Literature summary for 1.4.1.18 extracted from

Yoneda, K.; Fukuda, J.; Sakuraba, H.; Ohshima, T.
First crystal structure of L-lysine 6-dehydrogenase as an NAD-dependent amine dehydrogenase (2010), J. Biol. Chem., 285, 8444-8453.

Cloned(Commentary)

Cloned (Comment)	Organism
expression of His-tagged wild-type and mutant LysDHs in Escherichia coli strain Rosettagami (DE3)	Pyrococcus horikoshii

Crystallization (Commentary)

Crystallization (Comment)	Organism
purified recombinant enzyme complexed with NAD+ and a sulfate ion, sitting drop vapor diffusion method, 0.001 ml of 10 mg/ml protein in 100 mM citrate, pH 5.8, 2.1 M ammonium sulfate, and 1 mM NAD+, is mixed with 0.001 ml of reservoir solution, 20°C, X-ray diffraction structure determination and analysis at 2.44 A resolution	Pyrococcus horikoshii

Protein Variants

Protein Variants	Comment	Organism
E168A	site-directed mutagenesis	Pyrococcus horikoshii
E168Q	site-directed mutagenesis	Pyrococcus horikoshii
R226K	site-directed mutagenesis	Pyrococcus horikoshii
Y156F	site-directed mutagenesis	Pyrococcus horikoshii

Molecular Weight [Da]

Molecular Weight [Da]	Molecular Weight Maximum [Da]	Comment	Organism
42089	-	2 * 42600, recombinant enzyme, SDS-PAGE, 2 * 42089, amino acid sequence	Pyrococcus horikoshii
42600	-	2 * 42600, recombinant enzyme, SDS-PAGE, 2 * 42089, amino acid sequence	Pyrococcus horikoshii
97200	-	recombinant enzyme, gel filtration	Pyrococcus horikoshii

Natural Substrates/ Products (Substrates)

Natural Substrates	Organism	Comment (Nat. Sub.)	Natural Products	Comment (Nat. Pro.)	Rev.
(S)-2-aminoadipate 6-semialdehyde	Pyrococcus horikoshii	-	(S)-2,3,4,5-tetrahydropiperidine-2-carboxylate + H2O	i.e. DELTA1-piperidein-6-carboxylate, spontaneous nonenzymatical reaction	?
(S)-2-aminoadipate 6-semialdehyde	Pyrococcus horikoshii ATCC 700860	-	(S)-2,3,4,5-tetrahydropiperidine-2-carboxylate + H2O	i.e. DELTA1-piperidein-6-carboxylate, spontaneous nonenzymatical reaction	?
L-lysine + NAD(P)+ + H2O	Pyrococcus horikoshii	-	(S)-2-aminoadipate 6-semialdehyde + NAD(P)H + H+ + NH3	-	?
L-lysine + NAD(P)+ + H2O	Pyrococcus horikoshii ATCC 700860	-	(S)-2-aminoadipate 6-semialdehyde + NAD(P)H + H+ + NH3	-	?
additional information	Pyrococcus horikoshii	the enzyme catalyzes the reductive deamination of the epsilon-amino group and acts as a type of NAD+-dependent amine dehydrogenase producing DELTA1-piperideine-6-carboxylate	?	-	?
additional information	Pyrococcus horikoshii ATCC 700860	the enzyme catalyzes the reductive deamination of the epsilon-amino group and acts as a type of NAD+-dependent amine dehydrogenase producing DELTA1-piperideine-6-carboxylate	?	-	?

Organism

Organism	UniProt	Comment	Textmining
Pyrococcus horikoshii	O59312	-	-
Pyrococcus horikoshii ATCC 700860	O59312	-	-

Purification (Commentary)

Purification (Comment)	Organism
refolded recombinant His-tagged wild-type and mutant LysDHs from Escherichia coli strain Rosettagami (DE3) by gel filtration and nickel affinity chromatography	Pyrococcus horikoshii

Reaction

Reaction	Comment	Organism	Reaction ID
L-lysine + NAD+ + H2O = (S)-2-amino-6-oxohexanoate + NADH + H+ + NH3	reaction mechanism, overview	Pyrococcus horikoshii	a

Renatured (Commentary)

Renatured (Comment)	Organism
His-tagged wild-type and mutant LysDHs after recombinant expression in Escherichia coli inclusion bodies are suspended in 20 mM Tris-HCl, pH 7.5, containing 5 mM 2-mercaptoethanol and 4% Triton X-100, and disrupted by sonication, followed by solubilization in 50 mM Tris-HCl, pH 7.5, containing 5 mM 2-mercaptoethanol, 6 M guanidine HCl, 0.2 M NaCl, and 1 mM EDTA. Refolding in buffer containing 0.1 M Tris-HCl, pH 7.5, 5 mM 2-mercaptoethanol, 0.4 M arginine, 2 mM EDTA, and 0.1 mM phenylmethylsulfonyl fluoride and incubation for 36 h at 4°C. Refolded LysDH is heated at 80°C for 10 min and clarified by centrifugation	Pyrococcus horikoshii

Renatured (Comment)

