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Literature summary extracted from

  • Tchigvintsev, A.; Singer, A.; Brown, G.; Flick, R.; Evdokimova, E.; Tan, K.; Gonzalez, C.F.; Savchenko, A.; Yakunin, A.F.
    Biochemical and structural studies of uncharacterized protein PA0743 from Pseudomonas aeruginosa revealed NAD+-dependent L-serine dehydrogenase (2012), J. Biol. Chem., 287, 1874-1883.
    View publication on PubMedView publication on EuropePMC

Cloned(Commentary)

EC Number Cloned (Comment) Organism
1.1.1.387 overexpressed in Escherichia coli Pseudomonas aeruginosa
1.4.1.7 PA0743 open reading frame, expression of wild-type and mutant His-tagged enzymes in Escherichia coli strain BL21(DE3) Gold Pseudomonas aeruginosa

Crystallization (Commentary)

EC Number Crystallization (Comment) Organism
1.1.1.387 two crystal structures of the enzyme are solved at 2.2-2.3 A resolution and reveal an N-terminal Rossmann fold domain connected by a long alpha-helix to the C-terminal all-alpha-domain. The apostructure shows the presence of additional density modeled as HEPES bound in the interdomain cleft close to the predicted catalytic Lys171, revealing the molecular details of the enzyme substrate-binding site. The structure of the enzyme-NAD complex demonstrates that the opposite side of the enzyme active site accommodates the cofactor, which is also bound near Lys171. Crystals of the enzyme are grown at 21C by the hanging drop vapor diffusion method with 0.002 ml of protein sample mixed with an equal volume of the reservoir buffer. The crystals of the wild-type enzyme grew after 1 week in the presence of 4 M ammonium acetate and 0.1 M sodium acetate (pH 5.4). The crystals of the complex of the enzyme with NAD+ are obtained by soaking the crystals in 10 mM NAD+. For diffraction studies, the crystals are stabilized with the crystallization buffer supplemented with 12% ethylene glycol as a cryoprotectant and flash frozen in liquid nitrogen Pseudomonas aeruginosa
1.4.1.7 purified His-tagged recombinant protein PA0743 alone or in complex with cofactor NAD+, hanging drop vapor diffusion method, mixing of 0.002 ml protein solution with 0.002 ml reservoir solution containing 4 M ammonium acetate and 0.1 M sodium acetate, at pH 5.4, 1 week, soaking of crystals in 10 mM NAD+ solution for complexcystals, X-ray diffraction structure determination and analysis at 2.2 A resolution Pseudomonas aeruginosa

Protein Variants

EC Number Protein Variants Comment Organism
1.1.1.387 D247A inactive protein Pseudomonas aeruginosa
1.1.1.387 K171A inactive protein Pseudomonas aeruginosa
1.1.1.387 K246A inactive protein Pseudomonas aeruginosa
1.1.1.387 N175A mutant enzyme with very low activity Pseudomonas aeruginosa
1.1.1.387 S122A mutant enzyme with very low activity Pseudomonas aeruginosa
1.1.1.387 T96A mutant enzyme with very low activity Pseudomonas aeruginosa
1.1.1.387 W214A inactive protein Pseudomonas aeruginosa
1.1.1.387 Y219A mutant enzyme with very low activity Pseudomonas aeruginosa
1.4.1.7 D247A site-directed mutagenesis, inactive mutant Pseudomonas aeruginosa
1.4.1.7 E122A site-directed mutagenesis, the mutant shows alterations in cofactor binding and highly reduced activity compared to the wild-type enzyme Pseudomonas aeruginosa
1.4.1.7 K171A site-directed mutagenesis, inactive mutant Pseudomonas aeruginosa
1.4.1.7 K246A site-directed mutagenesis, inactive mutant Pseudomonas aeruginosa
1.4.1.7 additional information PA0743 deletion (PW2350) strain from a transposon library Pseudomonas aeruginosa
1.4.1.7 N175A site-directed mutagenesis, the mutant shows alterations in cofactor binding and highly reduced activity compared to the wild-type enzyme Pseudomonas aeruginosa
1.4.1.7 T96A site-directed mutagenesis, the mutant shows alterations in cofactor binding and highly reduced activity compared to the wild-type enzyme Pseudomonas aeruginosa
1.4.1.7 W214A site-directed mutagenesis, inactive mutant Pseudomonas aeruginosa
1.4.1.7 Y219A site-directed mutagenesis, the mutant shows alterations in cofactor binding and highly reduced activity compared to the wild-type enzyme Pseudomonas aeruginosa

