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  • 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

EC Number Application Comment Organism
2.6.1.B21 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
2.6.1.42 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)

EC Number Cloned (Comment) Organism
2.6.1.B21 recombinant expression of His-tagged enzyme in Escherichia coli strain Rosetta (DE3)pLysS Haliangium ochraceum

Crystallization (Commentary)

EC Number Crystallization (Comment) Organism
2.6.1.B21 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
2.6.1.B21 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
2.6.1.42 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

Protein Variants

EC Number Protein Variants Comment Organism
2.6.1.B21 H106Y/Y108R site-directed mutagenesis Haliangium ochraceum
2.6.1.42 H106Y/Y108R site-directed mutagenesis Haliangium ochraceum

Inhibitors

EC Number Inhibitors Comment Organism Structure
2.6.1.B21 acetonitril over 90% inhibition at 20-30%; strong inhibition at 20-30% Haliangium ochraceum
2.6.1.B21 DMSO 40% inhibition at 20%, 70% inhibition at 30%; slight to moderate inhibition at 20-30% Haliangium ochraceum
2.6.1.B21 methanol 65% inhibition at 20%, 85% inhibition at 30%; moderate inhibition at 20-30% Haliangium ochraceum
2.6.1.42 acetonitril over 90% inhibition at 20-30% Haliangium ochraceum
2.6.1.42 DMSO 40% inhibition at 20%, 70% inhibition at 30% Haliangium ochraceum
2.6.1.42 methanol 65% inhibition at 20%, 85% inhibition at 30% Haliangium ochraceum

KM Value [mM]

EC Number KM Value [mM] KM Value Maximum [mM] Substrate Comment Organism Structure
2.6.1.B21 additional information
-
additional information steady-state kinetics Haliangium ochraceum
2.6.1.B21 additional information
-
additional information Michaelis-Menten steady-state kinetics of the first half-reaction Haliangium ochraceum
2.6.1.B21 0.55
-
3-methyl-2-oxovalerate pH 10.0, 40°C Haliangium ochraceum
2.6.1.B21 0.55
-
3-methyl-2-oxovalerate recombinant enzyme, pH 10.0, 40°C, with (R)-alpha-methylbenzylamine Haliangium ochraceum
2.6.1.B21 2 3 (R)-alpha-methylbenzylamine pH 10.0, 40°C, with 2-oxoglutarate Haliangium ochraceum
2.6.1.B21 6
-
(R)-alpha-methylbenzylamine pH 10.0, 40°C, with 5 mM 3-methyl-2-oxovalerate Haliangium ochraceum
2.6.1.B21 6
-
(R)-alpha-methylbenzylamine recombinant enzyme, pH 10.0, 40°C, with 3-methyl-2-oxovalerate Haliangium ochraceum
2.6.1.B21 10.4
-
(R)-alpha-ethylbenzylamine pH 10.0, 40°C Haliangium ochraceum
2.6.1.B21 11.8
-
(R)-alpha-methylbenzylamine pH 10.0, 40°C, with 1.5 mM 3-methyl-2-oxovalerate Haliangium ochraceum
2.6.1.B21 11.8
-
(R)-alpha-methylbenzylamine recombinant enzyme, pH 9.0, 40°C, with 3-methyl-2-oxovalerate Haliangium ochraceum
2.6.1.42 additional information
-
additional information Michaelis-Menten steady-state kinetics of the first half-reaction Haliangium ochraceum
2.6.1.42 0.32
-
L-norvaline pH 10.0, 40°C Haliangium ochraceum
2.6.1.42 0.58
-
L-isoleucine pH 10.0, 40°C Haliangium ochraceum
2.6.1.42 0.65
-
L-leucine pH 10.0, 40°C Haliangium ochraceum
2.6.1.42 6.5
-
L-valine pH 10.0, 40°C Haliangium ochraceum

Metals/Ions

EC Number Metals/Ions Comment Organism Structure
2.6.1.B21 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
2.6.1.B21 NaCl the enzyme shows tolerance to high salt concentrations, up to 2 M NaCl Haliangium ochraceum
2.6.1.42 NaCl the enzyme shows tolerance to high salt concentrations up to 2 M NaCl. Addition of up to 400 mM NaCl results in a slight increase in the activity of enzyme Hoch3033 Haliangium ochraceum

Molecular Weight [Da]

EC Number Molecular Weight [Da] Molecular Weight Maximum [Da] Comment Organism
2.6.1.B21 70000
-
gel filtration Haliangium ochraceum
2.6.1.42 70000
-
gel filtration Haliangium ochraceum

Natural Substrates/ Products (Substrates)

