Succinic semialdehyde dehydrogenase (SSADH) is involved in the final degradation step of the inhibitory neurotransmitter gamma-aminobutyric acid by converting succinic semialdehyde to succinic acid in the mitochondrial matrix. SSADH deficiency, a rare autosomal recessive disease, exhibits variable clinical phenotypes, including psychomotor retardation, language delay, behaviour disturbance and convulsions. Here, we present crystal structures of both the oxidized and reduced forms of human SSADH. Interestingly, the structures show that the catalytic loop of the enzyme undergoes large structural changes depending on the redox status of the environment, which is mediated by a reversible disulphide bond formation between a catalytic Cys340 and an adjacent Cys342 residues located on the loop. Subsequent in vivo and in vitro studies reveal that the 'dynamic catalytic loop' confers a response to reactive oxygen species and changes in redox status, indicating that the redox-switch modulation could be a physiological control mechanism of human SSADH. Structural basis for the substrate specificity of the enzyme and the impact of known missense point mutations associated with the disease pathogenesis are presented as well.
The succinic semialdehyde dehydrogenase gene (SSADH; EC 1.2.1.24) from human brain was cloned and overexpressed in Escherichia coli. Based on SDS-PAGE, the apparent molecular mass of subunit was 54 kDa, in good agreement with the theoretical size. The purified SSADH appears to be a tetramer of identical subunits. The specific activity of the recombinant protein was 1.82 micromol NADH formedmin(-1)mg(-1) and the optimal pH was found to be 8.5. The Michaelis constants K(m) for succinic semialdehyde and NAD(+) were 6.3 and 125 microM, respectively. Initial velocity studies show NADH to be a competitive inhibitor with respect to NAD(+), but to be non-competitive inhibitor with respect to succinic semialdehyde. The overexpression of SSADH in E. coli and one-step purification of the highly active SSADH will facilitate further biochemical studies on this enzyme. In addition, an mRNA master dot-blot for multiple human tissues provided a complete map of the tissue distribution for SSADH. The major sites of SSADH expression are liver, skeletal muscle, kidney, and brain. The data indicate that mRNA expression of SSADH is ubiquitous, but highly regulated at the level of transcription in a tissue-specific manner.
J. Biol. Chem. 270, 461-467 (1995)[PubMed:7814412]
Three rat brain cDNA clones approximately 3500, 1465, and 1135 base pairs in length encoding succinic semialdehyde dehydrogenase (SSADH; EC 1.2.1.24) were isolated from two cDNA libraries using a polymerase chain reaction derived probe. Restriction mapping and DNA sequencing revealed that the 3.5-kilobase clone contained an 84-base pair (28 amino acid) insert in the coding region. Composite clones encoding mature SSADH predicted proteins with 488 amino acids (M(r) = 52,188) when including the insert and 460 amino acids (M(r) = 48,854) without the insert. The cDNA clones were confirmed by expression of enzyme activity in bacteria and protein sequence data obtained from sequencing purified rat brain SSADH. Two human liver SSADH cDNA clones of 1091 and 899 base pairs were also isolated. Human and rat SSADH share 83 and 91% identity in nucleotide and protein sequence, respectively. Northern blot analysis revealed two differentially expressed SSADH transcripts of approximately 2.0 and 6.0 kilobases in both rat and human tissues. Human genomic Southern blots indicate that the two SSADH transcripts are encoded by a greater than 20-kilobase single copy gene. Mammalian SSADH contains significant homology to bacterial NADP(+)-succinic semialdehyde dehydrogenase (EC 1.2.1.16) and conserved regions of general aldehyde dehydrogenases (EC 1.2.1.3), suggesting it is a member of the aldehyde dehydrogenase superfamily of proteins.