KM Value [mM]

EC Number KM Value [mM] KM Value Maximum [mM] Substrate Comment Organism Structure
1.1.1.387 2.4
-
 DL-threonine pH 11.0, 37°C, wild-type enzyme Pseudomonas aeruginosa
1.1.1.387 2.5
-
 L-serine pH 11.0, 37°C, wild-type enzyme Pseudomonas aeruginosa
1.1.1.387 3.4
-
 NAD+ pH 11.0, 37°C, wild-type enzyme Pseudomonas aeruginosa
1.1.1.387 4.2
-
 L-serine pH 11.0, 37°C, mutant enzyme N175A Pseudomonas aeruginosa
1.1.1.387 10.6
-
 L-serine pH 11.0, 37°C, mutant enzyme S122A Pseudomonas aeruginosa
1.1.1.387 10.8
-
 D-glycerate pH 11.0, 37°C, wild-type enzyme Pseudomonas aeruginosa
1.1.1.387 11
-
 L-serine pH 11.0, 37°C, mutant enzyme Y219A Pseudomonas aeruginosa
1.1.1.387 12.3
-
 L-serine pH 11.0, 37°C, mutant enzyme T96A Pseudomonas aeruginosa
1.1.1.387 17.4
-
 methyl 2,2-dimethyl-3-hydroxypropionate pH 11.0, 37°C, wild-type enzyme Pseudomonas aeruginosa
1.4.1.7 2.4
-
 3-methyl-DL-serine pH 11.0, 37°C, recombinant His-tagged wild-type enzyme Pseudomonas aeruginosa
1.4.1.7 2.5
-
 L-serine pH 11.0, 37°C, recombinant His-tagged wild-type enzyme Pseudomonas aeruginosa
1.4.1.7 3.4
-
 NAD+ pH 11.0, 37°C, recombinant His-tagged wild-type enzyme Pseudomonas aeruginosa
1.4.1.7 17.4
-
 methyl 2,2-dimethyl-3-hydroxypropionic acid pH 11.0, 37°C, recombinant His-tagged wild-type enzyme Pseudomonas aeruginosa
1.4.1.7 19.8
-
 DL-glycerate pH 11.0, 37°C, recombinant His-tagged wild-type enzyme Pseudomonas aeruginosa

Natural Substrates/ Products (Substrates)

EC Number Natural Substrates Organism Comment (Nat. Sub.) Natural Products Comment (Nat. Pro.) Rev. Reac.
1.1.1.387 L-serine + NAD+ Pseudomonas aeruginosa the enzyme might be involved in serine/threonine degradation. Since growth experiments with various nitrogen and carbon sources (including L-serine) reveal no difference between the Pseudomonas aeruginosa wild-type and PA0743 deletion strains, it is suggested hat this organism might contain other (complementing) serine dehydrogenases ?
-
 ?
1.4.1.7 L-serine + H2O + NAD+ Pseudomonas aeruginosa
-
 3-hydroxypyruvate + NH3 + NADH + H+
-
 ?
1.4.1.7 additional information Pseudomonas aeruginosa PA0743 from Pseudomonas aeruginosa catalyzes NAD-dependent oxidation of L-serine and methyl-L-serine, and exhibits low activity against beta-hydroxyisobutyrate ?
-
 ?

