Cloning and expression of succinic semialdehyde reductase from human brain. Identity with aflatoxin B1 aldehyde reductase.

The neuromodulator gamma-hydroxybutyrate is synthesized in vivo from gamma-aminobutyrate by transamination to succinic semialdehyde and subsequent reduction of the aldehyde group. In human brain, succinic semialdehyde reductase is thought to be responsible for the conversion of succinic semialdehyde to gamma-hydroxybutyrate. In the present work, we cloned the cDNA coding for succinic semialdehyde reductase and expressed it in Escherichia coli. A data bank search indicated that the enzyme is identical with aflatoxin B1-aldehyde reductase, an enzyme implicated in the detoxification of xenobiotic carbonyl compounds. Structurally, succinic semialdehyde reductase thus belongs to the aldo-keto reductase superfamily. The recombinant protein was indistinguishable from native human brain succinic semialdehyde reductase by SDS/PAGE. In addition to succinic semialdehyde, it readily catalyzed the reduction 9,10-phenanthrene quinone, phenylglyoxal and 4-nitrobenzaldehyde, typical substrates of aflatoxin B1 aldehyde reductase. The results suggest multiple functions of succinic semialdehyde reductase/aflatoxin B1 aldehyde reductase in the biosynthesis of gamma-hydroxybutyrate and the detoxification of xenobiotic carbonyl compounds, respectively.     

A single succinic semialdehyde dehydrogenase is involved in 4-hydroxyphenylacetate and 4-aminobutyrate catabolism in Klebsiella pneumoniae M5a1

Abstract Klebsiella pneumoniae M5a1 grows readily on two compounds, 4-hydroxyphenylacetate and 4-aminobutyrate, whose catabolism produces succinic semialdehyde. A single succinic semialdehyde dehydrogenase was detected, native molecular weight 52000, that has NAD as the preferred cofactor and is induced by succinic semialdehyde functions in the oxidation of succinic semialdehyde during growth on both 4-hydroxyphenyl-acetate and 4-aminobutyrate. This contrasts with the situation for Escherichia coli and Pseudomonas putida where two distinct forms of succinic semialdehyde dehydrogenase have been observed.     

Purification and characterization of methylmalonate-semialdehyde dehydrogenase from rat liver. Identity to malonate-semialdehyde dehydrogenase

Methylmalonate semialdehyde dehydrogenase was purified from rat liver in order to define the distal portion of valine catabolism and related pathways in mammals. The purified enzyme is active with malonate semialdehyde and consumes both stereoisomers of methylmalonate semialdehyde, implicating a single semialdehyde dehydrogenase in the catabolism of valine, thymine, and compounds catabolized by way of beta-alanine. The oxidation of malonate and methylmalonate semialdehydes by this enzyme is CoA-dependent, the products being acetyl-CoA and propionyl-CoA, respectively. Expected activity with ethylmalonate semialdehyde as substrate was not found. Methylmalonate semialdehyde dehydrogenase was separated on DEAE-Sephacel into two isoforms which differ in mobility during nondenaturing polyacrylamide gel electrophoresis. The two forms are immunologically cross-reactive and exhibit the same N-terminal sequence, suggesting that one form is the product of the other. The monomer molecular mass, determined by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate, was 58 kDa. The native molecular mass, estimated by gel filtration, was 250 kDa, suggesting a tetrameric structure.     

An Escherichia coli mutant defective in the NAD-dependent succinate semialdehyde dehydrogenase

Escherichia coli mutants, unable to grown on 4-hydroxyphenylacetate, have been isolated and found to be defective in the NAD-dependent succinate semialdehyde dehydrogenase. When the mutants are grown with 4-aminobutyrate as sole nitrogen source an NAD-dependent succinate semialdehyde dehydrogenase seen in the parental strain is absent but, as in the parental strain, an NADP-dependent enzyme is induced. Growth of the mutants is inhibited by 4-hydroxyphenylacetate due to the accumulation of succinate semialdehyde. The mutants are more sensitive to inhibition by exogenous succinate semialdehyde than is the parental strain. Secondary mutants able to grow in the presence of 4-hydroxyphenylacetate but still unable to use it as sole carbon source were defective in early steps of 4-hydroxyphenylacetate catabolism and so did not form succinate semialdehyde from 4-hydroxyphenylacetate. The gene encoding the NAD-dependent succinate semialdehyde dehydrogenase of Escherichia coli K-12 was located at min 34.1 on the genetic map.     

