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 (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 | 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 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 | 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 | UniProt | Comment | Textmining |
---|---|---|---|
Pseudomonas aeruginosa | - |
- |
- |
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 | 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 | 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 | Comment | Organism |
---|---|---|
NAD+-dependent L-serine dehydrogenase | - |
Pseudomonas aeruginosa |
protein PA0743 | - |
Pseudomonas aeruginosa |
Temperature Optimum [°C] | Temperature Optimum Maximum [°C] | Comment | Organism |
---|---|---|---|
37 | - |
assay at | Pseudomonas aeruginosa |
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 Minimum | pH Optimum Maximum | Comment | Organism |
---|---|---|---|
11 | - |
assay at | Pseudomonas aeruginosa |
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 | 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 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 |