Biochemical and molecular characterization of a succinate semialdehyde dehydrogenase involved in the catabolism of 4-hydroxybutyric acid inRalstonia eutropha

Abstract

A succinate semialdehyde dehydrogenase gene (gabD) was identified to be disrupted in a transposon-induced mutant of Ralstonia eutropha exhibiting the phenotype 4-hydroxybutyric acid-leaky. The native gabD gene was cloned by colony hybridization using a homologous gabD-specific DNA probe. DNA sequencing revealed an 1452-bp open reading frame, and the deduced amino acid sequence showed strong similarities to NADP⁺-dependent succinate semialdehyde dehydrogenases from Escherichia coli, Rhizobium sp., Homo sapiens and Rattus norvegicus. The gabD gene was heterologously expressed in a recombinant E. coli strain harboring plasmid pSK::EE6.8. Similar to the molecular organization of the gab cluster in E. coli, additional genes encoding enzymes for the degradation of γ-aminobutyrate are closely related to gabD in R. eutropha. Enzymatic studies indicated the existence of a second NAD⁺-dependent succinate semialdehyde dehydrogenase in R. eutropha.