Identification of a dehydrogenase acting on D-2-hydroxyglutarate

Abstract

Extracts of frozen rat liver were found to catalyse the formation of 3H2O from DL-2-hydroxy[2-3H]glutarate. Three peaks of enzyme activities were observed on separation by chromatography on DEAE-Sepharose. The first and second peaks corresponded to an enzyme acting on L-2-hydroxyglutarate and the third peak corresponded to an enzyme acting on D-2-hydroxyglutarate, as indicated by competitive inhibition of the detritiation of the racemic radioactive compound by the unlabelled L- and D-isomers respectively. The enzyme acting on the D-form was further characterized. It was independent of NAD or NADP and it converted D-2-hydroxyglutarate into a-ketoglutarate, transferring electrons to artificial electron acceptors. It also oxidized D-lactate, D-malate and meso-tartrate and was stimulated by Zn2+, Co2+ and Mn2+, but not by Mg2+ or Ca2+. Subcellular fractionation indicated that it was present in the mitochondrial fraction. The enzyme was further purified by chromatography on Blue Trisacryl and phenyl-Sepharose, up to a stage where only a few bands were still visible by SDS/PAGE. Among the four candidate polypeptides that were identified by MS, one corresponded to a predicted mitochondrial protein homologous with FAD-dependent D-lactate dehydrogenase. The corresponding human protein was expressed in HEK-293 cells and it was shown to catalyse the detritiation of DL-2-hydroxy[2-3H]glutarate with similar properties as the purified rat enzyme.

Figure 1. Separation of the enzymes acting on D- and L-2-hydroxyglutarate by chromatography on DEAE-Sepharose

Activity (•) was measured with radiolabelled DL-2-hydroxy[2-³H]glutarate on 25 μl of each fraction. The absorbance A₂₈₀ was also measured (▵). The oblique line indicates the NaCl gradient.

Figure 2. Co-elution of the enzyme utilizing radiolabelled D-2-hydroxyglutarate with a dehydrogenase acting on D-2-hydroxyglutarate, D-malate and D-lactate

A portion of the elution profile of a DEAE-Sepharose column is shown similar to the one shown in Figure 1. The activity was determined by the radiochemical assay in the presence of 2 μM unlabelled D-2-hydroxyglutarate (▴) or through the oxidation of DCIP in the presence of 1 mM D-2-hydroxyglutarate (▵), D-lactate (□) or D-malate (○). The approx. 50-fold difference in activity between the two conditions is due to a combination of several factors such as the use of subsaturating concentrations of substrate in the radiochemical assay, a primary isotopic effect and the stimulation exerted by electron acceptors (see the Discussion section).

Figure 3. Sensitivity of D-2-hydroxyglutarate dehydrogenase to several 2-hydroxy carboxylic acids

The partially purified enzyme was assayed through the release of tritium from DL-2-hydroxy[2-³H]glutarate in the presence of 2 μM unlabelled D-2-hydroxyglutarate, 50 μM CoCl₂ and the indicated concentrations of D-malate (○), mesotartrate (▵), D-lactate (□), L-malate (•), L-tartrate (▴), L-lactate (▪) and D-tartrate (▵).

Figure 4. Effect of bivalent cations and EGTA on the activity of D-2-hydroxyglutarate dehydrogenase

Radiochemical assay was performed on the enzyme in the presence of 2 μM unlabelled D-2-hydroxyglutarate and the indicated concentrations of CoCl₂ (○), MnCl₂ (▵), MgCl₂ (□), ZnCl₂ (▿), CaCl₂ (⋄) and EGTA (•).

Figure 5. Purification by chromatography on phenyl-Sepharose

Upper panel: a preparation (9 mg of protein) purified by chromatography on DEAE-Sepharose and Blue Trisacryl was applied on to a phenyl-Sepharose column and eluted with a decreasing gradient of NaCl and an increasing gradient of ethylene glycol. Fractions of 30 ml (fraction 1) and 2 ml (fractions 2–13) were collected. D-2-Hydroxyglutarate dehydrogenase (•) was measured by the radiochemical assay on 10 μl of each fraction. Lower panel: SDS/PAGE of the fractions. The indicated bands were found to co-elute with the activity both in the Blue Trisacryl (results not shown) and the phenyl-Sepharose columns. They were cut from the gel, digested with trypsin and their identity was determined by electrospray ionization–tandem MS.

Figure 6. Sequence alignment of human D-2-hydroxyglutarate dehydrogenase (HsHd) and mouse D-2-hydroxyglutarate dehydrogenase (MmHd) with S. cerevisiae actin-interacting protein 2 (CsD2) and human D-lactate dehydrogenase (HsLd)

The peptidic sequences identified in the rat protein are underlined in the mouse sequence. Except for the last peptide (NVLGYSKPPVAVK in the rat sequence), they are identical in the sequences from both species. The predicted cleavage site for the mitochondrial presequence is double underlined.

Figure 7. Effect of overexpression of the putative human D-2-hydroxyglutarate on the detritiation of radiolabelled 2-hydroxyglutarate

Cells were transfected with a vector containing the ORF of the putative D-2-hydroxyglutarate dehydrogenase (D-OHGD) or with an empty vector (control). The enzyme activity was determined by the radiochemical assay in the presence of 2 μM unlabelled D-2-hydroxyglutarate; 50 μM CoCl₂ or ZnCl₂ was present where indicated. Results are the means±S.E.M. for four independent transfections.