Plant succinic semialdehyde dehydrogenase. Cloning, purification, localization in mitochondria, and regulation by adenine nucleotides.
Succinic semialdehyde dehydrogenase (SSADH) is one of three enzymes constituting the gamma-aminobutyric acid shunt. We have cloned the cDNA for SSADH from Arabidopsis, which we designated SSADH1. SSADH1 cDNA encodes a protein of 528 amino acids (56 kD) with high similarity to SSADH from Escherichia coli and human (>59% identity). A sequence similar to a mitochondrial protease cleavage site is present 33 amino acids from the N terminus, indicating that the mature mitochondrial protein may contain 495 amino acids (53 kD). The native recombinant enzyme and the plant mitochondrial protein have a tetrameric molecular mass of 197 kD. Fractionation of plant mitochondria revealed its localization in the matrix. The purified recombinant enzyme showed maximal activity at pH 9.0 to 9.5, was specific for succinic semialdehyde (K(0.5) = 15 microM), and exclusively used NAD+ as a cofactor (Km = 130 +/- 77 microM). NADH was a competitive inhibitor with respect to NAD+ (Ki = 122 +/- 86 microM). AMP, ADP, and ATP inhibited the activity of SSADH (Ki = 2.5-8 mM). The mechanism of inhibition was competitive for AMP, noncompetitive for ATP, and mixed competitive for ADP with respect to NAD+. Plant SSADH may be responsive to mitochondrial energy charge and reducing potential in controlling metabolism of gamma-aminobutyric acid.
Busch
K B
KB
Department of Plant Sciences, The Weizmann Institute of Science, Rehovot 76100, Israel.
Fromm
H
H
D000227:Adenine Nucleotides / Q000378:metabolism*; D000255:Adenosine Triphosphate / Q000378:metabolism; D000445:Aldehyde Oxidoreductases / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism*; D000595:Amino Acid Sequence; D000818:Animals; D017360:Arabidopsis / Q000201:enzymology*; D016384:Consensus Sequence; D004926:Escherichia coli / Q000201:enzymology; D006801:Humans; D007700:Kinetics; D008322:Mammals; D008928:Mitochondria / Q000201:enzymology*; D008969:Molecular Sequence Data; D009243:NAD / Q000378:metabolism; D011994:Recombinant Proteins / Q000302:isolation & purification / Q000378:metabolism; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D050644:Succinate-Semialdehyde Dehydrogenase
D000227:Adenine Nucleotides; D011994:Recombinant Proteins; D009243:NAD; D000255:Adenosine Triphosphate; D000445:Aldehyde Oxidoreductases; D050644:Succinate-Semialdehyde Dehydrogenase