Organism

EC Number Organism UniProt Comment Textmining
1.1.1.387 Pseudomonas aeruginosa Q9I5I6
-
-
1.4.1.7 Pseudomonas aeruginosa
-
-
-

Purification (Commentary)

EC Number Purification (Comment) Organism
1.1.1.387
-
 Pseudomonas aeruginosa
1.4.1.7 recombinant wild-type and mutant His-tagged enzymes from Escherichia coli strain BL21(DE3) Gold by nickel affinity chromatography and gel filtration Pseudomonas aeruginosa

Reaction

EC Number Reaction Comment Organism Reaction ID
1.1.1.387 2-aminomalonate semialdehyde = 2-aminoacetaldehyde + CO2 (1b), spontaneous Pseudomonas aeruginosa
a
1.1.1.387 L-serine + NAD+ = 2-aminoacetaldehyde + CO2 + NADH + H+ overall reaction Pseudomonas aeruginosa
a
1.1.1.387 L-serine + NAD+ = 2-aminomalonate semialdehyde + NADH + H+ (1a) Pseudomonas aeruginosa
a

Substrates and Products (Substrate)

EC Number Substrates Comment Substrates Organism Products Comment (Products) Rev. Reac.
1.1.1.387 D-glycerate + NAD+
-
 Pseudomonas aeruginosa ?
-
 ?
1.1.1.387 DL-threonine + NAD+
-
 Pseudomonas aeruginosa ?
-
 ?
1.1.1.387 L-serine + NAD+ the enzyme might be involved in serine/threonine degradation. Since growth experiments with various nitrogen and carbon sources (including L-serine) reveal no difference between the Pseudomonas aeruginosa wild-type and PA0743 deletion strains, it is suggested hat this organism might contain other (complementing) serine dehydrogenases Pseudomonas aeruginosa ?
-
 ?
1.1.1.387 L-serine + NAD+ the enzyme might be involved in serine/methylserine degradation. Since growth experiments with various nitrogen and carbon sources (including L-serine) reveal no difference between the Pseudomonas aeruginosa wild-type and PA0743 deletion strains, it is suggested hat this organism might contain other (complementing) serine dehydrogenases Pseudomonas aeruginosa ?
-
 ?
1.1.1.387 methyl 2,2-dimethyl-3-hydroxypropionate + NAD+
-
 Pseudomonas aeruginosa ?
-
 ?
1.4.1.7 (R)-3-hydroxyisobutyric acid + H2O + NAD+ low activity Pseudomonas aeruginosa ? + NH3 + NADH + H+
-
 ?
1.4.1.7 (S)-3-hydroxyisobutyric acid + H2O + NAD+ low activity Pseudomonas aeruginosa ? + NH3 + NADH + H+
-
 ?
1.4.1.7 3-methyl-DL-serine + H2O + NAD+ high activity Pseudomonas aeruginosa 2-aminomethylmalonate semialdehyde + NH3 + NADH + H+
-
 ?
1.4.1.7 D-serine + H2O + NAD+ low activity Pseudomonas aeruginosa 3-hydroxypyruvate + NH3 + NADH + H+
-
 ?
1.4.1.7 DL-glycerate + H2O + NAD+ low activity Pseudomonas aeruginosa ? + NH3 + NADH + H+
-
 ?
1.4.1.7 L-serine + H2O + NAD+
-
 Pseudomonas aeruginosa 3-hydroxypyruvate + NH3 + NADH + H+
-
 ?
1.4.1.7 L-serine + H2O + NAD+ best substrate Pseudomonas aeruginosa 3-hydroxypyruvate + NH3 + NADH + H+
-
 ?
1.4.1.7 methyl (R)-3-hydroxy-2-methylpropionic acid + H2O + NAD+ low activity Pseudomonas aeruginosa ? + NH3 + NADH + H+
-
 ?
1.4.1.7 methyl (S)-3-hydroxy-2-methylpropionic acid + H2O + NAD+ low activity Pseudomonas aeruginosa ? + NH3 + NADH + H+
-
 ?
1.4.1.7 methyl 2,2-dimethyl-3-hydroxypropionic acid + H2O + NAD+ low activity Pseudomonas aeruginosa ? + NH3 + NADH + H+
-
 ?
1.4.1.7 additional information PA0743 from Pseudomonas aeruginosa catalyzes NAD-dependent oxidation of L-serine and methyl-L-serine, and exhibits low activity against beta-hydroxyisobutyrate Pseudomonas aeruginosa ?
-
 ?
1.4.1.7 additional information substrate specificity, overview. No activity with (S)-beta-hydroxybutyric acid, (R)-3-hydroxybutyric acid, 2-hydroxyisobutyric acid, methyl-2-hydroxyisobutyric acid, ethyl-2-hydroxyisobutyric acid, 2-hydroxybutyric acid, DL-malic acid, and DL-homoserine Pseudomonas aeruginosa ?
-
 ?
1.4.1.7 tert-butyl 3-hydroxypropionic acid + H2O + NAD+ low activity Pseudomonas aeruginosa ? + NH3 + NADH + H+
-
 ?

