Functional characterization of a Drosophila melanogaster succinic semialdehyde dehydrogenase and a non-specific aldehyde dehydrogenase.
The putative Drosophila (D.) melanogaster gene ortholog of mammalian succinic semialdehyde dehydrogenase (SSADH, EC1.2.1.24; NM_143151) that is involved in the degradation of the neurotransmitter GABA, and the putative D. melanogaster aldehyde dehydrogenase gene Aldh (NM_135441) were cloned and expressed as enzymatically active maltose binding protein (MalE) fusion products in Escherichia coli. The identities of the NM_143151 gene product as NAD+-dependent SSADH and of the Aldh gene product as NAD+-dependent non-specific aldehyde dehydrogenase (ALDH, EC1.2.1.3) were established by substrate specificity studies using 30 different aldehydes. In the case of D. melanogaster MalE-SSADH, the Michaelis constants (K(M)s) for the specific substrates succinic semialdehyde and NAD+ was 4.7 and 90.9 microM, respectively. For D. melanogaster MalE-ALDH the K(M) of the putative in vivo substrate acetaldehyde was 0.9 microM while for NAD+, a K(M) of 62.7 microM was determined. Site-directed mutagenesis studies on D. melanogaster MalE-SSADH suggest that cysteine 311 and glutamic acid 277 of this enzyme are likely candidates for the active site residues directly involved in catalysis.
Rothacker
Boris
B
Intervet Innovation GmbH, Zur Propstei, 55270 Schwabenheim, Germany.
Ilg
Thomas
T
D000444:Aldehyde Dehydrogenase / Q000737:chemistry* / Q000235:genetics; D000818:Animals; D001665:Binding Sites / Q000502:physiology; D004331:Drosophila melanogaster / Q000201:enzymology* / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics; D015870:Gene Expression; D007700:Kinetics; D016297:Mutagenesis, Site-Directed; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics; D013379:Substrate Specificity / Q000502:physiology; D050644:Succinate-Semialdehyde Dehydrogenase / Q000737:chemistry* / Q000235:genetics; D005680:gamma-Aminobutyric Acid / Q000235:genetics / Q000378:metabolism
D011993:Recombinant Fusion Proteins; D005680:gamma-Aminobutyric Acid; D050644:Succinate-Semialdehyde Dehydrogenase; D000444:Aldehyde Dehydrogenase