Plant succinic semialdehyde dehydrogenase. Cloning, purification, localization in mitochondria, and regulation by adenine nucleotides

Succinic semialdehyde dehydrogenase (SSADH) is one of three enzymes constit uting 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 prote ase 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 t etrameric molecular mass of 197 kD. Fractionation of plant mitochondria rev ealed its localization in the matrix. The purified recombinant enzyme showe d maximal activity at pH 9.0 to 9.5, was specific for succinic semialdehyde (K-0.5 = 15 mu M), and exclusively used NAD(+) as a cofactor (K-m = 130 +/ - 77 mu M). NADH was a competitive inhibitor with respect to NAD(+) (K-i = 122 +/- 86 mu M) AMP, ADP, and ATP inhibited the activity of SSADH (K-i = 2 .5-8 mM). The mechanism of inhibition was competitive for AMP, noncompetiti ve 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.