ASSAY AND PROPERTIES OF 4-AMINOBUTYRIC-2-OXOGLUTARIC ACID TRANSAMINASE AND SUCCINIC SEMIALDEHYDE DEHYDROGENASE IN RAT BRAIN TISSUE

Abstract— The activity of 4-aminobutyric-2-oxoglutaric acid transaminase (GABA transaminase) and succinic semialdehyde dehydrogenase was determined in total rat brain homogenate. GABA transaminase activity was measured using a coupled enzyme method which utilizes endogenous succinic semialdehyde dehydrogenase to convert the formed succinic semialdehyde into succinate. The concurrently produced NADH was used as an estimate of GABA transaminase activity. This method could be used since it was shown that the dehydrogenase was about twice as active as the transaminase and because no significant accumulation of the intermediate succinic semialdehyde could be detected. GABA transaminase was inhibited by high ionic strength. In contrast NaCl decreased the apparent K _m and increased V _max for succinic semialdehyde dehydrogenase at high but not al low tissue concentrations. Increasing tissue concentration also resulted in a decrease of the apparent K _m, but did not change the V_max of succinic semialdehyde dehydrogenase and it is suggested that this enzyme can exist in two distinct states of aggregation, one with a high and one with a low affinity for succinic semialdehyde. The high affinity form of the enzyme is thought to prevent succinic semialdehyde from accumulation in the GABA transaminase assay. It is concluded that within certain limits the coupled enzyme method described here can be used for the assay of GABA transaminase activity.     

Succinic Semialdehyde as a Substrate for the Formation of γ-Aminobutyric Acid

The conversion of succinic semialdehyde into gamma-aminobutyric acid (GABA) by GABA-transaminase was measured in rat brain homogenate in the presence of different concentrations of the cosubstrate glutamate. The calculated kinetic parameters of succinic semialdehyde for GABA-transaminase were a limiting Km value of 168 microM and a limiting Vmax value of 38 mumol g-1 h-1. Combination with previously obtained data for the conversion of GABA into succinic semialdehyde revealed a kEq value of 0.04, indicating that equilibrium of GABA-transaminase is biased toward the formation of GABA. The increased formation of GABA in the presence of succinic semialdehyde was not due to an increased conversion of glutamate into GABA by glutamic acid decarboxylase. Therefore these results indicate that succinic semialdehyde can act as a precursor for GABA synthesis.     

Activation of Glutamate Apodecarboxylase by Succinic Semialdehyde and Pyridoxamine 5''-Phosphate

Glutamate apodecarboxylase was activated by incubation with succinic semialdehyde and pyridoxamine 5'-phosphate. Activation required both compounds and was highly selective for succinic semialdehyde. Of 18 analogs tested, only glyoxylate, pyruvate, oxaloacetate, and 2-oxoglutarate activated the apoenzyme significantly, but much higher concentrations of these compounds than of succinic semialdehyde were required. In the presence of pyridoxamine 5'-phosphate, the concentration of succinic semialdehyde giving half-maximal activation of apoenzyme was 7 microM. In contrast, the Ki for succinic semialdehyde as a competitive inhibitor of glutamate decarboxylation was 1.2 mM, indicating that apoenzyme with bound pyridoxamine 5'-phosphate has a much higher affinity for succinic semialdehyde than does holoenzyme. The concentration of pyridoxamine 5'-phosphate giving half-maximal activation was 17 microM, which is more than an order of magnitude greater than the corresponding value for pyridoxal 5'-phosphate.     

Production and Characterization of Monoclonal Antibodies to Bovine Brain Succinic Semialdehyde Reductase

Abstract: Monoclonal antibodies against bovine brain succinic semialdehyde reductase were produced and characterized. A total of nine monoclonal antibodies recognizing different epitopes of the enzyme were obtained, of which two inhibited the enzyme activity and three stained cytosol of rat spinal cord neurons as observed by indirect immunofluorescence microscopy. When unfractionated total proteins of bovine brain homogenate were separated by gel electrophoresis and immunoblotted, the antibodies specifically recognized a single protein band of 34 kDa, which comigrates with purified bovine succinic semialdehyde reductase. Using the antisuccinic semialdehyde reductase antibodies as probes, we investigated the cross-reactivities of brain succinic semialdehyde reductases from some mammalian and an avian species. The immunoreactive bands on western blots appeared to be the same in molecular mass—34 kDa—in all animal species tested, including humans. The result indicates that brain succinic semialdehyde reductase is distinct from other aldehyde reductases and that mammalian brains contain only one succinic semialdehyde reductase. Moreover, the enzymes among the species are immunologically very similar, although some properties of the enzymes reported previously were different from one another.     

