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Literature summary for 1.4.1.7 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)

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

Crystallization (Commentary)

Crystallization (Comment) Organism
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

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

KM Value [mM] KM Value Maximum [mM] Substrate Comment Organism Structure
2.4
-
3-methyl-DL-serine pH 11.0, 37°C, recombinant His-tagged wild-type enzyme Pseudomonas aeruginosa
2.5
-
L-serine pH 11.0, 37°C, recombinant His-tagged wild-type enzyme Pseudomonas aeruginosa
3.4
-
NAD+ pH 11.0, 37°C, recombinant His-tagged wild-type enzyme Pseudomonas aeruginosa
17.4
-
methyl 2,2-dimethyl-3-hydroxypropionic acid pH 11.0, 37°C, recombinant His-tagged wild-type enzyme Pseudomonas aeruginosa
19.8
-
DL-glycerate pH 11.0, 37°C, recombinant His-tagged wild-type enzyme Pseudomonas aeruginosa

Natural Substrates/ Products (Substrates)

Natural Substrates Organism Comment (Nat. Sub.) Natural Products Comment (Nat. Pro.) Rev. Reac.
L-serine + H2O + NAD+ Pseudomonas aeruginosa
-
3-hydroxypyruvate + NH3 + NADH + H+
-
?
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

Organism UniProt Comment Textmining
Pseudomonas aeruginosa
-
-
-

Purification (Commentary)

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

Substrates and Products (Substrate)

Substrates Comment Substrates Organism Products Comment (Products) Rev. Reac.
(R)-3-hydroxyisobutyric acid + H2O + NAD+ low activity Pseudomonas aeruginosa ? + NH3 + NADH + H+
-
?
(S)-3-hydroxyisobutyric acid + H2O + NAD+ low activity Pseudomonas aeruginosa ? + NH3 + NADH + H+
-
?
3-methyl-DL-serine + H2O + NAD+ high activity Pseudomonas aeruginosa 2-aminomethylmalonate semialdehyde + NH3 + NADH + H+
-
?
D-serine + H2O + NAD+ low activity Pseudomonas aeruginosa 3-hydroxypyruvate + NH3 + NADH + H+
-
?
DL-glycerate + H2O + NAD+ low activity Pseudomonas aeruginosa ? + NH3 + NADH + H+
-
?
L-serine + H2O + NAD+
-
Pseudomonas aeruginosa 3-hydroxypyruvate + NH3 + NADH + H+
-
?
L-serine + H2O + NAD+ best substrate Pseudomonas aeruginosa 3-hydroxypyruvate + NH3 + NADH + H+
-
?
methyl (R)-3-hydroxy-2-methylpropionic acid + H2O + NAD+ low activity Pseudomonas aeruginosa ? + NH3 + NADH + H+
-
?
methyl (S)-3-hydroxy-2-methylpropionic acid + H2O + NAD+ low activity Pseudomonas aeruginosa ? + NH3 + NADH + H+
-
?
methyl 2,2-dimethyl-3-hydroxypropionic acid + H2O + NAD+ low activity Pseudomonas aeruginosa ? + NH3 + NADH + H+
-
?
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 ?
-
?
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 ?
-
?
tert-butyl 3-hydroxypropionic acid + H2O + NAD+ low activity Pseudomonas aeruginosa ? + NH3 + NADH + H+
-
?

Subunits

Subunits Comment Organism
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

Synonyms Comment Organism
NAD+-dependent L-serine dehydrogenase
-
Pseudomonas aeruginosa
protein PA0743
-
Pseudomonas aeruginosa

Temperature Optimum [°C]

Temperature Optimum [°C] Temperature Optimum Maximum [°C] Comment Organism
37
-
assay at Pseudomonas aeruginosa

Turnover Number [1/s]

Turnover Number Minimum [1/s] Turnover Number Maximum [1/s] Substrate Comment Organism Structure
1.6
-
NAD+ pH 11.0, 37°C, recombinant His-tagged wild-type enzyme Pseudomonas aeruginosa
5.8
-
DL-glycerate pH 11.0, 37°C, recombinant His-tagged wild-type enzyme Pseudomonas aeruginosa
9.6
-
3-methyl-DL-serine pH 11.0, 37°C, recombinant His-tagged wild-type enzyme Pseudomonas aeruginosa
10.4
-
L-serine pH 11.0, 37°C, recombinant His-tagged wild-type enzyme Pseudomonas aeruginosa
11.6
-
methyl 2,2-dimethyl-3-hydroxypropionic acid pH 11.0, 37°C, recombinant His-tagged wild-type enzyme Pseudomonas aeruginosa

pH Optimum

pH Optimum Minimum pH Optimum Maximum Comment Organism
11
-
assay at Pseudomonas aeruginosa

Cofactor

Cofactor Comment Organism Structure
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

General Information Comment Organism
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]

kcat/KM Value [1/mMs-1] kcat/KM Value Maximum [1/mMs-1] Substrate Comment Organism Structure
0.3
-
DL-glycerate pH 11.0, 37°C, recombinant His-tagged wild-type enzyme Pseudomonas aeruginosa
0.5
-
NAD+ pH 11.0, 37°C, recombinant His-tagged wild-type enzyme Pseudomonas aeruginosa
0.7
-
methyl 2,2-dimethyl-3-hydroxypropionic acid pH 11.0, 37°C, recombinant His-tagged wild-type enzyme Pseudomonas aeruginosa
40
-
L-serine pH 11.0, 37°C, recombinant His-tagged wild-type enzyme Pseudomonas aeruginosa
40
-
3-methyl-DL-serine pH 11.0, 37°C, recombinant His-tagged wild-type enzyme Pseudomonas aeruginosa