Publications

• Elucidation of the trigonelline degradation pathway reveals previously undescribed enzymes and metabolites.

  Perchat N., Saaidi P.L., Darii E., Pelle C., Petit J.L., Besnard-Gonnet M., de Berardinis V., Dupont M., Gimbernat A., Salanoubat M., Fischer C., Perret A.

  Trigonelline (TG; <i>N-</i>methylnicotinate) is a ubiquitous osmolyte. Although it is known that it can be degraded, the enzymes and metabolites have not been described so far. In this work, we challenged the laboratory model soil-borne, gram-negative bacterium <i>Acinetobacter baylyi</i> ADP1 (AD ... >> More

  Trigonelline (TG; <i>N-</i>methylnicotinate) is a ubiquitous osmolyte. Although it is known that it can be degraded, the enzymes and metabolites have not been described so far. In this work, we challenged the laboratory model soil-borne, gram-negative bacterium <i>Acinetobacter baylyi</i> ADP1 (ADP1) for its ability to grow on TG and we identified a cluster of catabolic, transporter, and regulatory genes. We dissected the pathway to the level of enzymes and metabolites, and proceeded to in vitro reconstruction of the complete pathway by six purified proteins. The four enzymatic steps that lead from TG to methylamine and succinate are described, and the structures of previously undescribed metabolites are provided. Unlike many aromatic compounds that undergo hydroxylation prior to ring cleavage, the first step of TG catabolism proceeds through direct cleavage of the C5-C6 bound, catalyzed by a flavin-dependent, two-component oxygenase, which yields (<i>Z</i>)-2-((<i>N-</i>methylformamido)methylene)-5-hydroxy-butyrolactone (MFMB). MFMB is then oxidized into (<i>E</i>)-2-((<i>N-</i>methylformamido) methylene) succinate (MFMS), which is split up by a hydrolase into carbon dioxide, methylamine, formic acid, and succinate semialdehyde (SSA). SSA eventually fuels up the TCA by means of an SSA dehydrogenase, assisted by a Conserved Hypothetical Protein. The cluster is conserved across marine, soil, and plant-associated bacteria. This emphasizes the role of TG as a ubiquitous nutrient for which an efficient microbial catabolic toolbox is available. << Less

  Proc. Natl. Acad. Sci. U.S.A. 115:E4358-E4367(2018) [PubMed] [EuropePMC]

  This publication is cited by 4 other entries.

• Final steps in the catabolism of nicotine.

  Chiribau C.B., Mihasan M., Ganas P., Igloi G.L., Artenie V., Brandsch R.

  New enzymes of nicotine catabolism instrumental in the detoxification of the tobacco alkaloid by Arthrobacter nicotinovorans pAO1 have been identified and characterized. Nicotine breakdown leads to the formation of nicotine blue from the hydroxylated pyridine ring and of gamma-N-methylaminobutyrat ... >> More

  New enzymes of nicotine catabolism instrumental in the detoxification of the tobacco alkaloid by Arthrobacter nicotinovorans pAO1 have been identified and characterized. Nicotine breakdown leads to the formation of nicotine blue from the hydroxylated pyridine ring and of gamma-N-methylaminobutyrate (CH(3)-4-aminobutyrate) from the pyrrolidine ring of the molecule. Surprisingly, two alternative pathways for the final steps in the catabolism of CH(3)-4-aminobutyrate could be identified. CH(3)-4-aminobutyrate may be demethylated to gamma-N-aminobutyrate by the recently identified gamma-N-methylaminobutyrate oxidase. In an alternative pathway, an amine oxidase with noncovalently bound FAD and of novel substrate specificity removed methylamine from CH(3)-4-aminobutyrate with the formation of succinic semialdehyde. Succinic semialdehyde was converted to succinate by a NADP(+)-dependent succinic semialdehyde dehydrogenase. Succinate may enter the citric acid cycle completing the catabolism of the pyrrolidine moiety of nicotine. Expression of the genes of these enzymes was dependent on the presence of nicotine in the growth medium. Thus, two enzymes of the nicotine regulon, gamma-N-methylaminobutyrate oxidase and amine oxidase share the same substrate. The K(m) of 2.5 mM and k(cat) of 1230 s(-1) for amine oxidase vs. K(m) of 140 microM and k(cat) of 800 s(-1) for gamma-N-methylaminobutyrate oxidase, determined in vitro with the purified recombinant enzymes, may suggest that demethylation predominates over deamination of CH(3)-4-aminobutyrate. However, bacteria grown on [(14)C]nicotine secreted [(14)C]methylamine into the medium, indicating that the pathway to succinate is active in vivo. << Less

  FEBS J. 273:1528-1536(2006) [PubMed] [EuropePMC]

  This publication is cited by 2 other entries.

