Publications

• Highly stable meso-diaminopimelate dehydrogenase from an Ureibacillus thermosphaericus strain A1 isolated from a Japanese compost: purification, characterization and sequencing.

  Akita H., Fujino Y., Doi K., Ohshima T.

  We screened various thermophiles for meso-diaminopimelate dehydrogenase (meso-DAPDH, EC 1.4.1.16), which catalyzes the NAD(P)-dependent oxidative deamination of meso-diaminopimelate, and found the enzyme in a thermophilic bacterium isolated from compost in Japan. The bacterium grew well aerobicall ... >> More

  We screened various thermophiles for meso-diaminopimelate dehydrogenase (meso-DAPDH, EC 1.4.1.16), which catalyzes the NAD(P)-dependent oxidative deamination of meso-diaminopimelate, and found the enzyme in a thermophilic bacterium isolated from compost in Japan. The bacterium grew well aerobically at around 55°C and was identified as Ureibacillus thermosphaericus strain A1. We purified the enzyme about 47-fold to homogeneity from crude cell extract using five successive purification steps. The molecular mass of the purified protein was about 80 kDa, and the molecule consists of a homodimer with the subunit molecular mass of about 40 kDa. The optimum pH and temperature for the catalytic activity of the enzyme are about 10.5 and 65°C, respectively. The enzyme is highly selective for meso-diaminopimelate as the electron donor, and NADP but not NAD can serve as the electron acceptor. The Km values for meso-diaminopimelate and NADP at 50°C and pH 10.5 are 1.6 mM and 0.13 mM, respectively. The nucleotide sequence of this meso-DAPDH gene encodes a 326-amino acid peptide. When the gene was cloned and overexpressed in Escherichia coli Rosetta (DE3), the specific activity in the crude extract of the recombinant cells was about 18.0-fold higher than in the extract from U. thermosphaericus strain A1. This made more rapid and simpler purification of the enzyme possible. << Less

  AMB Express 1:43-43(2011) [PubMed] [EuropePMC]

• Meso-alpha,epsilon-diaminopimelate D-dehydrogenase: distribution and the reaction product.

  Misono H., Togawa H., Yamamoto T., Soda K.

  A high activity of meso-alpha-epsilon-diaminopimelate dehydrogenase was found in extracts of Bacillus sphaericus, Brevibacterium sp., Corynebacterium glutamicum, and Proteus vulgaris among bacteria tested. B. sphaericus IFO 3525, in which the enzyme is most abundant, was chosen to study the enzyme ... >> More

  A high activity of meso-alpha-epsilon-diaminopimelate dehydrogenase was found in extracts of Bacillus sphaericus, Brevibacterium sp., Corynebacterium glutamicum, and Proteus vulgaris among bacteria tested. B. sphaericus IFO 3525, in which the enzyme is most abundant, was chosen to study the enzyme reaction. The enzyme was not induced by the addition of meso-alpha-epsilon-diaminopimelate to the growth medium. The reaction product was isolated and identified as alpha-amino-epsilon-ketopimelate by a comparison of the properties of its 2,4-dinitrophenylhydrazone with those of an authentic sample in silica gel thin-layer chromatography, absorption, infrared and proton nuclear magnetic resonance spectrometry, and elemental analyses. The alpha-amino-epsilon-ketopimelate formed enzymatically was decarboxylated by H2O2 to yield L-alpha-aminoadipate. This suggests that the amino group with D-configuration in the substrate is oxidatively deaminated; the enzyme is a D-amino acid dehydrogenase. L-alpha-Amino-epsilon-ketopimelate undergoes spontaneous dehydration to the cyclic delta1-piperideine-2,6-dicarboxylate. The enzyme reaction is reversible, and meso-alpha-epsilon-diaminopimelate was formed in the reductive amination of L-alpha-epsilon-ketopimelate. << Less

  J. Bacteriol. 137:22-27(1979) [PubMed] [EuropePMC]

  This publication is cited by 1 other entry.

