-KG, α-ketoglutarate; Enz, enzyme.

FIGURE 13.

d-2HG formation and degradation in yeast. A metabolic network, including the d-2HG formation and degradation reactions identified in this study, is represented. We showed here that yeast produces the d enantiomer of 2-hydroxyglutarate, and we identified Dld3 as the major d-2HG-degrading enzyme in S. cerevisiae. Unlike in other organisms, d-2HG is degraded via a transhydrogenase reaction in yeast in which Dld3 oxidizes d-2HG to α-ketoglutarate in the first half-reaction and reduces pyruvate to d-lactate in the second half-reaction. d-2HG is produced by the S. cerevisiae phosphoglycerate dehydrogenases Ser3 and Ser33, which catalyze the conversion of α-ketoglutarate to d-2HG in addition to their known activity in the serine biosynthesis pathway. Although we found that Dld2 acts as a d-2HG dehydrogenase in vitro, deletion mutants of this gene displayed only a moderate increase in intracellular d-2HG levels if any, which, together with the subcellular localization information available for Dld2 (mitochondrial) and Dld3, Ser3, and Ser33 (cytosolic), indicates that d-2HG is mainly produced and degraded in the cytosol of yeast cells. The newly identified reactions seem to couple d-2HG degradation to the shuttling of reducing equivalents from cytosolic NADH to the mitochondrial respiratory chain via the d-lactate dehydrogenase Dld1. DLD3 expression is induced together with CIT2, ACO1, IDH1, and IDH2 by the retrograde transcription factors Rtg1 and Rtg3 under conditions of mitochondrial dysfunction to preserve intracellular α-ketoglutarate pools. Idh1 and Idh2, but also cytosolic Idp2, may be additional sources of d-2HG formation.