Unraveling the function of paralogs of the aldehyde dehydrogenase super family from Sulfolobus solfataricus

Aldehyde dehydrogenases (ALDHs) have been well established in all three domains of life and were shown to play essential roles, e.g., in intermediary metabolism and detoxification. In the genome of Sulfolobus solfataricus, five paralogs of the aldehyde dehydrogenases superfamily were identified, however, so far only the non-phosphorylating glyceraldehyde-3-phosphate dehydrogenase (GAPN) and α-ketoglutaric semialdehyde dehydrogenase (α-KGSADH) have been characterized. Detailed biochemical analyses of the remaining three ALDHs revealed the presence of two succinic semialdehyde dehydrogenase (SSADH) isoenzymes catalyzing the NAD(P)⁺-dependent oxidation of succinic semialdehyde. Whereas SSO1629 (SSADH-I) is specific for NAD⁺, SSO1842 (SSADH-II) exhibits dual cosubstrate specificity (NAD(P)⁺). Physiological significant activity for both SSO-SSADHs was only detected with succinic semialdehyde and α-ketoglutarate semialdehyde. Bioinformatic reconstructions suggest a major function of both enzymes in γ-aminobutyrate, polyamine as well as nitrogen metabolism and they might additionally also function in pentose metabolism. Phylogenetic studies indicated a close relationship of SSO-SSALDHs to GAPNs and also a convergent evolution with the SSADHs from E. coli. Furthermore, for SSO1218, methylmalonate semialdehyde dehydrogenase (MSDH) activity was demonstrated. The enzyme catalyzes the NAD⁺- and CoA-dependent oxidation of methylmalonate semialdehyde, malonate semialdehyde as well as propionaldehyde (PA). For MSDH, a major function in the degradation of branched chain amino acids is proposed which is supported by the high sequence homology with characterized MSDHs from bacteria. This is the first report of MSDH as well as SSADH isoenzymes in Archaea.     

Human brain "high Km" aldehyde dehydrogenase: purification, characterization, and identification as NAD+ -dependent succinic semialdehyde dehydrogenase

NAD-dependent succinic semialdehyde dehydrogenase (EC 1.2.1.24) has been purified to homogeneity from human brain via ion-exchange chromatography and affinity chromatography employing Blue Sepharose and 5'-AMP Sepharose. Succinic semialdehyde dehydrogenase was never previously purified to homogeneity from any species; this preparation therefore allows the determination of its molecular weight, subunit molecular weight, subunit composition, isoelectric points, and substrate specificity for the first time. The enzyme is a tetramer of Mr230,000 to 245,000 and consists of weight-nonidentical subunits (Mr 61,000 and 63,000). On isoelectric focusing the enzyme separates into five bands with the following isoelectric points: 6.3, 6.6, 6.8, 6.95, and 7.15. Its substrates include glutaric semialdehyde, nitrobenzaldehyde, and short chain aliphatic aldehydes in addition to succinic semialdehyde which is the best substrate. The Km values for succinic semialdehyde, acetaldehyde, and propionaldehyde are 1,875, and 580 microM, respectively. The enzyme is inactive with 3,4-dihydroxyphenylacetaldehyde and indole-3-acetaldehyde as substrates. Its subcellular localization is in the mitochondrial fraction. Succinic semialdehyde dehydrogenase is sensitive to inhibition by disulfiram (a drug used therapeutically to produce alcohol aversion) resembling, in this respect, aldehyde dehydrogenase (EC 1.2.1.3). It does not, however, interact with the antibody developed in the rabbit vs aldehyde dehydrogenase, suggesting that the two enzymes are structurally distinct.     

Enzymatic preparation of radiolabeled succinic semialdehyde

[U-14C]Succinic semialdehyde was prepared with yields of 30-40% by oxidation of purified [U-14C]4-aminobutyric acid with commercially available bovine plasma monoamine oxidase. [U-14C]Succinic semialdehyde was purified by cation-exchange chromatography and quantified as the oxime and methoxime derivatives using liquid partition chromatography on silicic acid. The availability of [U-14C]succinic semialdehyde permits the reliable assay of succinic semialdehyde dehydrogenase in crude cell extracts of lymphocytes isolated from human blood, cultured human lymphoblasts, and other tissues where 4-aminobutyric acid metabolism is known to occur.     

