- First crystal structure of L-lysine 6-dehydrogenase as an NAD-dependent amine dehydrogenase
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A gene encoding an L-lysine dehydrogenase was identified in the hyperthermophilic archaeon Pyrococcus horikoshii. The gene was overexpressed in Escherichia coli, and its product was purified and characterized. The expressed enzyme is the most thermostable L-lysine dehydrogenase yet described, with a half-life of 180 min at 100 °C. The product of the enzyme's catalytic activity is Δ1-piperideine-6-carboxylate, which makes this enzyme an L-lysine 6-dehydrogenase (EC 1.4.1.18) that catalyzes the reductive deamination of the ∈-amino group and a type of NAD-dependent amine dehydrogenase. The three-dimensional structure of the enzyme was determined using the mercury-based multiple-wavelength anomalous dispersion method at a resolution of 2.44 A in the presence of NAD and sulfate ion. The asymmetric unit consisted of two subunits, and a crystallographic 2-fold axis generated the functional dimer. Each monomer consisted of a Rossmann fold domain and a C-terminal catalytic domain, and the fold of the catalytic domain showed similarity to that of saccharopine reductase. Notably, the structures of subunits A and B differed significantly. In subunit A, the active site contained a sulfate ion that was not seen in subunit B. Consequently, subunit A adopted a closed conformation, whereas subunit B adopted an open one. In each subunit, one NAD molecule was bound to the active site in an anti-conformation, indicating that the enzyme makes use of pro-R-specific hydride transfer between the two hydrides at C-4 of NADH (type A specificity). This is the first description of the three-dimensional structure of L-lysine 6-dehydrogenase as an NAD-dependent amine dehydrogenase.
- Yoneda, Kazunari,Fukuda, Junya,Sakuraba, Haruhiko,Ohshima, Toshihisa
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experimental part
p. 8444 - 8453
(2011/04/16)
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- Synthesis of 1-piperideine-6-carboxylic acid produced by L-lysine-ε-aminotransferase from the Streptomyces clavuligerus gene expressed in Escherichia coli
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The gene (laf) encoding L-lysine ε-aminotransferase (LAT) in Streptomyces clavuligerus was cloned and expressed in Escherichia coli. Nucleotide sequence analysis of lat predicted a single open reading frame (ORF) of 1371 bp, encoding a polypeptide of 457 amino acids with calculated molecular mass of 49.89 kDa. S. clavuligerus LAT was grouped into aminotransferase subfamily II of a family on the basis of sequence homology. A model system composed of the recombinant LAT in phosphate buffer was set up to study the biosynthesis of 2-acetyltetrahydropyridine. Lysine was found to be transformed to 1-piperideine-6-carboxylic acid. 2-Acetyltetrahydropyridine was characterized from the mixture of 1-piperideine-6-carboxylic acid and methylglyoxal. For the first time, we demonstrated that the L-lysine ε-aminotransferase is responsible for the formation of 1-piperideine-6-carboxylic acid, which may react with methylglyoxal to generate the acylated N-heterocyclic odorant 2-acetyltetrahydropyridine.
- Wu, Mei-Li,Chen, Jun-Hao,Ho, Chi-Tang,Huang, Tzou-Chi
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p. 1767 - 1772
(2008/02/04)
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- VITAMIN COMPRISING PYROLOQUINOLINE QUINONE AND USE THEREOF
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It is an object of the present invention to clarify the biochemical role of pyrroloquinoline quinone (PQQ) in living bodies by identifying an enzyme that uses PQQ as a coenzyme in mammals and then by clarifying the oxidation-reduction reaction, with which PQQ is associated as a coenzyme in living bodies. The present invention provides a method of using pyrroloquinoline quinone as a coenzyme for 2-aminoadipate 6-semialdehyde dehydrogenase.
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Page/Page column 20
(2008/06/13)
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