A Novel γ-Hydroxybutyrate Dehydrogenase

Abstract

In plants, ␥-aminobutyrate (GABA), a non-protein amino acid, accumulates rapidly in response to a variety of abiotic stresses such as oxygen deficiency. Under normoxia, GABA is catabolized to succinic semialdehyde and then to succinate with the latter reaction being catalyzed by succinic semialdehyde dehydrogenase (SSADH). Complementation of an SSADH-deficient yeast mutant with an Arabidopsis cDNA library enabled the identification of a novel cDNA (designated as AtGH-BDH for Arabidopsis thaliana ␥-hydroxybutyrate dehydrogenase), which encodes a 289-amino acid polypeptide containing an NADP-binding domain. Constitutive expression of AtGHBDH in the mutant yeast enabled growth on 20 mM GABA and significantly enhanced the cellular concentrations of ␥-hydroxybutyrate, the product of the GHDBH reaction. These data confirm that the cDNA encodes a polypeptide with GHBDH activity. Arabidopsis plants subjected to flooding-induced oxygen deficiency for up to 4 h possessed elevated concentrations of ␥-hydroxybutyrate as well as GABA and alanine. RNA expression analysis revealed that GHBDH transcription was not up-regulated by oxygen deficiency. These findings suggest that GHBDH activity is regulated by the supply of succinic semialdehyde or by redox balance. It is proposed that GHBDH and SSADH activities in plants are regulated in a complementary fashion and that GH-BDH and ␥-hydroxybutyrate function in oxidative stress tolerance.␥-Aminobutyrate (GABA) 1 is a four-carbon non-protein amino acid that is present in virtually all of the prokaryotic and eukaryotic organisms as a significant component of the free amino acid pool (1, 2). In bacteria, it is involved in carbon and nitrogen metabolism (3), whereas in mammals, it functions as an inhibitory neurotransmitter (4). The role of GABA in plants is uncertain; however, GABA accumulates rapidly in response to a variety of abiotic stresses such as oxygen deficiency or cold temperature (1, 2, 5, 6). These stresses initiate a signal transduction pathway in which increased cytosolic Ca 2ϩ stimulates Ca 2ϩ /calmodulin-dependent activity of the anabolic enzyme, glutamate decarboxylase (Fig. 1). Under normoxia, GABA is catabolized via GABA transaminase (GABA-T, EC 2.6.1.19) to succinic semialdehyde (SSA), which in turn is oxidized via an NAD-dependent succinic semialdehyde dehydrogenase (SSADH, EC 1.2.2.16) to succinate. Under oxygen deficiency, SSADH activity is probably restricted by increases in reducing potential and adenylate energy charge (7,8), thereby contributing to the accumulation of GABA.Research on bacterial and animal systems indicates the existence of an alternative pathway for SSA catabolism to ␥-hydroxybutyrate (GHB) that involves the enzyme ␥-hydroxybutyrate dehydrogenase (GHBDH, might also be designated as succinic semialdehyde reductase, EC 1.1.1.61) (GenBank TM accession numbers AJ250267, L21902, and AAC41425) (9, 10). Mamelak (11) reviewed evidence for elevated GHB levels in mammalian tissues in response to anoxia or excessive metabolic demand and suggested that ...

Characterization of Human DHRS6, an Orphan Short Chain Dehydrogenase/Reductase Enzyme

“…The similarities observed are not only found in the regular SDR sequence motifs defined earlier (6,9,16,17) but also extend especially into the substrate and active site region, pointing to essentially the same substrate specificities and mechanisms. The enzymes described in this report are thus clearly different from mammalian type 1 (mitochondrial) BDH (sharing about 20% sequence identities), mammalian 3-hydroxyisobutyrate dehydrogenases involved in valine catabolism (18,19) (sharing about 15% sequence identities), or the unrelated plant hydroxybutyrate dehydrogenases (20).…”

Evaluation of Metabolite Alteration under Flooding Stress in Soybeans

“…These results suggest that AlaAT activity in soybean under hypoxia would have only a limited role in the accumulation of Ala and indicated the presence of another mechanism responsible for the hypoxia-induced Ala accumulation. However, gamma-hydroxybutyrate (GHB) accumulation in anaerobically stressed plant tissues has been reported 7,49 . GHB was synthesized via succinic semialdehyde from GABA and catalyzed by GABA transaminase (GABA-T), which also synthesizes Ala from GABA in the GABA shunt pathway 7 ; Breitkreuz et al 7 also suggested that GABA-T is operative and that its reaction involves Ala accumulation under hypoxia in flooding stress.…”

“…However, gamma-hydroxybutyrate (GHB) accumulation in anaerobically stressed plant tissues has been reported 7,49 . GHB was synthesized via succinic semialdehyde from GABA and catalyzed by GABA transaminase (GABA-T), which also synthesizes Ala from GABA in the GABA shunt pathway 7 ; Breitkreuz et al 7 also suggested that GABA-T is operative and that its reaction involves Ala accumulation under hypoxia in flooding stress. Thus, the GABA shunt would be involved in Ala accumulation in response under flooding stress; Miyashita and Good 33 partially proved this using gad1 and gaba-t1 mutants of Arabidopsis.…”

AtGAT1, a High Affinity Transporter for γ-Aminobutyric Acid in Arabidopsis thaliana

“…As in other organisms, GABA is synthesized in plants primarily by decarboxylation of glutamate and degraded via succinic semialdehyde to succinate, a pathway that is also called the GABA shunt (12). Alternatively, succinic semialdehyde can be further catabolized to ␥-hydroxybutyrate (14). In plants GABA and the GABA shunt have been discussed as important for regulation of cytosolic pH, nitrogen storage and metabolism, protection against oxidative stress, development, and deterrence of insects (12,13,15).…”