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Literature summary for 2.6.1.42 extracted from

  • Stekhanova, T.N.; Rakitin, A.L.; Mardanov, A.V.; Bezsudnova, E.Y.; Popov, V.O.
    A novel highly thermostable branched-chain amino acid aminotransferase from the crenarchaeon Vulcanisaeta moutnovskia (2017), Enzyme Microb. Technol., 96, 127-134 .
    View publication on PubMed

Cloned(Commentary)

Cloned (Comment) Organism
gene Vmut_0738, DNA and amino acid sequence determination and analysis, sequence comparisons, recombinant expression of His6-tagged enzyme in Escherichia coli strain DLT1270 from vector pQE30_Vmut0738 Vulcanisaeta moutnovskia

KM Value [mM]

KM Value [mM] KM Value Maximum [mM] Substrate Comment Organism Structure
0.84
-
L-norvaline pH 8.0, 65°C Vulcanisaeta moutnovskia
1.17
-
L-valine pH 8.0, 65°C Vulcanisaeta moutnovskia
7.95
-
L-ornithine pH 8.0, 65°C Vulcanisaeta moutnovskia

Molecular Weight [Da]

Molecular Weight [Da] Molecular Weight Maximum [Da] Comment Organism
145000
-
recombinant His6-tagged enzyme, gel filtration Vulcanisaeta moutnovskia

Organism

Organism UniProt Comment Textmining
Vulcanisaeta moutnovskia F0QW25 isolated from a hot spring in Kamchatka, Russia
-
Vulcanisaeta moutnovskia 768-28 F0QW25 isolated from a hot spring in Kamchatka, Russia
-

Oxidation Stability

Oxidation Stability Organism
the enzyme activity in the presence of 15% v/v organic solvents showes significant tolerance of VMUT0738 to solvents Vulcanisaeta moutnovskia

Purification (Commentary)

Purification (Comment) Organism
recombinant His6-tagged enzyme from Escherichia coli strain DLT1270 by nickel affinity chromatography and gel filtration Vulcanisaeta moutnovskia

Source Tissue

Source Tissue Comment Organism Textmining
additional information Vulcanisaeta moutnovskia strain 768-28 is an obligately anaerobic acidophile with a pH range of growth from pH 3.5 to 6.5 and a temperature range of growth from 60 to 98°C Vulcanisaeta moutnovskia
-

Specific Activity [micromol/min/mg]

Specific Activity Minimum [µmol/min/mg] Specific Activity Maximum [µmol/min/mg] Comment Organism
0.0076
-
substrate 2-methylbenzylamine, purified recombinant His-tagged enzyme, pH 8.0, 65°C Vulcanisaeta moutnovskia
0.23
-
substrate pyruvate, with valine, purified recombinant His-tagged enzyme, pH 8.0, 65°C Vulcanisaeta moutnovskia
0.83
-
substrate 3-methyl-2-oxovalerate, purified recombinant His-tagged enzyme, pH 8.0, 65°C Vulcanisaeta moutnovskia
1.1
-
substrate DL-tryptophan, purified recombinant His-tagged enzyme, pH 8.0, 65°C Vulcanisaeta moutnovskia
1.6
-
substrate L-cysteine, purified recombinant His-tagged enzyme, pH 8.0, 65°C Vulcanisaeta moutnovskia
1.6
-
substrate L-phenylalanine, purified recombinant His-tagged enzyme, pH 8.0, 65°C Vulcanisaeta moutnovskia
1.65
-
substrate 4-methyl-2-oxovalerate, purified recombinant His-tagged enzyme, pH 8.0, 65°C Vulcanisaeta moutnovskia
1.65
-
substrate L-norleucine, purified recombinant His-tagged enzyme, pH 8.0, 65°C Vulcanisaeta moutnovskia
1.72
-
substrate L-leucine, purified recombinant His-tagged enzyme, pH 8.0, 65°C Vulcanisaeta moutnovskia
1.76
-
substrate L-histidine, purified recombinant His-tagged enzyme, pH 8.0, 65°C Vulcanisaeta moutnovskia
1.76
-
substrate L-isoleucine, purified recombinant His-tagged enzyme, pH 8.0, 65°C Vulcanisaeta moutnovskia
1.88
-
substrate indole-3-pyruvate, purified recombinant His-tagged enzyme, pH 8.0, 65°C Vulcanisaeta moutnovskia
2.64
-
substrate L-arginine, purified recombinant His-tagged enzyme, pH 8.0, 65°C Vulcanisaeta moutnovskia
2.96
-
substrate L-norvaline, purified recombinant His-tagged enzyme, pH 8.0, 65°C Vulcanisaeta moutnovskia
2.97
-
substrate 2-aminobutyrate, purified recombinant His-tagged enzyme, pH 8.0, 65°C Vulcanisaeta moutnovskia
3
-
substrate L-valine, purified recombinant His-tagged enzyme, pH 8.0, 65°C Vulcanisaeta moutnovskia
3.1
-
substrate 2-oxobutyrate, purified recombinant His-tagged enzyme, pH 8.0, 65°C Vulcanisaeta moutnovskia
3.56
-
substrate L-threonine, purified recombinant His-tagged enzyme, pH 8.0, 65°C Vulcanisaeta moutnovskia
3.9
-
substrate L-ornithine, purified recombinant His-tagged enzyme, pH 8.0, 65°C Vulcanisaeta moutnovskia
3.93
-
substrate DL-lysine, purified recombinant His-tagged enzyme, pH 8.0, 65°C Vulcanisaeta moutnovskia
4.37
-
substrate L-methionine, purified recombinant His-tagged enzyme, pH 8.0, 65°C Vulcanisaeta moutnovskia

