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

BRENDA support

Literature summary for 2.6.1.B21 extracted from

  • Zeifman, Y.S.; Boyko, K.M.; Nikolaeva, A.Y.; Timofeev, V.I.; Rakitina, T.V.; Popov, V.O.; Bezsudnova, E.Y.
    Functional characterization of PLP fold type IV transaminase with a mixed type of activity from Haliangium ochraceum (2019), Biochim. Biophys. Acta, 1867, 575-585 .
    View publication on PubMed

Application

Application Comment Organism
synthesis pyridoxal-5'-phosphate (PLP)-dependent transaminases are industrially important enzymes catalyzing the stereoselective amination of ketones and keto acids. Transaminases of PLP fold type IV are characterized by (R)- or (S)-stereoselective transfer of amino groups, depending on the substrate profile of the enzyme Haliangium ochraceum

Cloned(Commentary)

Cloned (Comment) Organism
recombinant expression of His-tagged enzyme in Escherichia coli strain Rosetta (DE3)pLysS Haliangium ochraceum

Crystallization (Commentary)

Crystallization (Comment) Organism
purified native enzyme, hanging drop vapor diffusion technique, 20°C, mixing of 10 mg/ml protein in 15 mM Tris, pH 8.0, 50 mM NaCl, and 0.02 mM PLP, with reservoir solution containing 0.1 M MES, pH 6.0, 0.2 M CaCl2, and 50% methyl-2,4-pentanediol, method screening and optimization, X-ray diffraction structure determination and analysis at 2.35 A resolution, molecular replacement method using the structure of the branched-chain amino acid aminotransferase from Burkholderia pseudomallei (PDB ID 3U0G) as a template, modeling Haliangium ochraceum
purified recombinant detagged enzyme, hanging drop vapor diffusion method, mixing of 10 mg/ml protein solution with reservoir solution containing 0.1 M MES, pH 6.0, 0.2 M CaCl2, and 50% methyl-2,4-pentanediol, 20°C, method optimization, X-ray diffraction structure determination and analysis at 2.35 A resolution, molecular replacement method using the structure of the branched-chain amino acid aminotransferase from Burkholderia pseudomallei (PDB ID 3U0G) as a template, modelling Haliangium ochraceum

Protein Variants

Protein Variants Comment Organism
H106Y/Y108R site-directed mutagenesis Haliangium ochraceum

Inhibitors

Inhibitors Comment Organism Structure
acetonitril over 90% inhibition at 20-30%; strong inhibition at 20-30% Haliangium ochraceum
DMSO 40% inhibition at 20%, 70% inhibition at 30%; slight to moderate inhibition at 20-30% Haliangium ochraceum
methanol 65% inhibition at 20%, 85% inhibition at 30%; moderate inhibition at 20-30% Haliangium ochraceum

KM Value [mM]

KM Value [mM] KM Value Maximum [mM] Substrate Comment Organism Structure
additional information
-
additional information steady-state kinetics Haliangium ochraceum
additional information
-
additional information Michaelis-Menten steady-state kinetics of the first half-reaction Haliangium ochraceum
0.55
-
3-methyl-2-oxovalerate pH 10.0, 40°C Haliangium ochraceum
0.55
-
3-methyl-2-oxovalerate recombinant enzyme, pH 10.0, 40°C, with (R)-alpha-methylbenzylamine Haliangium ochraceum
2 3 (R)-alpha-methylbenzylamine pH 10.0, 40°C, with 2-oxoglutarate Haliangium ochraceum
6
-
(R)-alpha-methylbenzylamine pH 10.0, 40°C, with 5 mM 3-methyl-2-oxovalerate Haliangium ochraceum
6
-
(R)-alpha-methylbenzylamine recombinant enzyme, pH 10.0, 40°C, with 3-methyl-2-oxovalerate Haliangium ochraceum
10.4
-
(R)-alpha-ethylbenzylamine pH 10.0, 40°C Haliangium ochraceum
11.8
-
(R)-alpha-methylbenzylamine pH 10.0, 40°C, with 1.5 mM 3-methyl-2-oxovalerate Haliangium ochraceum
11.8
-
(R)-alpha-methylbenzylamine recombinant enzyme, pH 9.0, 40°C, with 3-methyl-2-oxovalerate Haliangium ochraceum

