Cloned (Comment) | Organism |
---|---|
gene ald, DNA and amino acid sequence determination and analysis, sequence comparisons, recombinant expression of His-tagged wild-type and mutant enzymes in Escherichia coli strain BL21(DE3) | Alkalihalophilus pseudofirmus |
Protein Variants | Comment | Organism |
---|---|---|
D269A | site-directed mutagenesis | Alkalihalophilus pseudofirmus |
H96A | site-directed mutagenesis | Alkalihalophilus pseudofirmus |
K73A | site-directed mutagenesis, the mutant shows 6fold improvement in kcat/Km towards L-alanine as compared to the wild-type enzyme, the mutant is slightly more temperture-sensitive compared to wild-type | Alkalihalophilus pseudofirmus |
K73E | site-directed mutagenesis | Alkalihalophilus pseudofirmus |
K73F | site-directed mutagenesis | Alkalihalophilus pseudofirmus |
K73Q | site-directed mutagenesis | Alkalihalophilus pseudofirmus |
K73R | site-directed mutagenesis | Alkalihalophilus pseudofirmus |
K73S | site-directed mutagenesis | Alkalihalophilus pseudofirmus |
K73Y | site-directed mutagenesis | Alkalihalophilus pseudofirmus |
K75A | site-directed mutagenesis | Alkalihalophilus pseudofirmus |
additional information | mutations at four conserved residue Arg15, Lys75, His-6 and Asp269 (except residue Lys73) result in a complete loss in enzymatic activity, which signifies that these predicted active sites are indispensable for OF4Ald activity | Alkalihalophilus pseudofirmus |
R15A | site-directed mutagenesis | Alkalihalophilus pseudofirmus |
Inhibitors | Comment | Organism | Structure |
---|---|---|---|
Ca2+ | 80% inhibition at 10 mM, 71% at 1 mM, 21% at 0.1 mM | Alkalihalophilus pseudofirmus | |
Co2+ | complete inhibition at 10 mM, 27% at 0.1 mM | Alkalihalophilus pseudofirmus | |
Cu2+ | complete inhibition at 10 mM, 34% at 0.1 mM | Alkalihalophilus pseudofirmus | |
DTT | 39% inhibition at 10 mM | Alkalihalophilus pseudofirmus | |
EDTA | 34% inhibition at 10 mM | Alkalihalophilus pseudofirmus | |
Mn2+ | complete inhibition at 10 mM, 25% at 1 mM | Alkalihalophilus pseudofirmus | |
Pb2+ | protein OF4Ald completely loses its activity in the presence of 0.1-10 mM Pb2+ | Alkalihalophilus pseudofirmus |
KM Value [mM] | KM Value Maximum [mM] | Substrate | Comment | Organism | Structure |
---|---|---|---|---|---|
0.2 | - |
NAD+ | recombinant His-tagged mutant K73Q, pH 10.5, 40°C | Alkalihalophilus pseudofirmus | |
0.3 | - |
NAD+ | recombinant His-tagged mutant K73A, pH 10.5, 40°C | Alkalihalophilus pseudofirmus | |
0.3 | - |
NAD+ | recombinant His-tagged mutant K73F, pH 10.5, 40°C | Alkalihalophilus pseudofirmus | |
0.3 | - |
NAD+ | recombinant His-tagged wild-type enzyme, pH 10.5, 40°C | Alkalihalophilus pseudofirmus | |
0.4 | - |
NAD+ | recombinant His-tagged mutant K73E, pH 10.5, 40°C | Alkalihalophilus pseudofirmus | |
0.4 | - |
NAD+ | recombinant His-tagged mutant K73R, pH 10.5, 40°C | Alkalihalophilus pseudofirmus | |
0.4 | - |
NAD+ | recombinant His-tagged mutant K73Y, pH 10.5, 40°C | Alkalihalophilus pseudofirmus | |
1 | - |
L-alanine | recombinant His-tagged mutant K73A, pH 10.5, 40°C | Alkalihalophilus pseudofirmus | |
1 | - |