Organism

His-tagged wild-type and mutant LysDHs after recombinant expression in Escherichia coli inclusion bodies are suspended in 20 mM Tris-HCl, pH 7.5, containing 5 mM 2-mercaptoethanol and 4% Triton X-100, and disrupted by sonication, followed by solubilization in 50 mM Tris-HCl, pH 7.5, containing 5 mM 2-mercaptoethanol, 6 M guanidine HCl, 0.2 M NaCl, and 1 mM EDTA. Refolding in buffer containing 0.1 M Tris-HCl, pH 7.5, 5 mM 2-mercaptoethanol, 0.4 M arginine, 2 mM EDTA, and 0.1 mM phenylmethylsulfonyl fluoride and incubation for 36 h at 4°C. Refolded LysDH is heated at 80°C for 10 min and clarified by centrifugation

Pyrococcus horikoshii

Substrates and Products (Substrate)

Substrates	Comment Substrates	Organism	Products	Comment (Products)	Rev.
(S)-2-aminoadipate 6-semialdehyde	-	Pyrococcus horikoshii	(S)-2,3,4,5-tetrahydropiperidine-2-carboxylate + H2O	i.e. DELTA1-piperidein-6-carboxylate, spontaneous nonenzymatical reaction	?
(S)-2-aminoadipate 6-semialdehyde	-	Pyrococcus horikoshii	(S)-2,3,4,5-tetrahydropiperidine-2-carboxylate + H2O	spontaneous reaction	?
(S)-2-aminoadipate 6-semialdehyde	-	Pyrococcus horikoshii ATCC 700860	(S)-2,3,4,5-tetrahydropiperidine-2-carboxylate + H2O	i.e. DELTA1-piperidein-6-carboxylate, spontaneous nonenzymatical reaction	?
(S)-2-aminoadipate 6-semialdehyde	-	Pyrococcus horikoshii ATCC 700860	(S)-2,3,4,5-tetrahydropiperidine-2-carboxylate + H2O	spontaneous reaction	?
L-lysine + NAD(P)+ + H2O	-	Pyrococcus horikoshii	(S)-2-aminoadipate 6-semialdehyde + NAD(P)H + H+ + NH3	-	?
L-lysine + NAD(P)+ + H2O	-	Pyrococcus horikoshii ATCC 700860	(S)-2-aminoadipate 6-semialdehyde + NAD(P)H + H+ + NH3	-	?
additional information	active site structure, overview	Pyrococcus horikoshii	?	-	?
additional information	the enzyme catalyzes the reductive deamination of the epsilon-amino group and acts as a type of NAD+-dependent amine dehydrogenase producing DELTA1-piperideine-6-carboxylate	Pyrococcus horikoshii	?	-	?
additional information	active site structure, overview	Pyrococcus horikoshii ATCC 700860	?	-	?
additional information	the enzyme catalyzes the reductive deamination of the epsilon-amino group and acts as a type of NAD+-dependent amine dehydrogenase producing DELTA1-piperideine-6-carboxylate	Pyrococcus horikoshii ATCC 700860	?	-	?

Subunits

Subunits	Comment	Organism
dimer	2 * 42600, recombinant enzyme, SDS-PAGE, 2 * 42089, amino acid sequence	Pyrococcus horikoshii
More	each monomer consists 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, three-dimensional structure of the enzyme, structure comparisons, overview. Subunit A active site contains a sulfate ion not seen in subunit B. Consequently, subunit A adopts a closed conformation, whereas subunit B adopts 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 with type A specificity	Pyrococcus horikoshii

Synonyms

Synonyms	Comment	Organism
L-lysine 6-dehydrogenase	-	Pyrococcus horikoshii
LysDH	-	Pyrococcus horikoshii

Temperature Optimum [°C]

Temperature Optimum [°C]	Temperature Optimum Maximum [°C]	Comment	Organism
95	-	about, recombinant enzyme	Pyrococcus horikoshii

Temperature Range [°C]

Temperature Minimum [°C]	Temperature Maximum [°C]	Comment	Organism
50	95	maximum activity at about 95°C and an extraordinary drop off in activity with reduction in temperature, the activity at 50°C is only about 1/170th of that at 95°C	Pyrococcus horikoshii

Temperature Stability [°C]

Temperature Stability Minimum [°C]	Temperature Stability Maximum [°C]	Comment	Organism
100	-	purified recombinant enzyme, half-life is 180 min, the enzyme is highly thermostable and retaining full activity even after incubation for 50 min at 100°C at pH 7.5	Pyrococcus horikoshii

pH Optimum

pH Optimum Minimum	pH Optimum Maximum	Comment	Organism
10	-	assay at	Pyrococcus horikoshii

pH Stability

pH Stability	pH Stability Maximum	Comment	Organism
5	12	the purified recombinant enzyme is stable over a wide range of pH values, losing no activity when incubated at pH values between 5.0 and 12 for 30 min at 50°C, most stable at pH 10.0	Pyrococcus horikoshii

Cofactor

Cofactor	Comment	Organism
additional information	both NAD+ and NADP+ are able to serve as the coenzyme for Pyrococcus horikoshii LysDH, although the reaction rate with NADP+ is only 7.8% of that with NAD+, when L-lysine is the substrate	Pyrococcus horikoshii
NAD+	preferred cofactor, binding structure, overview. The enzyme makes use of pro-R-specific hydride transfer between the two hydrides at C-4 of NADH with type A specificity	Pyrococcus horikoshii
NADP+	-	Pyrococcus horikoshii