Metals/Ions

Metals/Ions Comment Organism Structure
NaCl the enzyme shows tolerance to high salt concentrations up to 2 M NaCl. Addition of up to 400 mM NaCl in the reaction mixture results in a slight increase in the activity of enzyme Hoch3033 Haliangium ochraceum
NaCl the enzyme shows tolerance to high salt concentrations, up to 2 M NaCl Haliangium ochraceum

Molecular Weight [Da]

Molecular Weight [Da] Molecular Weight Maximum [Da] Comment Organism
70000
-
gel filtration Haliangium ochraceum

Organism

Organism UniProt Comment Textmining
Haliangium ochraceum
-
-
-
Haliangium ochraceum D0LR31
-
-
Haliangium ochraceum DSM 14365 D0LR31
-
-
Haliangium ochraceum JCM 11303 D0LR31
-
-
Haliangium ochraceum SMP-2 D0LR31
-
-

Purification (Commentary)

Purification (Comment) Organism
native enzyme to homogeneity Haliangium ochraceum
recombinant His-tagged enzyme from Escherichia coli strain Rosetta (DE3)pLysS by nickel affinity chromatgraphy, tag cleavage through TEV protease, another step of nickel affinity chromatgraphy, followed by gel filtration Haliangium ochraceum

Reaction

Reaction Comment Organism Reaction ID
(R)-alpha-methylbenzylamine + 2-oxo acid = acetophenone + L-amino acid first transaminase half-reaction via internal aldimine, external aldimine, ketimine, and pyridoxamine (PMP)-bound form Haliangium ochraceum

Specific Activity [micromol/min/mg]

Specific Activity Minimum [µmol/min/mg] Specific Activity Maximum [µmol/min/mg] Comment Organism
0.0042
-
purified enzyme, pH 10.0, 40°C, substrate 2-oxoglutarate Haliangium ochraceum
0.0042
-
purified recombinant enzyme, pH 10.0, 40°C, substrate (R)-alpha-methylbenzylamine and 2-oxoglutarate Haliangium ochraceum
0.007
-
purified enzyme, pH 10.0, 40°C, substrate pyruvate Haliangium ochraceum
0.007
-
purified recombinant enzyme, pH 10.0, 40°C, substrate (R)-alpha-methylbenzylamine and pyruvate Haliangium ochraceum
0.013
-
purified recombinant enzyme, pH 10.0, 40°C, substrate (R)-alpha-ethylbenzylamine and 3-methyl-2-oxovalerate Haliangium ochraceum
0.025
-
purified enzyme, pH 10.0, 40°C, substrate 2-oxobutyrate Haliangium ochraceum
0.025
-
purified recombinant enzyme, pH 10.0, 40°C, substrate (R)-alpha-methylbenzylamine and 2-oxobutyrate Haliangium ochraceum
0.12
-
purified enzyme, pH 10.0, 40°C, substrate 3-methyl-2-oxobutyrate Haliangium ochraceum
0.12
-
purified recombinant enzyme, pH 10.0, 40°C, substrate (R)-alpha-methylbenzylamine and 3-methyl-2-oxobutyrate Haliangium ochraceum
0.135
-
purified enzyme, pH 10.0, 40°C, substrate 2-oxohexanoate Haliangium ochraceum
0.135
-
purified recombinant enzyme, pH 10.0, 40°C, substrate (R)-alpha-methylbenzylamine and 2-oxohexanoate Haliangium ochraceum
0.15
-
purified enzyme, pH 10.0, 40°C, substrate 4-methyl-2-oxovalerate Haliangium ochraceum
0.15
-
purified recombinant enzyme, pH 10.0, 40°C, substrate (R)-alpha-methylbenzylamine and 4-methyl-2-oxovalerate Haliangium ochraceum
0.19
-
purified enzyme, pH 10.0, 40°C, substrate 3-methyl-2-oxovalerate Haliangium ochraceum
0.19
-
purified recombinant enzyme, pH 10.0, 40°C, substrate (R)-alpha-methylbenzylamine and 3-methyl-2-oxovalerate Haliangium ochraceum
0.23
-
purified enzyme, pH 10.0, 40°C, substrate 2-oxovalerate Haliangium ochraceum
0.23
-
purified recombinant enzyme, pH 10.0, 40°C, substrate (R)-alpha-methylbenzylamine and 2-oxovalerate Haliangium ochraceum