NAD+ | recombinant His-tagged mutant K73S, pH 10.5, 40°C | Alkalihalophilus pseudofirmus | |
2 | - |
L-alanine | recombinant His-tagged mutant K73E, pH 10.5, 40°C | Alkalihalophilus pseudofirmus | |
3 | - |
L-alanine | recombinant His-tagged mutant K73Q, pH 10.5, 40°C | Alkalihalophilus pseudofirmus | |
3 | - |
L-alanine | recombinant His-tagged mutant K73Y, pH 10.5, 40°C | Alkalihalophilus pseudofirmus | |
4 | - |
L-alanine | recombinant His-tagged mutant K73F, pH 10.5, 40°C | Alkalihalophilus pseudofirmus | |
4 | - |
L-alanine | recombinant His-tagged mutant K73R, pH 10.5, 40°C | Alkalihalophilus pseudofirmus | |
4 | - |
L-alanine | recombinant His-tagged mutant K73S, pH 10.5, 40°C | Alkalihalophilus pseudofirmus | |
4 | - |
L-alanine | recombinant His-tagged wild-type enzyme, pH 10.5, 40°C | Alkalihalophilus pseudofirmus |
Metals/Ions | Comment | Organism | Structure |
---|---|---|---|
Mg2+ | results in a slight increase in the enzyme activity at concentrations between 0.1-10 mM, 22% activation at 10 mM | Alkalihalophilus pseudofirmus |
Natural Substrates | Organism | Comment (Nat. Sub.) | Natural Products | Comment (Nat. Pro.) | Rev. | Reac. |
---|---|---|---|---|---|---|
L-alanine + H2O + NAD+ | Alkalihalophilus pseudofirmus | - |
pyruvate + NH3 + NADH + H+ | - |
r | |
L-alanine + H2O + NAD+ | Alkalihalophilus pseudofirmus OF4 | - |
pyruvate + NH3 + NADH + H+ | - |
r | |
L-alanine + H2O + NAD+ | Alkalihalophilus pseudofirmus ATCC BAA-2126 | - |
pyruvate + NH3 + NADH + H+ | - |
r | |
L-alanine + H2O + NAD+ | Alkalihalophilus pseudofirmus JCM 17055 | - |
pyruvate + NH3 + NADH + H+ | - |
r |
Organism | UniProt | Comment | Textmining |
---|---|---|---|
Alkalihalophilus pseudofirmus | D3FSP2 | Bacillus pseudofirmus | - |
Alkalihalophilus pseudofirmus ATCC BAA-2126 | D3FSP2 | Bacillus pseudofirmus | - |
Alkalihalophilus pseudofirmus JCM 17055 | D3FSP2 | Bacillus pseudofirmus | - |
Alkalihalophilus pseudofirmus OF4 | D3FSP2 | Bacillus pseudofirmus | - |
Purification (Comment) | Organism |
---|---|
recombinant His-tagged wild-type and mutant enzymes from Escherichia coli strain BL21(DE3) by nickel affinity chromatography and gel filtration | Alkalihalophilus pseudofirmus |
Specific Activity Minimum [µmol/min/mg] | Specific Activity Maximum [µmol/min/mg] | Comment | Organism |
---|---|---|---|
2 | - |
purified recombinant His-tagged mutant K73Q, pH 10.5, 40°C | Alkalihalophilus pseudofirmus |
5 | - |
purified recombinant His-tagged mutant K73E, pH 10.5, 40°C | Alkalihalophilus pseudofirmus |
6 | - |
purified recombinant His-tagged mutants K73S, K73R, K73Y, and K73F, pH 10.5, 40°C | Alkalihalophilus pseudofirmus |
15 | - |
purified recombinant His-tagged wild-type enzyme, pH 10.5, 40°C | Alkalihalophilus pseudofirmus |
27 | - |
purified recombinant His-tagged mutant K73A, pH 10.5, 40°C | Alkalihalophilus pseudofirmus |
Substrates | Comment Substrates | Organism | Products | Comment (Products) | Rev. | Reac. |
---|---|---|---|---|---|---|
L-alanine + H2O + NAD+ | - |
Alkalihalophilus pseudofirmus | pyruvate + NH3 + NADH + H+ | - |
r | |
L-alanine + H2O + NAD+ | - |
Alkalihalophilus pseudofirmus OF4 | pyruvate + NH3 + NADH + H+ | - |
r | |
L-alanine + H2O + NAD+ | - |
