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

Abstract

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.

Figure 1

Comparison of Arabidopsis SSADH1 with SSADH from bacteria and mammals. Alignment of the deduced amino acid sequences of the full-length SSADH from Arabidopsis (A), E. coli (E), and human (H) (Chambliss et al., 1998) (GenBank accession nos. AF117335, M8834, and Y11192, respectively), and a partial rat (R) SSADH (Chambliss et al., 1995) (GenBank accession no. P51650). Identical amino acids are boxed with a gray background. The ADH motifs discussed in the text are underlined. Arrows indicate the predicted cleavage sites of plant and human (Chambliss et al., 1998) SSADH by mitochondrial proteases.

Figure 2

Purification of recombinant Arabidopsis SSADH1. Coomassie Blue-stained SDS-PAGE of different chromatography fractions during purification of Arabidopsis SSADH1. Lane 1, Total soluble fraction; lane 2, pooled active fractions of the first Q-Sepharose; lane 3, pooled active fractions of the second Q-Sepharose; lane 4, pooled active fractions of the Blue Sepharose. Molecular mass markers are shown on the right.

Figure 3

Arabidopsis SSADH1 is a homotetramer under nondenaturing conditions. Molecular mass of the purified recombinant SSADH1 (rec.SSADH) was determined by gel filtration. Inset, Nondenaturing-PAGE separation of the purified recombinant SSADH1 (Rec.) and mitochondrial SSADH (Mit.) from potato tubers. Proteins were immunodetected with polyclonal antibodies raised against the purified recombinant Arabidopsis SSADH1 protein.

Figure 4

Detection of a 53-kD SSADH in the plant mitochondrial matrix. Matrix and membrane fractions of mitochondria from potato tubers (P) and Arabidopsis cell cultures (A), and a sample of the recombinant Arabidopsis SSADH1 (Rec. SSADH) were separated on SDS-PAGE and either stained with Coomassie Blue or tested with antibodies against Arabidopsis SSADH1 or prohibitin (Snedden and Fromm, 1997).

Figure 5

Control of SSADH1 activity by the NADH/NAD⁺ ratio. SSADH1 activity was determined in the presence of increasing NADH concentrations, while the NAD⁺ concentration was held constant at 500 μm. The SSA concentration was 100 μm.

Figure 6

Inhibition of SSADH1 by ADP determined by Hanes-plot (A) and secondary plots (B). The concentration of NAD⁺ varied between 0.01 and 0.3 mm, while the ADP concentration was held constant at the following values: 1 mm ADP (○); 2 mm ADP (▵); 3 mm ADP (▿); 4 mm ADP (⋄). The SSA concentration was 100 μm.

Figure 7

Inhibition of SSADH1 by ATP determined by Hanes-plot (A) and secondary plots (B). The concentration of NAD⁺ varied between 0.05 and 0.5 mm, while the ATP concentration was held constant at the following values: 0 mm ATP (□); 1 mm ATP (○); 2 mm ATP (▵); 3 mm ATP (▿); 5 mm ATP (⋄). The SSA concentration was 100 μm.

Figure 8

Model for the regulation of the GABA shunt in plants. The three enzymes of the GABA shunt, GAD (cytosolic), GABA-T (mitochondrial), and SSADH (mitochondrial) are in bold letters. Dashed lines, Effectors; solid lines, substrates and products. Phosphoryl., Phosphorylation.