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

Karin B. Busch, Hillel Fromm

Research output: Contribution to journalArticlepeer-review

Abstract

Succinic semialdehyde dehydrogenase (SSADH) is one of three enzymes constituting the γ-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 coil 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 semiaidehyde (K0.5 = 15 μM), and exclusively used NAD+ as a cofactor (Km = 130 ± 77 μM). NADH was a competitive inhibitor with respect to NAD+ (K(i)= 122 ± 86 μM). AMP, ADP, and ATP inhibited the activity of SSADH (K(i) = 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 γ-aminobutyric acid.

Original languageEnglish
Pages (from-to)589-597
Number of pages9
JournalPlant Physiology
Volume121
Issue number2
DOIs
StatePublished - Oct 1999
Externally publishedYes

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