Alkalihalophilus pseudofirmus ATCC BAA-2126 | pyruvate + NH3 + NADH + H+ | - |
r | |
L-alanine + H2O + NAD+ | - |
Alkalihalophilus pseudofirmus JCM 17055 | pyruvate + NH3 + NADH + H+ | - |
r | |
additional information | the activity assay includes L-alanine, NAD+, phenazine methosulfate and 0.24 mM nitroblue tetrazolium in carbonate buffer (50 mn, pH 10.5) | Alkalihalophilus pseudofirmus | ? | - |
- |
|
additional information | the activity assay includes L-alanine, NAD+, phenazine methosulfate and 0.24 mM nitroblue tetrazolium in carbonate buffer (50 mn, pH 10.5) | Alkalihalophilus pseudofirmus OF4 | ? | - |
- |
|
additional information | the activity assay includes L-alanine, NAD+, phenazine methosulfate and 0.24 mM nitroblue tetrazolium in carbonate buffer (50 mn, pH 10.5) | Alkalihalophilus pseudofirmus ATCC BAA-2126 | ? | - |
- |
|
additional information | the activity assay includes L-alanine, NAD+, phenazine methosulfate and 0.24 mM nitroblue tetrazolium in carbonate buffer (50 mn, pH 10.5) | Alkalihalophilus pseudofirmus JCM 17055 | ? | - |
- |
Synonyms | Comment | Organism |
---|---|---|
ALD | - |
Alkalihalophilus pseudofirmus |
L-alanine dehydrogenase | - |
Alkalihalophilus pseudofirmus |
OF4Ald | - |
Alkalihalophilus pseudofirmus |
Temperature Optimum [°C] | Temperature Optimum Maximum [°C] | Comment | Organism |
---|---|---|---|
40 | - |
with L-alanine, deamination reaction, recombinant enzyme | Alkalihalophilus pseudofirmus |
Temperature Minimum [°C] | Temperature Maximum [°C] | Comment | Organism |
---|---|---|---|
- |
60 | over 65% of maxmal activity within this range for the wild-type enzyme | Alkalihalophilus pseudofirmus |
Turnover Number Minimum [1/s] | Turnover Number Maximum [1/s] | Substrate | Comment | Organism | Structure |
---|---|---|---|---|---|
2 | - |
NAD+ | recombinant His-tagged mutant K73Q, pH 10.5, 40°C | Alkalihalophilus pseudofirmus | |
2 | - |
L-alanine | recombinant His-tagged mutant K73Q, pH 10.5, 40°C | Alkalihalophilus pseudofirmus | |
3 | - |
L-alanine | recombinant His-tagged mutant K73E, pH 10.5, 40°C | Alkalihalophilus pseudofirmus | |
4 | - |
L-alanine | recombinant His-tagged mutant K73F, pH 10.5, 40°C | Alkalihalophilus pseudofirmus | |
4 | - |
L-alanine | recombinant His-tagged mutant K73R, pH 10.5, 40°C | Alkalihalophilus pseudofirmus | |
4 | - |
L-alanine | recombinant His-tagged mutant K73S, pH 10.5, 40°C | Alkalihalophilus pseudofirmus | |
4 | - |
L-alanine | recombinant His-tagged mutant K73Y, pH 10.5, 40°C | Alkalihalophilus pseudofirmus | |
5 | - |
NAD+ | recombinant His-tagged mutant K73E, pH 10.5, 40°C | Alkalihalophilus pseudofirmus | |
5 | - |
NAD+ | recombinant His-tagged mutant K73Y, pH 10.5, 40°C | Alkalihalophilus pseudofirmus | |
6 | - |
NAD+ | recombinant His-tagged mutant K73F, pH 10.5, 40°C | Alkalihalophilus pseudofirmus | |
6 | - |
NAD+ | recombinant His-tagged wild-type enzyme, pH 10.5, 40°C | Alkalihalophilus pseudofirmus | |
7 | - |
NAD+ | recombinant His-tagged mutant K73R, pH 10.5, 40°C | Alkalihalophilus pseudofirmus | |
7 | - |
NAD+ | recombinant His-tagged mutant K73S, pH 10.5, 40°C | Alkalihalophilus pseudofirmus | |
8 | - |
