Identification of a dehydrogenase acting on D-2-hydroxyglutarate.
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.
Achouri
Younes
Y
Laboratory of Physiological Chemistry, Université catholique de Louvain and Christian de Duve Institute of Cellular Pathology (ICP), Avenue Hippocrate 75, B-1200 Brussels, Belgium.
Noël
Gaëtane
G
Vertommen
Didier
D
Rider
Mark H
MH
Veiga-Da-Cunha
Maria
M
Van Schaftingen
Emile
E
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
D004983:Ethanolamines; D005977:Glutarates; D008110:Liver Extracts; D029701:Saccharomyces cerevisiae Proteins; C019417:alpha-hydroxyglutarate; D010088:Oxidoreductases; C007369:2-diethylaminoethanol