Succinic semialdehyde dehydrogenase (SSADH) deficiency, a rare metabolic disorder of 4-aminobutyric acid degradation, has been identified in approximately 150 patients. Affected individuals accumulate large quantities of 4-hydroxybutyric acid, a compound with a wide range of neuropharmacological activities, in physiological fluids. As a first step in beginning an investigation of the molecular genetics of SSADH deficiency, we have utilized SSADH cDNA and genomic sequences to identify two point mutations in the SSADH genes derived from four patients. These mutations, identified by standard methods of reverse transcription, PCR, dideoxy-chain termination, and cycle sequencing, alter highly conserved sequences at intron/exon boundaries and prevent the RNA-splicing apparatus from properly recognizing the normal splice junction. Each family segregated a mutation in a different splice site, resulting in exon skipping and, in one case, a frameshift and premature termination and, in the other case, an in-frame deletion in the resulting protein. Family members, including parents and siblings of these patients, were shown to be heterozygotes for the splicing abnormality, providing additional evidence for autosomal recessive inheritance. Our results provide the first evidence that 4-hydroxybutyric aciduria, resulting from SSADH deficiency, is the result of genetic defects in the human SSADH gene.
The process whose specific outcome is the progression of the central nervous system over time, from its formation to the mature structure. The central nervous system is the core nervous system that serves an integrating and coordinating function. In vertebrates it consists of the brain, spinal cord and spinal nerves. In those invertebrates with a central nervous system it typically consists of a brain, cerebral ganglia and a nerve cord.
Evidence
1:
Inferred from Mutant PhenotypeUniProtKB
Succinic semialdehyde dehydrogenase (SSADH) deficiency, a rare metabolic disorder of 4-aminobutyric acid degradation, has been identified in approximately 150 patients. Affected individuals accumulate large quantities of 4-hydroxybutyric acid, a compound with a wide range of neuropharmacological activities, in physiological fluids. As a first step in beginning an investigation of the molecular genetics of SSADH deficiency, we have utilized SSADH cDNA and genomic sequences to identify two point mutations in the SSADH genes derived from four patients. These mutations, identified by standard methods of reverse transcription, PCR, dideoxy-chain termination, and cycle sequencing, alter highly conserved sequences at intron/exon boundaries and prevent the RNA-splicing apparatus from properly recognizing the normal splice junction. Each family segregated a mutation in a different splice site, resulting in exon skipping and, in one case, a frameshift and premature termination and, in the other case, an in-frame deletion in the resulting protein. Family members, including parents and siblings of these patients, were shown to be heterozygotes for the splicing abnormality, providing additional evidence for autosomal recessive inheritance. Our results provide the first evidence that 4-hydroxybutyric aciduria, resulting from SSADH deficiency, is the result of genetic defects in the human SSADH gene.
The chemical reactions and pathways involving galactosylceramides, any compound formed by the replacement of the glycosidic hydroxyl group of a cyclic form of galactose by a ceramide group.
The chemical reactions and pathways resulting in the breakdown of gamma-aminobutyric acid (GABA, 4-aminobutyrate), an amino acid which acts as a neurotransmitter in some organisms.
Succinic semialdehyde dehydrogenase (SSADH) deficiency, a rare metabolic disorder of 4-aminobutyric acid degradation, has been identified in approximately 150 patients. Affected individuals accumulate large quantities of 4-hydroxybutyric acid, a compound with a wide range of neuropharmacological activities, in physiological fluids. As a first step in beginning an investigation of the molecular genetics of SSADH deficiency, we have utilized SSADH cDNA and genomic sequences to identify two point mutations in the SSADH genes derived from four patients. These mutations, identified by standard methods of reverse transcription, PCR, dideoxy-chain termination, and cycle sequencing, alter highly conserved sequences at intron/exon boundaries and prevent the RNA-splicing apparatus from properly recognizing the normal splice junction. Each family segregated a mutation in a different splice site, resulting in exon skipping and, in one case, a frameshift and premature termination and, in the other case, an in-frame deletion in the resulting protein. Family members, including parents and siblings of these patients, were shown to be heterozygotes for the splicing abnormality, providing additional evidence for autosomal recessive inheritance. Our results provide the first evidence that 4-hydroxybutyric aciduria, resulting from SSADH deficiency, is the result of genetic defects in the human SSADH gene.
Evidence
3:
Inferred from Mutant PhenotypeUniProtKB
Succinic semialdehyde dehydrogenase (SSADH) deficiency is a rare hereditary disorder of the CNS catabolism of gamma-aminobutyric acid (GABA), leading to accumulation of the metabolite 4-hydroxybutyrate (GHB). Here the authors report on 1.5 and 3.0 T proton MR spectroscopy in a patient with SSADH deficiency. A characteristic pattern with clearly elevated GABA levels and traces of GHB was found in both the white and the gray matter of the brain. In vivo spectroscopy may be useful for diagnosis and monitoring SSADH deficiency.