• Substrate and inhibitor binding sites in Corynebacterium glutamicum diaminopimelate dehydrogenase.

  Scapin G., Cirilli M., Reddy S.G., Gao Y., Vederas J.C., Blanchard J.S.

  The three-dimensional structures of Corynebacterium glutamicum diaminopimelate dehydrogenase as a binary complex with the substrate meso-diaminopimelate (meso-DAP) and a ternary complex with NADP+ and an isoxazoline inhibitor [Abbot, S.D., Lane-Bell, P., Kanwar, P.S.S., and Vederas, J. C. (1994) J ... >> More

  The three-dimensional structures of Corynebacterium glutamicum diaminopimelate dehydrogenase as a binary complex with the substrate meso-diaminopimelate (meso-DAP) and a ternary complex with NADP+ and an isoxazoline inhibitor [Abbot, S.D., Lane-Bell, P., Kanwar, P.S.S., and Vederas, J. C. (1994) J. Am. Chem. Soc. 116, 6513-6520] have been solved and refined against X-ray diffraction data to 2.2 A. Diaminopimelate dehydrogenase is a homodimer of approximately 35,000 molecular weight subunits and is the only dehydrogenase present in the bacterial diaminopimelate/lysine biosynthetic pathway. Inhibitors of the enzymes of L-lysine biosynthesis have been proposed as potential antibiotics or herbicides, since mammals lack this metabolic pathway. Diaminopimelate dehydrogenase catalyzes the unique, reversible, pyridine dinucleotide-dependent oxidative deamination of the D-amino acid stereocenter of meso-diaminopimelate to generate L-2-amino-6-oxopimelate. The enzyme is absolutely specific for the meso stereoisomer of DAP and must distinguish between two opposite chiral amino acid centers on the same symmetric substrate. The determination of the three-dimensional structure of the enzyme--meso-diaminopimelate complex allows a description of the molecular basis of this stereospecific discrimination. The substrate is bound in an elongated cavity, in which the distribution of residues that act as hydrogen bond donors or acceptors defines a single orientation in which the substrate may bind in order to position the D-amino acid center of meso-DAP near the oxidized nucleotide. The previously described isoxazoline inhibitor binds at the same site as DAP but has its L-amino acid center positioned where the D-amino acid center of meso-DAP would normally be located, thereby generating a nonproductive inhibitor complex. The relative positions of the N-terminal dinucleotide and C-terminal substrate-binding domains in the diaminopimelate dehydrogenase--NADP+, diaminopimelate dehydrogenase--DAP, and diaminopimelate dehydrogenase--NADP(+)--inhibitor complexes confirm our previous observations that the enzyme undergoes significant conformational changes upon binding of both dinucleotide and substrate. << Less

  Biochemistry 37:3278-3285(1998) [PubMed] [EuropePMC]

• Dual diaminopimelate biosynthesis pathways in Bacteroides fragilis and Clostridium thermocellum.

  Hudson A.O., Klartag A., Gilvarg C., Dobson R.C., Marques F.G., Leustek T.

  Bacteroides fragilis and Clostridium thermocellum were recently found to synthesize diaminopimelate (DAP) by way of LL-DAP aminotransferase. Both species also contain an ortholog of meso-diaminopimelate dehydrogenase (Ddh), suggesting that they may have redundant pathways for DAP biosynthesis. The ... >> More