Substrates and Products (Substrate)

Substrates Comment Substrates Organism Products Comment (Products) Rev. Reac.
(R)-alpha-ethylbenzylamine + 3-methyl-2-oxovalerate reaction of (R)-amine:pyruvate transaminase, EC 2.6.1.B21 Haliangium ochraceum alpha-ethylbenzaldehyde + L-isoleucine
-
r
(R)-alpha-ethylbenzylamine + pyruvate R-EtBA Haliangium ochraceum alpha-ethylbenzaldehyde + D-alanine
-
r
(R)-alpha-methylbenzylamine + 2-oxobutyrate
-
Haliangium ochraceum acetophenone + L-alpha-aminobutyric acid
-
r
(R)-alpha-methylbenzylamine + 2-oxoglutarate
-
Haliangium ochraceum acetophenone + L-glutamate
-
r
(R)-alpha-methylbenzylamine + 2-oxoglutarate reaction of (R)-amine:pyruvate transaminase, EC 2.6.1.B21 Haliangium ochraceum acetophenone + L-glutamate
-
r
(R)-alpha-methylbenzylamine + 2-oxohexanoate
-
Haliangium ochraceum acetophenone + L-norleucine
-
r
(R)-alpha-methylbenzylamine + 2-oxovalerate
-
Haliangium ochraceum acetophenone + L-norvaline
-
r
(R)-alpha-methylbenzylamine + 3-methyl-2-oxobutyrate
-
Haliangium ochraceum acetophenone + L-valine
-
r
(R)-alpha-methylbenzylamine + 3-methyl-2-oxovalerate
-
Haliangium ochraceum acetophenone + D-isoleucine
-
r
(R)-alpha-methylbenzylamine + 3-methyl-2-oxovalerate
-
Haliangium ochraceum acetophenone + L-isoleucine
-
r
(R)-alpha-methylbenzylamine + 3-methyl-2-oxovalerate reaction of (R)-amine:pyruvate transaminase, EC 2.6.1.B21 Haliangium ochraceum acetophenone + L-isoleucine
-
r
(R)-alpha-methylbenzylamine + 4-methyl-2-oxovalerate
-
Haliangium ochraceum acetophenone + L-leucine
-
r
(R)-alpha-methylbenzylamine + pyruvate R-MBA, specific substrate for (R)-amine:pyruvate transaminases Haliangium ochraceum acetophenone + D-alanine
-
r
2-oxohexanoate + L-glutamate reaction of branched-chain amino acid transaminase, EC 2.6.1.42 Haliangium ochraceum norleucine + 2-oxoglutarate
-
r
L-alanine + 2-oxoglutarate reaction of branched-chain amino acid transaminase, EC 2.6.1.42 Haliangium ochraceum pyruvate + L-glutamate
-
r
L-isoleucine + 2-oxoglutarate reaction of branched-chain amino acid transaminase, EC 2.6.1.42 Haliangium ochraceum 3-methyl-2-oxopentanoate + L-glutamate
-
r
L-leucine + 2-oxoglutarate reaction of branched-chain amino acid transaminase, EC 2.6.1.42 Haliangium ochraceum 4-methyl-2-oxopentanoate + L-glutamate
-
r
L-valine + 2-oxoglutarate reaction of branched-chain amino acid transaminase, EC 2.6.1.42 Haliangium ochraceum 3-methyl-2-oxobutanoate + L-glutamate
-
r
additional information enzyme Hoch3033 is a transaminases with a n enzyme with mixed type of activity showing branched-chain amino acid amintransferase activity (EC 2.6.1.42) and (R)-amine:pyruvate transaminase (EC 2.6.1). The mixed type of activity of Hoch3033 is implemented within the BCAT-like active site, but in the active site of Hoch3033, substitutions of specificity-determining residues, that are important for substrate binding in canonical BCATs, are observed. In the standard assay, the specific activity towards (S)-alpha-methylbenzylamine (S-MBA) is only 0.32% of the activity towards R-MBA. A much lower value of activity is obtained with (R)-alpha-ethylbenzylamine (R-EtBA), the closest homologue of R-MBA. No activity towards S-EtBA is observed. Substrate specificity overview Haliangium ochraceum ?
-
-
additional information the bifunctional transaminase from myxobacterium Haliangium ochraceum, encoded by gene Hoch3033, is active towards keto analogues of branched-chain amino acids (specific substrates for BCATs, EC 2.6.1.42) and (R)-alpha-methylbenzylamine (specific substrate for (R)-amine:pyruvate transaminases, EC 2.6.1.B21). The enzyme shows no activity with (S)-2-methylbenzylamine, 2-amino-5-methylhexane, 1-methyl-3-phenyl-propylamine, (R)-2-aminohexane, D-alanine, L-beta-leucine, and beta-alanine Haliangium ochraceum ?
-
-
norvaline + 2-oxoglutarate reaction of branched-chain amino acid transaminase, EC 2.6.1.42 Haliangium ochraceum 2-oxovalerate + L-glutamate
-
r

