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 |
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 |
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 |
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 |
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 | ? | - |
? |
EC Number | Organism | UniProt | Comment | Textmining |
---|---|---|---|---|
1.1.1.387 | Pseudomonas aeruginosa | Q9I5I6 | - |
- |
1.4.1.7 | Pseudomonas aeruginosa | - |
- |
- |
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 |
EC Number | Reaction | Comment | Organism | Reaction ID |
---|---|---|---|---|
1.1.1.387 | 2-aminomalonate semialdehyde = 2-aminoacetaldehyde + CO2 | (1b), spontaneous | Pseudomonas aeruginosa | |
1.1.1.387 | L-serine + NAD+ = 2-aminoacetaldehyde + CO2 + NADH + H+ | overall reaction | Pseudomonas aeruginosa | |
1.1.1.387 | L-serine + NAD+ = 2-aminomalonate semialdehyde + NADH + H+ | (1a) | Pseudomonas aeruginosa |
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+ | - |
? |
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 |
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 |
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 |
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 |
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 |
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+ (46%) | 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+ (46%) | 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 |
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 |
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 |