Any feedback?
Please rate this page
(literature.php)
(0/150)

BRENDA support

Literature summary for 1.4.1.1 extracted from

  • He, G.; Xu, S.; Wang, S.; Zhang, Q.; Liu, D.; Chen, Y.; Ju, J.; Zhao, B.
    A conserved residue of L-alanine dehydrogenase from Bacillus pseudofirmus, Lys-73, participates in the catalytic reaction through hydrogen bonding (2018), Enzyme Microb. Technol., 110, 61-68 .
    View publication on PubMed

Cloned(Commentary)

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

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

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 [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

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/ Products (Substrates)

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

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 (Commentary)

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 [micromol/min/mg]

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 and Products (Substrate)

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

Synonyms Comment Organism
ALD
-
Alkalihalophilus pseudofirmus
L-alanine dehydrogenase
-
Alkalihalophilus pseudofirmus
OF4Ald
-
Alkalihalophilus pseudofirmus

Temperature Optimum [°C]

Temperature Optimum [°C] Temperature Optimum Maximum [°C] Comment Organism
40
-
with L-alanine, deamination reaction, recombinant enzyme Alkalihalophilus pseudofirmus

Temperature Range [°C]

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 [1/s]

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

pH Optimum Minimum pH Optimum Maximum Comment Organism
10.5
-
with L-alanine, deamination reaction, recombinant enzyme Alkalihalophilus pseudofirmus

pH Range

pH Minimum pH Maximum Comment Organism
9 11.5 over 50% of maximal activity within this range wild-type enzyme Alkalihalophilus pseudofirmus

Cofactor

Cofactor Comment Organism Structure
NAD+
-
Alkalihalophilus pseudofirmus
NADH
-
Alkalihalophilus pseudofirmus

General Information

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 [mM/s]

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