α-Hydroxyglutarate Oxidoreductase of Pseudomonas putida (original) (raw)

1969, Journal of Bacteriology

Oxidation of D-a-hydroxyglutarate to ct-ketoglutarate is catalyzed by D-ca-hydroxyglutarate oxidoreductase, an inducible membrane-bound enzyme of the electron transport particle [ETP; a comminuted cytoplasmic membrane preparation with enzymic properties and chemical composition resembling beef heart mitochondrial ETP (1)] of Pseudomonas putida P2 (P2-ETP). Treatment of P2-ETP with a nonionic detergent yields a preparation with the sedimentation characteristics of a soluble enzyme, but which retains an intact electron transport chain. Oxygen acts solely as a terminal electron acceptor and may be replaced by ferricyanide, 2,6dichlorophenol indophenol, or mammalian cytochrome c. The oxidoreductase is specific for the D-isomer (Km = 4.0 X 10-4 Mfor DL-a-hydroxyglutarate) and is distinct both from Land D-malate dehydrogenases. Spectral studies suggest that the carrier sequence is substrate-k flavine or nonheme iron-* cyt b-[cyt c]-* oxygen. Our interest in ca-hydroxyglutarate metabolism derived initially from isotope distribution patterns observed during pipecolate and at-aminoadipate oxidation by Pseudomonas putida P2 (Fig. 1). Both pipecolate (13) and a-aminoadipate (R. A. Hartline, Ph.D. Thesis, Univ. of California, San Francisco, 1965) give rise to glutamate, and carbon 6 of a-aminoadipate becomes carbon 5 of glutamate (Hartline, 1965). Although the intermediates between a-ketoadipate and glutamate are not known, it may be inferred that at least one oxidative reaction occurs. Symmetrical intermediates such as glutarate probably are not involved, although asymmetrical glutaryl derivatives such as glutaryl-CoA, glutarate semialdehyde, and a-hydroxyglutarate are likely intermediates. Although a-hydroxyglutarate is a likely intermediate in pipecolate and a-aminoadipate metabolism, attempts to demonstrate its formation have not succeeded (Hartline, 1965). We therefore asked whether an enzyme catalyzing conversion of a-hydroxyglutarate to a-ketoglutarate (reaction 6, Fig. 1) occurred in this organism and whether its synthesis was induced by compounds known to induce formation of enzymes of pipecolate catabolism. This paper reports the properties of Pseudomonas putida ct-hydroxyglutarate oxidoreductase and discusses its possible metabolic role. MATERIALS AND METHODS Chemicals. Materials obtained commercially included: nicotinamide adenine dinucleotide (NAD), nicotinamide adenine dinucleotide phosphate (NADP), hemoglobin (type I, bovine), and horse heart cytochrome c (type II, 67% pure) from Sigma Chemical Co., St. Louis, Mo.; flavine mononucleotide (FMN) and flavine adenine dinucleotide (FAD) from Calbiochem, Los Angeles, Calif.; D-and L-malate, L-glutamate, L-lysine, a-ketoglutarate, pyruvate, oxalacetate, and tris(hydroxymethyl)aminomethane (Tris)-hydrochloride from Nutritional Biochemicals Corp., Cleveland, Ohio; 2,4-dinitrophenylhydrazine and 2, 6-dichlorophenol indophenol from Eastman Organic Chemicals, Rochester, N.Y.; DL-pipecolic acid from Aldrich Chemical Co., Milwaukee, Wisc.;