title: 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.
authors.affiliation: Laboratory of Physiological Chemistry, Université catholique de Louvain and Christian de Duve Institute of Cellular Pathology (ICP), Avenue Hippocrate 75, B-1200 Brussels, Belgium.
authors.forename: Younes
authors.initials: Y
authors.lastname: Achouri
authors.forename: Gaëtane
authors.initials: G
authors.lastname: Noël
authors.forename: Didier
authors.initials: D
authors.lastname: Vertommen
authors.forename: Mark H
authors.initials: MH
authors.lastname: Rider
authors.forename: Maria
authors.initials: M
authors.lastname: Veiga-Da-Cunha
authors.forename: Emile
authors.initials: E
authors.lastname: Van Schaftingen
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D002847:Chromatography, Agarose / Q000379:methods; D004983:Ethanolamines / Q000378:metabolism; D005977:Glutarates / Q000378:metabolism*; D006801:Humans; D007668:Kidney / Q000166:cytology / Q000196:embryology / Q000201:enzymology; D008099:Liver / Q000737:chemistry / Q000201:enzymology; D008110:Liver Extracts / Q000737:chemistry; D051379:Mice; D008969:Molecular Sequence Data; D010088:Oxidoreductases / Q000096:biosynthesis / Q000737:chemistry / Q000378:metabolism*; D051381:Rats; D029701:Saccharomyces cerevisiae Proteins / Q000737:chemistry; D016415:Sequence Alignment / Q000379:methods
chemical_list: D004983:Ethanolamines; D005977:Glutarates; D008110:Liver Extracts; D029701:Saccharomyces cerevisiae Proteins; C019417:alpha-hydroxyglutarate; D010088:Oxidoreductases; C007369:2-diethylaminoethanol