Functional characterization of a Drosophila melanogaster succinic semialdehyde dehydrogenase and a non-specific aldehyde dehydrogenase

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• 作者：Boris Rothacker ; Thomas Ilg
• 关键词：Succinic semialdehyde dehydrogenase ; Aldehyde dehydrogenase ; Site-directed mutagenesis ; Drosophila melanogaster ; GABA catabolism
• 刊名：Insect Biochemistry and Molecular Biology
• 出版年：2008
• 期刊代码：80_09651748
• 类别：bio
• 出版时间：March 2008
• 卷：38
• 期：3
• 页码：354-366
• 文件大小：663 K

摘要

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_Ms) for the specific substrates succinic semialdehyde and NAD⁺ was 4.7 and 90.9 μM, respectively. For D. melanogaster MalE-ALDH the K_M of the putative in vivo substrate acetaldehyde was 0.9 μM while for NAD⁺, a K_M of 62.7 μM 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.