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

  Busch K.B., Fromm H.

  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 Escherichi ... >> More

  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. << Less

  Plant Physiol. 121:589-597(1999) [PubMed] [EuropePMC]

• Computational prediction and experimental verification of the gene encoding the NAD+/NADP+-dependent succinate semialdehyde dehydrogenase in Escherichia coli.

  Fuhrer T., Chen L., Sauer U., Vitkup D.

  Although NAD(+)-dependent succinate semialdehyde dehydrogenase activity was first described in Escherichia coli more than 25 years ago, the responsible gene has remained elusive so far. As an experimental proof of concept for a gap-filling algorithm for metabolic networks developed earlier, we dem ... >> More

  Although NAD(+)-dependent succinate semialdehyde dehydrogenase activity was first described in Escherichia coli more than 25 years ago, the responsible gene has remained elusive so far. As an experimental proof of concept for a gap-filling algorithm for metabolic networks developed earlier, we demonstrate here that the E. coli gene yneI is responsible for this activity. Our biochemical results demonstrate that the yneI-encoded succinate semialdehyde dehydrogenase can use either NAD(+) or NADP(+) to oxidize succinate semialdehyde to succinate. The gene is induced by succinate semialdehyde, and expression data indicate that yneI plays a unique physiological role in the general nitrogen metabolism of E. coli. In particular, we demonstrate using mutant growth experiments that the yneI gene has an important, but not essential, role during growth on arginine and probably has an essential function during growth on putrescine as the nitrogen source. The NADP(+)-dependent succinate semialdehyde dehydrogenase activity encoded by the functional homolog gabD appears to be important for nitrogen metabolism under N limitation conditions. The yneI-encoded activity, in contrast, functions primarily as a valve to prevent toxic accumulation of succinate semialdehyde. Analysis of available genome sequences demonstrated that orthologs of both yneI and gabD are broadly distributed across phylogenetic space. << Less

  J. Bacteriol. 189:8073-8078(2007) [PubMed] [EuropePMC]

  This publication is cited by 1 other entry.

• Properties and functions of two succinic-semialdehyde dehydrogenases from Pseudomonas putida.

  Sanchez M., Alvarez M.A., Balana R., Garrido-Pertierra A.

  Two forms of succinic-semialdehyde dehydrogenase have been isolated in Pseudomonas putida. The two enzymes could be separated by filtration on Sephacryl S-300 and their apparent molecular weights were approx. 200,000 and 100,000. The smaller enzyme, which is induced by growth on 4-hydroxyphenylace ... >> More

  Two forms of succinic-semialdehyde dehydrogenase have been isolated in Pseudomonas putida. The two enzymes could be separated by filtration on Sephacryl S-300 and their apparent molecular weights were approx. 200,000 and 100,000. The smaller enzyme, which is induced by growth on 4-hydroxyphenylacetate, has been purified to 88% homogeneity by anion-exchange and affinity chromatography. Electrophoresis in sodium dodecyl sulphate gave rise to a molecular weight of 53,000, indicating that the native enzyme is dimeric. Under standard assay conditions this enzyme acts preferentially with NAD but reduces NADP at 9% of the rate observed for NAD. The large enzyme, which is dependent on NADP, is induced by growth on putrescine and its induction is highly coordinated with putrescine: 2-oxoglutarate transaminase, gamma-amino-butyraldehyde dehydrogenase and gamma-aminobutyrate: 2-oxoglutarate transaminase activities. Activity and stability conditions and true Km values for substrate and cosubstrates of the two enzymes were determined. << Less

  Biochim Biophys Acta 953:249-257(1988) [PubMed] [EuropePMC]

• A novel alpha-ketoglutaric semialdehyde dehydrogenase: evolutionary insight into an alternative pathway of bacterial L-arabinose metabolism.