  Bacteroides fragilis and Clostridium thermocellum were recently found to synthesize diaminopimelate (DAP) by way of LL-DAP aminotransferase. Both species also contain an ortholog of meso-diaminopimelate dehydrogenase (Ddh), suggesting that they may have redundant pathways for DAP biosynthesis. The B. fragilis Ddh ortholog shows low homology with other examples of Ddh and this species belongs to a phylum, the Bacteriodetes, not previously known to contain this enzyme. By contrast, the C. thermocellum ortholog is well conserved with known examples of Ddh. Using in vitro and in vivo assays both the B. fragilis and C. thermocellum enzymes were found to be authentic examples of Ddh, displaying kinetic properties typical of this enzyme. The result indicates that B. fragilis contains a sequence diverged form of Ddh. Phylogenomic analysis of the microbial genome database revealed that 77% of species with a Ddh ortholog also contain a second pathway for DAP biosynthesis suggesting that Ddh evolved as an ancillary mechanism for DAP biosynthesis. << Less

  Biochim. Biophys. Acta 1814:1162-1168(2011) [PubMed] [EuropePMC]

• Expression, purification, and crystallization of meso-diaminopimelate dehydrogenase from Corynebacterium glutamicum.

  Reddy S.G., Scapin G., Blanchard J.S.

  The gene encoding the meso-diaminopimelate dehydrogenase (DAPDH) from Corynebacterium glutamicum was over-expressed and purified to homogeneity. Crystals of the binary DAPDH-NADP+ complex were obtained from solutions of polyethylene glycol 8000, 100 mM sodium cacodylate, pH 6.5, and 150-300 mM Mg( ... >> More

  The gene encoding the meso-diaminopimelate dehydrogenase (DAPDH) from Corynebacterium glutamicum was over-expressed and purified to homogeneity. Crystals of the binary DAPDH-NADP+ complex were obtained from solutions of polyethylene glycol 8000, 100 mM sodium cacodylate, pH 6.5, and 150-300 mM Mg(OAc)2. The crystals diffract to 2.2 A, belong to the orthorhombic space group P2(1), and contain two molecules per asymmetric unit. << Less

  Proteins 25:514-516(1996) [PubMed] [EuropePMC]

• Properties of meso-alpha,epsilon-diaminopimelate D-dehydrogenase from Bacillus sphaericus.

  Misono H., Soda K.

  meso-alpha,epsilon-Diaminopimelate D-dehydrogenase, which has been purified to homogeneity from the extract of Bacillus sphaericus IFO 3525, has a molecular weight of about 80,000 and consists of two subunits identical in molecular weight (approximately 40,000). The enzyme has a high substrate spe ... >> More

  meso-alpha,epsilon-Diaminopimelate D-dehydrogenase, which has been purified to homogeneity from the extract of Bacillus sphaericus IFO 3525, has a molecular weight of about 80,000 and consists of two subunits identical in molecular weight (approximately 40,000). The enzyme has a high substrate specificity. In addition to meso-alpha,epsilon-diaminopimelate, lanthionine is deaminated by the enzyme to a far lesser extent. NADP+ is the exclusive cofactor. The pH optima were at about 10.5 for the deamination of meso-alpha,epsilon-diaminopimelate and at 7.5 for its amination. L and D isomers of alpha,epsilon-diaminopimelate and meso-alpha,delta-diaminoadipate competitively inhibit the oxidation of meso-alpha,epsilon-diaminopimelate. Initial velocity and product inhibition studies show that the reductive amination proceeds through a sequential ordered ternary-binary mechanism. NADPH binds first to the enzyme followed by L-alpha-amino-epsilon-ketopimelate and ammonia, and the products are released in the order of meso-alpha,epsilon-diaminopimelate and NADP+. The Michaelis constants are as follows: meso-alpha,epsilon-diaminopimelate (2.5 mM), NADP+ (83 micro M), NADPH (0.2 mM), L-alpha-amino-epsilon-ketopimelate (0.24 mM), and ammonia (12.5 mM). The pro-S hydrogen at C-4 of the dihydronicotinamide ring of NADPH is transferred to the substrate; the enzyme is B-stereospecific. Fluorometric study on binding of NADPH to the enzyme revealed that the enzyme contains two coenzyme binding sites per molecule. << Less

  J. Biol. Chem. 255:10599-10605(1980) [PubMed] [EuropePMC]