Substrates and Products (Substrate)

Substrates Comment Substrates Organism Products Comment (Products) Rev. Reac.
2-methylbenzylamine + 4-methyl-2-oxovalerate
-
Vulcanisaeta moutnovskia acetophenone + L-leucine
-
r
2-methylbenzylamine + 4-methyl-2-oxovalerate
-
Vulcanisaeta moutnovskia 768-28 acetophenone + L-leucine
-
r
2-oxo-3-indolylpropanoate + L-leucine
-
Vulcanisaeta moutnovskia L-tryptophan + 4-methyl-2-oxovalerate
-
r
2-oxo-3-indolylpropanoate + L-leucine
-
Vulcanisaeta moutnovskia 768-28 L-tryptophan + 4-methyl-2-oxovalerate
-
r
2-oxobutyrate + L-leucine
-
Vulcanisaeta moutnovskia 2-aminobutyrate + 4-methyl-2-oxovalerate
-
r
2-oxobutyrate + L-leucine
-
Vulcanisaeta moutnovskia 768-28 2-aminobutyrate + 4-methyl-2-oxovalerate
-
r
3-methyl-2-oxopentanoate + L-leucine
-
Vulcanisaeta moutnovskia L-isoleucine + 4-methyl-2-oxovalerate
-
r
3-methyl-2-oxopentanoate + L-leucine
-
Vulcanisaeta moutnovskia 768-28 L-isoleucine + 4-methyl-2-oxovalerate
-
r
4-methyl-2-oxopentanoate + L-leucine
-
Vulcanisaeta moutnovskia L-leucine + 4-methyl-2-oxovalerate
-
r
DL-lysine + 4-methyl-2-oxovalerate
-
Vulcanisaeta moutnovskia 2-oxo-6-aminohexanoate + L-leucine
-
r
DL-tryptophan + pyruvate
-
Vulcanisaeta moutnovskia 2-oxo-3-indolylpropanoate + L-alanine
-
r
L-alanine + 4-methyl-2-oxovalerate
-
Vulcanisaeta moutnovskia pyruvate + L-leucine
-
r
L-cysteine + 4-methyl-2-oxovalerate
-
Vulcanisaeta moutnovskia 2-oxo-3-thiobutyrate + L-leucine
-
r
L-isoleucine + 4-methyl-2-oxovalerate
-
Vulcanisaeta moutnovskia 3-methyl-2-oxopentanoate + L-leucine
-
r
L-leucine + 4-methyl-2-oxovalerate
-
Vulcanisaeta moutnovskia 4-methyl-2-oxopentanoate + L-leucine
-
r
L-methionine + 4-methyl-2-oxovalerate
-
Vulcanisaeta moutnovskia 4-methylsulfanyl-2-oxobutanoate + L-leucine
-
r
L-norleucine + 4-methyl-2-oxovalerate
-
Vulcanisaeta moutnovskia 2-oxohexanoate + L-leucine
-
r
L-norvaline + 4-methyl-2-oxovalerate
-
Vulcanisaeta moutnovskia 2-oxovalerate + L-leucine
-
r
L-ornithine + 4-methyl-2-oxovalerate
-
Vulcanisaeta moutnovskia ? + L-leucine
-
r
L-phenylalanine + 4-methyl-2-oxovalerate
-
Vulcanisaeta moutnovskia phenylpyruvate + L-leucine
-
r
L-threonine + 4-methyl-2-oxovalerate
-
Vulcanisaeta moutnovskia 2-oxo-3-hydroxybutyrate + L-leucine
-
r
L-valine + 4-methyl-2-oxovalerate
-
Vulcanisaeta moutnovskia 3-methyl-2-oxobutanoate + L-leucine
-
r
additional information enzyme VMUT0738 displays broad and atypical substrate specificity. It shows absence of activity toward acidic substrates, high activity toward basic ones, and low but detectable activity toward the (R)-enantiomer of 2-methylbenzylamine. VMUT0738 shows specificity for typical BCAT substrates, L-branched-chain amino acids (BCAAs) and corresponding keto acids. The same or higher level of activity is observed in the overall reaction with aromatic amino acids, methionine, threonine and 2-aminobuturic acid. But activity with 2-oxoglutarate, which is a typical BCAT amino acceptor, is not detected at a concentration range from 0.5 to 20 mM. VMUT0738 is not active with both the corresponding amino acid Glu and another acidic amino acid (Asp). On the contrary, the specific activity of VMUT0738 with the basic amino acids ornithine, lysine and arginine is high. Enzyme VMUT0738 shows high stereospecificity for L-amino acids, no preference for BCAAs, exhibits unusual substrate specificity toward substrates with a basic side chain, and has no activity toward substrates with an acidic side chain Vulcanisaeta moutnovskia ?
-
-
additional information enzyme VMUT0738 displays broad and atypical substrate specificity. It shows absence of activity toward acidic substrates, high activity toward basic ones, and low but detectable activity toward the (R)-enantiomer of 2-methylbenzylamine. VMUT0738 shows specificity for typical BCAT substrates, L-branched-chain amino acids (BCAAs) and corresponding keto acids. The same or higher level of activity is observed in the overall reaction with aromatic amino acids, methionine, threonine and 2-aminobuturic acid. But activity with 2-oxoglutarate, which is a typical BCAT amino acceptor, is not detected at a concentration range from 0.5 to 20 mM. VMUT0738 is not active with both the corresponding amino acid Glu and another acidic amino acid (Asp). On the contrary, the specific activity of VMUT0738 with the basic amino acids ornithine, lysine and arginine is high. Enzyme VMUT0738 shows high stereospecificity for L-amino acids, no preference for BCAAs, exhibits unusual substrate specificity toward substrates with a basic side chain, and has no activity toward substrates with an acidic side chain Vulcanisaeta moutnovskia 768-28 ?
-
-
pyruvate + L-alanine
-
Vulcanisaeta moutnovskia L-alanine + pyruvate
-
r
pyruvate + L-leucine
-
Vulcanisaeta moutnovskia L-alanine + 4-methyl-2-oxovalerate
-
r

