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

Citation
Kb. Busch et H. Fromm, Plant succinic semialdehyde dehydrogenase. Cloning, purification, localization in mitochondria, and regulation by adenine nucleotides, PLANT PHYSL, 121(2), 1999, pp. 589-597
Citations number
37
Categorie Soggetti
Plant Sciences","Animal & Plant Sciences
Journal title
PLANT PHYSIOLOGY
ISSN journal
00320889 → ACNP
Volume
121
Issue
2
Year of publication
1999
Pages
589 - 597
Database
ISI
SICI code
0032-0889(199910)121:2<589:PSSDCP>2.0.ZU;2-5
Abstract
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.