Subunits

EC Number Subunits Comment Organism
1.4.1.7 More the enzyme has an N-terminal Rossmann fold domain connected by a long alpha-helix to the C-terminal all-alpha domain. Critical role of four amino acid residues in catalysis including the primary catalytic residue Lys171, PA0743 substrate-binding site structure, and molecular mechanisms of substrate selectivity, overview. The structure of the PA0743-NAD+ complex demonstrates that the opposite side of the enzyme active site accommodates the cofactor, which is also bound near Lys171, with dinucleotide cofactor binding GLGXMG motif-1, substrate binding DAPVSGG motif-2, catalysis GXXGXGXXXKXXN motif-3, and cofactor binding motif-4 Pseudomonas aeruginosa

Synonyms

EC Number Synonyms Comment Organism
1.1.1.387 NAD+-dependent L-serine dehydrogenase
-
 Pseudomonas aeruginosa
1.1.1.387 PA0743
-
 Pseudomonas aeruginosa
1.4.1.7 NAD+-dependent L-serine dehydrogenase
-
 Pseudomonas aeruginosa
1.4.1.7 protein PA0743
-
 Pseudomonas aeruginosa

Temperature Optimum [°C]

EC Number Temperature Optimum [°C] Temperature Optimum Maximum [°C] Comment Organism
1.1.1.387 37
-
 assay at Pseudomonas aeruginosa
1.4.1.7 37
-
 assay at Pseudomonas aeruginosa

Turnover Number [1/s]

EC Number Turnover Number Minimum [1/s] Turnover Number Maximum [1/s] Substrate Comment Organism Structure
1.1.1.387 0.4
-
 L-serine pH 11.0, 37°C, mutant enzyme S122A Pseudomonas aeruginosa
1.1.1.387 0.8
-
 L-serine pH 11.0, 37°C, mutant enzyme Y219A Pseudomonas aeruginosa
1.1.1.387 1.4
-
 L-serine pH 11.0, 37°C, mutant enzyme T96A Pseudomonas aeruginosa
1.1.1.387 1.6
-
 NAD+ pH 11.0, 37°C, wild-type enzyme Pseudomonas aeruginosa
1.1.1.387 2.7
-
 L-serine pH 11.0, 37°C, mutant enzyme N175A Pseudomonas aeruginosa
1.1.1.387 5.8
-
 D-glycerate pH 11.0, 37°C, wild-type enzyme Pseudomonas aeruginosa
1.1.1.387 9.6
-
 DL-threonine pH 11.0, 37°C, wild-type enzyme Pseudomonas aeruginosa
1.1.1.387 10.4
-
 L-serine pH 11.0, 37°C, wild-type enzyme Pseudomonas aeruginosa
1.1.1.387 11.6
-
 methyl 2,2-dimethyl-3-hydroxypropionate pH 11.0, 37°C, wild-type enzyme Pseudomonas aeruginosa
1.4.1.7 1.6
-
 NAD+ pH 11.0, 37°C, recombinant His-tagged wild-type enzyme Pseudomonas aeruginosa
1.4.1.7 5.8
-
 DL-glycerate pH 11.0, 37°C, recombinant His-tagged wild-type enzyme Pseudomonas aeruginosa
1.4.1.7 9.6
-
 3-methyl-DL-serine pH 11.0, 37°C, recombinant His-tagged wild-type enzyme Pseudomonas aeruginosa
1.4.1.7 10.4
-
 L-serine pH 11.0, 37°C, recombinant His-tagged wild-type enzyme Pseudomonas aeruginosa
1.4.1.7 11.6
-
 methyl 2,2-dimethyl-3-hydroxypropionic acid pH 11.0, 37°C, recombinant His-tagged wild-type enzyme Pseudomonas aeruginosa

pH Optimum

EC Number pH Optimum Minimum pH Optimum Maximum Comment Organism
1.1.1.387 10 11
-
 Pseudomonas aeruginosa
1.4.1.7 11
-
 assay at Pseudomonas aeruginosa

