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.