The chemical reactions and pathways involving glucose, the aldohexose gluco-hexose. D-glucose is dextrorotatory and is sometimes known as dextrose; it is an important source of energy for living organisms and is found free as well as combined in homo- and hetero-oligosaccharides and polysaccharides.
The chemical reactions and pathways involving glutathione, the tripeptide glutamylcysteinylglycine, which acts as a coenzyme for some enzymes and as an antioxidant in the protection of sulfhydryl groups in enzymes and other proteins; it has a specific role in the reduction of hydrogen peroxide (H2O2) and oxidized ascorbate, and it participates in the gamma-glutamyl cycle.
The chemical reactions and pathways involving glycerophospholipids, any derivative of glycerophosphate that contains at least one O-acyl, O-alkyl, or O-alkenyl group attached to the glycerol residue.
The chemical reactions and pathways resulting in the breakdown of any of a group of substances that are released on excitation from the axon terminal of a presynaptic neuron of the central or peripheral nervous system and travel across the synaptic cleft to either excite or inhibit the target cell.
The succinic semialdehyde dehydrogenase gene (SSADH; EC 1.2.1.24) from human brain was cloned and overexpressed in Escherichia coli. Based on SDS-PAGE, the apparent molecular mass of subunit was 54 kDa, in good agreement with the theoretical size. The purified SSADH appears to be a tetramer of identical subunits. The specific activity of the recombinant protein was 1.82 micromol NADH formedmin(-1)mg(-1) and the optimal pH was found to be 8.5. The Michaelis constants K(m) for succinic semialdehyde and NAD(+) were 6.3 and 125 microM, respectively. Initial velocity studies show NADH to be a competitive inhibitor with respect to NAD(+), but to be non-competitive inhibitor with respect to succinic semialdehyde. The overexpression of SSADH in E. coli and one-step purification of the highly active SSADH will facilitate further biochemical studies on this enzyme. In addition, an mRNA master dot-blot for multiple human tissues provided a complete map of the tissue distribution for SSADH. The major sites of SSADH expression are liver, skeletal muscle, kidney, and brain. The data indicate that mRNA expression of SSADH is ubiquitous, but highly regulated at the level of transcription in a tissue-specific manner.
A process in which a series of electron carriers operate together to transfer electrons from donors such as NADH and FADH2 to any of several different terminal electron acceptors to generate a transmembrane electrochemical gradient.
The chemical reactions and pathways involving succinate, also known as butanedioate or ethane dicarboxylate, the dianion of succinic acid. Succinate is an important intermediate in metabolism and a component of the TCA cycle.
ISSOrtholog Curator
Enzymatic activity
This protein acts as an enzyme. It is known to catalyze the following reaction
Succinic semialdehyde dehydrogenase (SSADH) is involved in the final degradation step of the inhibitory neurotransmitter gamma-aminobutyric acid by converting succinic semialdehyde to succinic acid in the mitochondrial matrix. SSADH deficiency, a rare autosomal recessive disease, exhibits variable clinical phenotypes, including psychomotor retardation, language delay, behaviour disturbance and convulsions. Here, we present crystal structures of both the oxidized and reduced forms of human SSADH. Interestingly, the structures show that the catalytic loop of the enzyme undergoes large structural changes depending on the redox status of the environment, which is mediated by a reversible disulphide bond formation between a catalytic Cys340 and an adjacent Cys342 residues located on the loop. Subsequent in vivo and in vitro studies reveal that the 'dynamic catalytic loop' confers a response to reactive oxygen species and changes in redox status, indicating that the redox-switch modulation could be a physiological control mechanism of human SSADH. Structural basis for the substrate specificity of the enzyme and the impact of known missense point mutations associated with the disease pathogenesis are presented as well.
A reference proteome is a set of protein sequences derived from a complete proteome which constitutes a defined standard for a particular user community. Reference proteomes are manually defined according to a number of criteria. They cover the proteomes of well- studied model organisms and other proteomes of interest for biomedical and biotechnological research. Reference proteomes have been selected to provide broad coverage of the tree of life, and constitute a representative cross-section of the taxonomic diversity to be found within UniProtKB.
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