Cofactor

EC Number Cofactor Comment Organism Structure
1.1.1.387 NAD+ the enzyme can also use NADP+ as a cofactor for the oxidation of L-serine, but this activity is significantly lower than that with NAD+ (4–6%) Pseudomonas aeruginosa
1.1.1.387 NADP+ the enzyme can also use NADP+ as a cofactor for the oxidation of L-serine, but this activity is significantly lower than that with NAD+ (4–6%) Pseudomonas aeruginosa
1.4.1.7 NAD+ binding GLGXMG motif-1 and motif-4, the nicotinamide moiety of NAD+ is mostly buried in the interdomain cleft of the PA0743 protomer Pseudomonas aeruginosa

General Information

EC Number General Information Comment Organism
1.1.1.387 metabolism the enzyme might be involved in serine/threonine degradation. Since growth experiments with various nitrogen and carbon sources (including L-serine) reveal no difference between the Pseudomonas aeruginosa wild-type and PA0743 deletion strains, it is suggested hat this organism might contain other (complementing) serine dehydrogenases Pseudomonas aeruginosa
1.4.1.7 additional information the enzyme has an N-terminal Rossmann fold domain connected by a long alpha-helix to the C-terminal all-alpha domain. Critical role of four amino acid residues in catalysis including the primary catalytic residue Lys171, PA0743 substrate-binding site structure and molecular mechanisms of substrate selectivity, overview. The structure of the PA0743-NAD+ complex demonstrates that the opposite side of the enzyme active site accommodates the cofactor, which is also bound near Lys171 Pseudomonas aeruginosa

kcat/KM [mM/s]

EC Number kcat/KM Value [1/mMs-1] kcat/KM Value Maximum [1/mMs-1] Substrate Comment Organism Structure
1.1.1.387 0.04
-
 L-serine pH 11.0, 37°C, mutant enzyme S122A Pseudomonas aeruginosa
1.1.1.387 0.07
-
 L-serine pH 11.0, 37°C, mutant enzyme Y219A Pseudomonas aeruginosa
1.1.1.387 0.1
-
 L-serine pH 11.0, 37°C, mutant enzyme T96A Pseudomonas aeruginosa
1.1.1.387 0.3
-
 D-glycerate pH 11.0, 37°C, wild-type enzyme Pseudomonas aeruginosa
1.1.1.387 0.5
-
 NAD+ pH 11.0, 37°C, wild-type enzyme Pseudomonas aeruginosa
1.1.1.387 0.6
-
 L-serine pH 11.0, 37°C, mutant enzyme N175A Pseudomonas aeruginosa
1.1.1.387 0.7
-
 methyl 2,2-dimethyl-3-hydroxypropionate pH 11.0, 37°C, wild-type enzyme Pseudomonas aeruginosa
1.1.1.387 4
-
 L-serine pH 11.0, 37°C, wild-type enzyme Pseudomonas aeruginosa
1.1.1.387 4
-
 DL-threonine pH 11.0, 37°C, wild-type enzyme Pseudomonas aeruginosa
1.4.1.7 0.3
-
 DL-glycerate pH 11.0, 37°C, recombinant His-tagged wild-type enzyme Pseudomonas aeruginosa
1.4.1.7 0.5
-
 NAD+ pH 11.0, 37°C, recombinant His-tagged wild-type enzyme Pseudomonas aeruginosa
1.4.1.7 0.7
-
 methyl 2,2-dimethyl-3-hydroxypropionic acid pH 11.0, 37°C, recombinant His-tagged wild-type enzyme Pseudomonas aeruginosa
1.4.1.7 40
-
 L-serine pH 11.0, 37°C, recombinant His-tagged wild-type enzyme Pseudomonas aeruginosa
1.4.1.7 40
-
 3-methyl-DL-serine pH 11.0, 37°C, recombinant His-tagged wild-type enzyme Pseudomonas aeruginosa