Subunits

Subunits Comment Organism
homodimer
-
Haliangium ochraceum

Synonyms

Synonyms Comment Organism
(R)-amine:pyruvate transaminase
-
Haliangium ochraceum
halotolerant R-TA
-
Haliangium ochraceum
Hoch3033
-
Haliangium ochraceum
IlvE
-
Haliangium ochraceum
More see also branched-chain amino acid transaminase, EC 2.6.1.42 Haliangium ochraceum
R-TA
-
Haliangium ochraceum

Temperature Optimum [°C]

Temperature Optimum [°C] Temperature Optimum Maximum [°C] Comment Organism
40 45
-
Haliangium ochraceum
45
-
-
Haliangium ochraceum

Temperature Range [°C]

Temperature Minimum [°C] Temperature Maximum [°C] Comment Organism
40 60 maximal activity at 40-45°C, 40% of maximal activity at 60°C Haliangium ochraceum

Temperature Stability [°C]

Temperature Stability Minimum [°C] Temperature Stability Maximum [°C] Comment Organism
60
-
the purified recombinant enzyme retains about 40% of the maximum activity at pH 11.0 Haliangium ochraceum

Turnover Number [1/s]

Turnover Number Minimum [1/s] Turnover Number Maximum [1/s] Substrate Comment Organism Structure
0.0113
-
(R)-alpha-ethylbenzylamine pH 10.0, 40°C, with 2-oxoglutarate Haliangium ochraceum
0.15
-
3-methyl-2-oxovalerate pH 10.0, 40°C Haliangium ochraceum
0.15
-
3-methyl-2-oxovalerate recombinant enzyme, pH 10.0, 40°C, with (R)-alpha-methylbenzylamine Haliangium ochraceum
0.19
-
(R)-alpha-methylbenzylamine pH 10.0, 40°C, with 1.5 mM 3-methyl-2-oxovalerate Haliangium ochraceum
0.19
-
(R)-alpha-methylbenzylamine recombinant enzyme, pH 9.0, 40°C, with 3-methyl-2-oxovalerate Haliangium ochraceum
0.22
-
(R)-alpha-methylbenzylamine pH 10.0, 40°C, with 5 mM 3-methyl-2-oxovalerate Haliangium ochraceum
0.22
-
(R)-alpha-methylbenzylamine recombinant enzyme, pH 10.0, 40°C, with 3-methyl-2-oxovalerate Haliangium ochraceum
0.23
-
(R)-alpha-methylbenzylamine pH 10.0, 40°C, with 2-oxoglutarate Haliangium ochraceum