  Watanabe S., Kodaki T., Makino K.

  Azospirillum brasilense possesses an alternative pathway of l-arabinose metabolism, which is different from the known bacterial and fungal pathways. In a previous paper (Watanabe, S., Kodaki, T., and Makino, K. (2006) J. Biol. Chem. 281, 2612-2623), we identified and characterized l-arabinose 1-de ... >> More

  Azospirillum brasilense possesses an alternative pathway of l-arabinose metabolism, which is different from the known bacterial and fungal pathways. In a previous paper (Watanabe, S., Kodaki, T., and Makino, K. (2006) J. Biol. Chem. 281, 2612-2623), we identified and characterized l-arabinose 1-dehydrogenase, which catalyzes the first reaction step in this pathway, and we cloned the corresponding gene. Here we focused on the fifth enzyme, alpha-ketoglutaric semialdehyde (alphaKGSA) dehydrogenase, catalyzing the conversion of alphaKGSA to alpha-ketoglutarate. alphaKGSA dehydrogenase was purified tentatively as a NAD(+)-preferring aldehyde dehydrogenase (ALDH) with high activity for glutaraldehyde. The gene encoding this enzyme was cloned and shown to be located on the genome of A. brasilense separately from a gene cluster containing the l-arabinose 1-dehydrogenase gene, in contrast with Burkholderia thailandensis in which both genes are located in the same gene cluster. Higher catalytic efficiency of ALDH was found with alphaKGSA and succinic semialdehyde among the tested aldehyde substrates. In zymogram staining analysis with the cell-free extract, a single active band was found at the same position as the purified enzyme. Furthermore, a disruptant of the gene did not grow on l-arabinose. These results indicated that this ALDH gene was the only gene of the NAD(+)-preferring alphaKGSA dehydrogenase in A. brasilense. In the phylogenetic tree of the ALDH family, alphaKGSA dehydrogenase from A. brasilense falls into the succinic semialdehyde dehydrogenase (SSALDH) subfamily. Several putative alphaKGSA dehydrogenases from other bacteria belong to a different ALDH subfamily from SSALDH, suggesting strongly that their substrate specificities for alphaKGSA are acquired independently during the evolutionary stage. This is the first evidence of unique "convergent evolution" in the ALDH family. << Less

  J. Biol. Chem. 281:28876-28888(2006) [PubMed] [EuropePMC]

  This publication is cited by 2 other entries.

• Purification and properties of rat brain succinic semialdehyde dehydrogenase.

  Cash C., Ciesielski L., Maitre M., Mandel P.

  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 ... >> More

  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. << Less

  Biochimie 59:257-268(1977) [PubMed] [EuropePMC]

• Kinetic studies with rat-brain succinic-semialdehyde dehydrogenase.

  Rivett A.J., Tipton K.F.

  A simple procedure is described that gives an approximately 100-fold purification of rat brain succinic-semialdehyde dehydrogenase with a high yield. The enzyme exhibits a relatively low Km value for succinic semialdehyde (2.5 microM) and is inhibited by high concentrations of that substrate in an ... >> More

  A simple procedure is described that gives an approximately 100-fold purification of rat brain succinic-semialdehyde dehydrogenase with a high yield. The enzyme exhibits a relatively low Km value for succinic semialdehyde (2.5 microM) and is inhibited by high concentrations of that substrate in an uncompetitive manner with respect to NAD+ (Ki = 150 microM). p-Hydroxybenzaldehyde was shown to give competitive inhibition with respect to succinic semialdehyde and uncompetitive inhibition with respect to NAD+. Initial rate studies in the presence of a fixed concentration of this inhibitor allowed a more accurate estimation of the kinetic parameters for the uninhibited reaction. The results of these studies, together with analysis of the dead-end inhibition by AMP and the effects of NAD+ and 3-acetylpyridine--adenine dinucleotide as alternative acceptors in the reaction, were consistent with the enzyme-catalysed reaction obeying a compulsory-order mechanism in which NAD+ was the first substrate to bind to the enzyme and NADH was the last product to dissociate from it. << Less

  Eur J Biochem 117:187-193(1981) [PubMed] [EuropePMC]