Inhibition of Succinic Semialdehyde Dehydrogenase by N-Formylglycine

Abstract

N-formylglycine was developed as a dead-end inhibitor of the succinic semialdehyde dehydro-genase reaction. At 4mM, it inhibited Aspergillus niger succinic semialdehyde dehydrogenase by 40%. N-formylglycine is a reversible, complete inhibitor; the inhibition is competitive with succinic semialdehyde and uncompetitive with respect to NAD⁺ and the K_i values are 4.9 and 10.4 mM respectively. Potato succinic semialdehyde dehydrogenase is also inhibited by N-formylglycine to a similar extent, the nature of the inhibition being identical to that observed with the A. niger enzyme.     

Enzymatic and Immunologic Identification of Succinic Semialdehyde Dehydrogenase in Rat and Human Neural and Nonneural Tissues

Abstract: We have identified succinic semialdehyde dehydrogenase protein in rat and human neural and nonneural tissues. Tissue localization was determined by enzymatic assay and by western immunoblotting using polyclonal antibodies raised in rabbit against the purified rat brain protein. Although brain shows the highest level of succinic semialdehyde dehydrogenase activity, substantial amounts of enzyme activity occur in mammalian liver, pituitary, heart, and ovary. We further demonstrate the absence of succinic semialdehyde dehydrogenase enzyme activity and protein in brain, liver, and kidney tissue samples from an individual affected with succinic semialdehyde dehydrogenase deficiency, thereby verifying the specificity of our antibodies.     

Propionate assimilation in the flagellate Polytomella caeca. An inducible mitochondrial enzyme system

1. The assimilation of propionate by Polytomella caeca involves the beta-oxidation of this fatty acid. 2. Propionate-grown cells immediately oxidize propionate, beta-hydroxypropionate, malonic semialdehyde and acetate; acetate-grown cells oxidize propionate rapidly only after a lag of 2hr., and this adaptation of resting cells to propionate involves the formation of the enzymes of beta-oxidation. 3. The beta-hydroxypropionate dehydrogenase and malonic semialdehyde dehydrogenase activities of both propionate-grown and propionate-adapted cells are partly located in mitochondrial fractions. 4. Mitochondria isolated from propionate-grown cells, and also those from acetate-grown cells fully adapted to propionate, oxidize succinate, alpha-oxoglutarate, beta-hydroxypropionate and malonic semialdehyde; oxidation of these substrates is tightly coupled to the phosphorylation of ADP. 5. Mitochondria from acetate-grown cells exhibit ADP-dependent oxidation of succinate and alpha-oxoglutarate, but do not oxidize beta-hydroxypropionate or malonic semialdehyde. Mitochondria isolated from acetate-grown cells adapted to propionate for 5hr. slowly oxidize beta-hydroxypropionate and malonic semialdehyde, but no tightly coupled phosphorylation is detectable. 6. Two of the inducible enzymes of propionate oxidation are located within the NAD-impermeable barrier and appear to be membrane-bound. 7. The formation of the inducible enzymes is inhibited by cycloheximide and actinomycin D, but not by chloramphenicol.     

Novel beta -hydroxyacid dehydrogenases in Escherichia coli and Haemophilus influenzae

Our laboratory has previously reported a structurally and mechanistically related family of beta-hydroxyacid dehydrogenases with significant homology to beta-hydroxyisobutyrate dehydrogenase. A large number of the members of this family are hypothetical proteins of bacterial origin with unknown identity in terms of their substrate specificities and metabolic roles. The Escherichia coli beta-hydroxyacid dehydrogenase homologue corresponding to the locus was cloned and expressed with a 6-histidine tag for specific purification. The purified recombinant protein very specifically catalyzed the NAD(+)-dependent oxidation of d-glycerate and the NADH-dependent reduction of tartronate semialdehyde, identifying this protein as a tartronate semialdehyde reductase. Further evidence for identification as tartronate semialdehyde reductase is the observation that the coding region for this protein is directly preceded by genes coding for hydroxypyruvate isomerase and glyoxylate carboligase, two enzymes that synthesize tartronate semialdehyde, producing an operon clearly designed for d-glycerate biosynthesis from tartronate semialdehyde. The single beta-hydroxyacid dehydrogenase homologue from Haemophilus influenzae was also cloned, expressed, and purified with a 6-histidine tag. This protein also catalyzed the NAD(+)-dependent oxidation of d-glycerate but was significantly more efficient in the oxidation of four-carbon beta-hydroxyacids like d-hydroxybutyrate and d-threonine. This enzyme differs from all the presently known beta-hydroxybutyrate dehydrogenases which are well established members of the short chain dehydrogenase/reductase superfamily.     