pH Optimum

pH Optimum Minimum pH Optimum Maximum Comment Organism
10
-
-
Haliangium ochraceum
10 10.5
-
Haliangium ochraceum

pH Range

pH Minimum pH Maximum Comment Organism
10 11 maximal activity at pH 10.0, 70% of maximal activity at pH 11.0 Haliangium ochraceum

pH Stability

pH Stability pH Stability Maximum Comment Organism
11
-
the purified recombinant enzyme retains more than 70% of the maximum activity at pH 11.0 Haliangium ochraceum

Cofactor

Cofactor Comment Organism Structure
pyridoxal 5'-phosphate PLP, dependent on Haliangium ochraceum

General Information

General Information Comment Organism
evolution the enzyme belongs to the PLP fold type IV transaminases. PLP fold type IV transaminases include branched-chain amino acid transaminases (BCATs), D-amino acid transaminases, and (R)-amine:pyruvate transaminases Haliangium ochraceum
evolution the enzyme is a PLP fold type IV transaminase. PLP fold type IV transaminases include branched-chain amino acid transaminases (BCATs), D-amino acid transaminases, and (R)-amine:pyruvate transaminases. Enzyme Hoch3033 is a transaminases with mixed type of activity. The mixed type of activity of Hoch3033 is implemented within the BCAT-like active site, but in the active site of Hoch3033, substitutions of specificity-determining residues, that are important for substrate binding in canonical BCATs, are observed Haliangium ochraceum
additional information enzyme Hoch3033 active site structure and substrate binding. Hoch3033 has changes in the specificity determining key residues, which constitute characteristic sequence motifs, overview Haliangium ochraceum
additional information the mixed type of activity of the enzyme is implemented within the BCAT-like active site. In the active site of the enzyme, substitutions of specificity-determining residues, that are important for substrate binding in canonical BCATs, are observed. These changes result in the loss of activity towards 2-oxoglutarate and increase the affinity towards (R)-alpha-methylbenzylamine. Active structure analysis Haliangium ochraceum
physiological function enzyme Hoch3033 is a transaminases with mixed type of activity. The mixed type of activity of Hoch3033 is implemented within the BCAT-like active site, but in the active site of Hoch3033, substitutions of specificity-determining residues, that are important for substrate binding in canonical BCATs, are observed. These changes (H106Y/Y108R) result in the loss of activity towards 2-oxoglutarate and increase the affinity towards (R)-alpha-methylbenzylamine Haliangium ochraceum

kcat/KM [mM/s]

kcat/KM Value [1/mMs-1] kcat/KM Value Maximum [1/mMs-1] Substrate Comment Organism Structure
0.0011
-
(R)-alpha-ethylbenzylamine pH 10.0, 40°C, with 2-oxoglutarate Haliangium ochraceum
0.01
-
(R)-alpha-methylbenzylamine pH 10.0, 40°C, with 2-oxoglutarate Haliangium ochraceum
0.016
-
(R)-alpha-methylbenzylamine pH 10.0, 40°C, with 1.5 mM 3-methyl-2-oxovalerate Haliangium ochraceum
0.016
-
(R)-alpha-methylbenzylamine recombinant enzyme, pH 9.0, 40°C, with 3-methyl-2-oxovalerate Haliangium ochraceum
0.037
-
(R)-alpha-methylbenzylamine pH 10.0, 40°C, with 5 mM 3-methyl-2-oxovalerate Haliangium ochraceum
0.037
-
(R)-alpha-methylbenzylamine recombinant enzyme, pH 10.0, 40°C, with 3-methyl-2-oxovalerate Haliangium ochraceum
0.27
-
3-methyl-2-oxovalerate pH 10.0, 40°C Haliangium ochraceum
0.27
-
3-methyl-2-oxovalerate recombinant enzyme, pH 10.0, 40°C, with (R)-alpha-methylbenzylamine Haliangium ochraceum