Subunits

Subunits Comment Organism
homotetramer 4 * 36800, a dimer of dimers, recombinant His6-tagged enzyme, SDS-PAGE Vulcanisaeta moutnovskia
More the catalytically competent unit of BCATs is a dimer Vulcanisaeta moutnovskia

Synonyms

Synonyms Comment Organism
BcaT
-
Vulcanisaeta moutnovskia
branched-chain amino acid aminotransferase
-
Vulcanisaeta moutnovskia
VMUT0738
-
Vulcanisaeta moutnovskia
VMUT_0738 locus name Vulcanisaeta moutnovskia

Temperature Optimum [°C]

Temperature Optimum [°C] Temperature Optimum Maximum [°C] Comment Organism
65 70 assay at Vulcanisaeta moutnovskia

Temperature Range [°C]

Temperature Minimum [°C] Temperature Maximum [°C] Comment Organism
60 90 activity of VMUT0738 increases with a rise in the temperature from 60°C to 90°C Vulcanisaeta moutnovskia

Temperature Stability [°C]

Temperature Stability Minimum [°C] Temperature Stability Maximum [°C] Comment Organism
70
-
purified enzyme VMUT0738, 24 h, 73% of the initial activity remains Vulcanisaeta moutnovskia

Turnover Number [1/s]

Turnover Number Minimum [1/s] Turnover Number Maximum [1/s] Substrate Comment Organism Structure
2.07
-
L-norvaline pH 8.0, 65°C Vulcanisaeta moutnovskia
2.1
-
L-valine pH 8.0, 65°C Vulcanisaeta moutnovskia
2.44
-
L-ornithine pH 8.0, 65°C Vulcanisaeta moutnovskia

pH Optimum

pH Optimum Minimum pH Optimum Maximum Comment Organism
8
-
-
Vulcanisaeta moutnovskia

Cofactor

Cofactor Comment Organism Structure
pyridoxal 5'-phosphate PLP, dependent on Vulcanisaeta moutnovskia

General Information

General Information Comment Organism
evolution branched-chain amino acid aminotransferases (BCATs) belong to fold-type IV class of PLP enzymes and are referred to as alpha-aminotransferases. Sequence alignment reveals two motifs (V/I)xLDxR and PFG(K/H)YL characteristic of BCATs from species of the related genera Vulcanisaeta, Pyrobaculum and Thermoproteus that might be responsible for the unique substrate recognition profile of the enzyme Vulcanisaeta moutnovskia
additional information enzyme structure comparisons, overview Vulcanisaeta moutnovskia

kcat/KM [mM/s]

kcat/KM Value [1/mMs-1] kcat/KM Value Maximum [1/mMs-1] Substrate Comment Organism Structure
0.31
-
L-ornithine pH 8.0, 65°C Vulcanisaeta moutnovskia
1.8
-
L-valine pH 8.0, 65°C Vulcanisaeta moutnovskia
2.46
-
L-norvaline pH 8.0, 65°C Vulcanisaeta moutnovskia