A mitochondrial NADP+-dependent reductase related to the 4-aminobutyrate shunt. Purification, characterization, and mechanism

Succinic semialdehyde reductase, a NADP+-dependent enzyme, was purified from whole pig brain homogenates. The enzyme preparation migrates as a single protein and activity band on analytical gel electrophoresis. Succinic semialdehyde reductase (Mr 110,000) catalyzes the reduction of succinic semialdehyde to 4-hydroxybutyrate. The equilibrium constant of the reaction is Keq = 5.8 X 10(7) M-1 at pH 7 and 25 degrees C. The inhibition kinetic patterns obtained when 4-hydroxybutyrate or substrate analogs are used as inhibitors of the reaction catalyzed by the reductase are consistent with an ordered sequential mechanism, in which the coenzyme NADPH adds to the enzyme before the aldehyde substrate. A specific aldehyde reductase was also purified to homogeneity from brain mitochondria preparations. Its catalytic properties are identical to those of the enzyme isolated from whole brain homogenates. It is postulated that two enzymes, i.e. a NAD+-dependent dehydrogenase and a NADP+-dependent reductase, participate in the metabolism of succinic semialdehyde in the mitochondria matrix.     

Purification and properties of rat brain succinic semialdehyde dehydrogenase.

Succinic semialdehyde dehydrogenase from rat brain has been purified to electrophoretic homogeneity. It has a molecular weight of about 140, 000 and is composed of two apparently identical subunits. The reaction catalized by the pure protein is entirely dependent on endogenous --SH groups. The Kim (limits) for NAD and succinic semialdehyde are 2 X 10(-5) M and 1 X 10(-4) M respectively at the optimum pH of 8.6. Inhibition studies show that the reaction mechanism is a compulsory ordered on where NAD binds first followed by succinic semialdehyde.     

PURIFICATION FROM HUMAN BRAIN AND SOME PROPERTIES OF TWO NADPH-LINKED ALDEHYDE REDUCTASES WHICH REDUCE SUCCINIC SEMIALDEHYDE TO 4-HYDROXYBUTYRATE

Abstract— Two NADPH-linked aldehyde reductases (alcohol:NADP⁺oxidoreductase, EC 1.1.1.2) capable of reducing succinic semialdehyde to the anaesthetic Chydroxybutyrate have been purified from human brain to electrophoretic homogeneity. The first of these enzymes, which is typical of its category, is not specific for succinic semialdehyde and can reduce some aromatic aldehydes at a high rate. It is a monomer of molecular weight about 45,000 and is strongly inhibited by various hypnotics and anticonvulsants. The second enzyme is, in contrast, fairly specific for succinic semialdehyde. It is a dimer of molecular weight about 90,000 and is not inhibited by the hypnotics and anticonvulsants which inhibit the first enzyme. It is thus different from previously described aldehyde reductases from human brain.     

Purification and properties of two succinic semialdehyde dehydrogenases from Klebsiella pneumoniae

Two forms of succinic semialdehyde dehydrogenase have been isolated in Klebsiella pneumoniae M5a1. The two enzymes could be separated by filtration on Sephacryl S-300 and their apparent molecular weights were approx. 275,000 and 300,000. The large enzyme is specific for NADP. The smaller enzyme, which is induced by growth on 3-hydroxyphenylacetic acid, 4-hydroxyphenylacetic acid, 3,4-dihydroxyphenylacetic acid and gamma-aminobutyrate, has been purified to 96% homogeneity by affinity chromatography. The NAD-linked succinic semialdehyde dehydrogenase was able to use NADP as cofactor. Its induction is coordinated with 3- and 4-hydroxylase, the enzymes which initiate degradation of 3- and 4-hydroxyphenylacetic acid. The NAD-linked form is also induced by exogenous succinic semialdehyde. The large enzyme is specific for NADP and has been isolated from a defective mutant which lacked the activity of the NAD-linked succinic semialdehyde dehydrogenase. Activity and stability conditions and true K m values for substrates and cosubstrates of the two enzymes were determined. Some aspects of the induction of the NAD-linked enzyme participating in the metabolism of 4-hydroxyphenylacetic and gamma-aminobutyrate were studied.