NAD+ | recombinant His-tagged mutant K73A, pH 10.5, 40°C | Alkalihalophilus pseudofirmus | |
10 | - |
L-alanine | recombinant His-tagged wild-type enzyme, pH 10.5, 40°C | Alkalihalophilus pseudofirmus | |
18 | - |
L-alanine | recombinant His-tagged mutant K73A, pH 10.5, 40°C | Alkalihalophilus pseudofirmus |
pH Optimum Minimum | pH Optimum Maximum | Comment | Organism |
---|---|---|---|
10.5 | - |
with L-alanine, deamination reaction, recombinant enzyme | Alkalihalophilus pseudofirmus |
pH Minimum | pH Maximum | Comment | Organism |
---|---|---|---|
9 | 11.5 | over 50% of maximal activity within this range wild-type enzyme | Alkalihalophilus pseudofirmus |
Cofactor | Comment | Organism | Structure |
---|---|---|---|
NAD+ | - |
Alkalihalophilus pseudofirmus | |
NADH | - |
Alkalihalophilus pseudofirmus |
General Information | Comment | Organism |
---|---|---|
malfunction | mutations at four conserved residue Arg15, Lys75, His-6, and Asp269 (except residue Lys73) result in a complete loss in enzymatic activity, which signifies that these predicted active sites are indispensable for OF4Ald activity | Alkalihalophilus pseudofirmus |
additional information | five highly conserved amino acid residues Arg15, Lys73, Lys75, His96, and Asp269 are potential catalytic residues of L-alanine dehydrogenase from Bacillus pseudofirmus strain OF4. Enzyme structure homology modeling and structure-function analysis, comparison of the partial potential active site of OF4Ald and its mutants based on the structure of Mycobacterium tuberculosis MtAld (PDB ID 2VHY)., overview. Residue Lys73 is involved in substrate binding | Alkalihalophilus pseudofirmus |
kcat/KM Value [1/mMs-1] | kcat/KM Value Maximum [1/mMs-1] | Substrate | Comment | Organism | Structure |
---|---|---|---|---|---|
0.67 | - |
L-alanine | recombinant His-tagged mutant K73Q, pH 10.5, 40°C | Alkalihalophilus pseudofirmus | |
1 | - |
L-alanine | recombinant His-tagged mutants K73R, K73S, and K73F, pH 10.5, 40°C | Alkalihalophilus pseudofirmus | |
1.33 | - |
L-alanine | recombinant His-tagged mutant K73Y, pH 10.5, 40°C | Alkalihalophilus pseudofirmus | |
1.5 | - |
L-alanine | recombinant His-tagged mutant K73E, pH 10.5, 40°C | Alkalihalophilus pseudofirmus | |
2.5 | - |
L-alanine | recombinant His-tagged wild-type enzyme, pH 10.5, 40°C | Alkalihalophilus pseudofirmus | |
7 | - |
NAD+ | recombinant His-tagged mutant K73S, pH 10.5, 40°C | Alkalihalophilus pseudofirmus | |
12.5 | - |
NAD+ | recombinant His-tagged mutant K73E and K73Y, pH 10.5, 40°C | Alkalihalophilus pseudofirmus | |
17.5 | - |
NAD+ | recombinant His-tagged mutant K73R, pH 10.5, 40°C | Alkalihalophilus pseudofirmus | |
18 | - |
L-alanine | recombinant His-tagged mutant K73A, pH 10.5, 40°C | Alkalihalophilus pseudofirmus | |
20 | - |
NAD+ | recombinant His-tagged mutant K73F, pH 10.5, 40°C | Alkalihalophilus pseudofirmus | |
20 | - |
NAD+ | recombinant His-tagged wild-type enzyme, pH 10.5, 40°C | Alkalihalophilus pseudofirmus | |
25 | - |
NAD+ | recombinant His-tagged mutant K73Q, pH 10.5, 40°C | Alkalihalophilus pseudofirmus | |
26.67 | - |
NAD+ | recombinant His-tagged mutant K73A, pH 10.5, 40°C | Alkalihalophilus pseudofirmus |