EC Number Natural Substrates Organism Comment (Nat. Sub.) Natural Products Comment (Nat. Pro.) Rev. Reac.
2.6.1.42 L-isoleucine + 2-oxoglutarate Haliangium ochraceum
-
3-methyl-2-oxopentanoate + L-glutamate
-
r
2.6.1.42 L-isoleucine + 2-oxoglutarate Haliangium ochraceum JCM 11303
-
3-methyl-2-oxopentanoate + L-glutamate
-
r
2.6.1.42 L-isoleucine + 2-oxoglutarate Haliangium ochraceum DSM 14365
-
3-methyl-2-oxopentanoate + L-glutamate
-
r
2.6.1.42 L-isoleucine + 2-oxoglutarate Haliangium ochraceum SMP-2
-
3-methyl-2-oxopentanoate + L-glutamate
-
r
2.6.1.42 L-leucine + 2-oxoglutarate Haliangium ochraceum
-
4-methyl-2-oxopentanoate + L-glutamate
-
r
2.6.1.42 L-leucine + 2-oxoglutarate Haliangium ochraceum JCM 11303
-
4-methyl-2-oxopentanoate + L-glutamate
-
r
2.6.1.42 L-leucine + 2-oxoglutarate Haliangium ochraceum DSM 14365
-
4-methyl-2-oxopentanoate + L-glutamate
-
r
2.6.1.42 L-leucine + 2-oxoglutarate Haliangium ochraceum SMP-2
-
4-methyl-2-oxopentanoate + L-glutamate
-
r
2.6.1.42 L-valine + 2-oxoglutarate Haliangium ochraceum
-
3-methyl-2-oxobutanoate + L-glutamate
-
r

Organism

EC Number Organism UniProt Comment Textmining
2.6.1.B21 Haliangium ochraceum
-
-
-
2.6.1.B21 Haliangium ochraceum D0LR31
-
-
2.6.1.B21 Haliangium ochraceum DSM 14365 D0LR31
-
-
2.6.1.B21 Haliangium ochraceum JCM 11303 D0LR31
-
-
2.6.1.B21 Haliangium ochraceum SMP-2 D0LR31
-
-
2.6.1.42 Haliangium ochraceum D0LR31
-
-
2.6.1.42 Haliangium ochraceum DSM 14365 D0LR31
-
-
2.6.1.42 Haliangium ochraceum JCM 11303 D0LR31
-
-
2.6.1.42 Haliangium ochraceum SMP-2 D0LR31
-
-

Purification (Commentary)

EC Number Purification (Comment) Organism
2.6.1.B21 native enzyme to homogeneity Haliangium ochraceum
2.6.1.B21 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
2.6.1.42 native enzyme to homogeneity Haliangium ochraceum

Reaction

EC Number Reaction Comment Organism Reaction ID
2.6.1.B21 (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
2.6.1.42 L-leucine + 2-oxoglutarate = 4-methyl-2-oxopentanoate + L-glutamate first transaminase half-reaction via internal aldimine, external aldimine, ketimine, and pyridoxamine (PMP)-bound form Haliangium ochraceum

Specific Activity [micromol/min/mg]

EC Number Specific Activity Minimum [µmol/min/mg] Specific Activity Maximum [µmol/min/mg] Comment Organism
2.6.1.B21 0.0042
-
purified enzyme, pH 10.0, 40°C, substrate 2-oxoglutarate Haliangium ochraceum
2.6.1.B21 0.0042
-
purified recombinant enzyme, pH 10.0, 40°C, substrate (R)-alpha-methylbenzylamine and 2-oxoglutarate Haliangium ochraceum
2.6.1.B21 0.007
-
purified enzyme, pH 10.0, 40°C, substrate pyruvate Haliangium ochraceum
2.6.1.B21 0.007
-
purified recombinant enzyme, pH 10.0, 40°C, substrate (R)-alpha-methylbenzylamine and pyruvate Haliangium ochraceum
2.6.1.B21 0.013
-
purified recombinant enzyme, pH 10.0, 40°C, substrate (R)-alpha-ethylbenzylamine and 3-methyl-2-oxovalerate Haliangium ochraceum
2.6.1.B21 0.025
-
purified enzyme, pH 10.0, 40°C, substrate 2-oxobutyrate Haliangium ochraceum
2.6.1.B21 0.025
-
purified recombinant enzyme, pH 10.0, 40°C, substrate (R)-alpha-methylbenzylamine and 2-oxobutyrate Haliangium ochraceum
2.6.1.B21 0.12
-
purified enzyme, pH 10.0, 40°C, substrate 3-methyl-2-oxobutyrate Haliangium ochraceum
2.6.1.B21 0.12
-
purified recombinant enzyme, pH 10.0, 40°C, substrate (R)-alpha-methylbenzylamine and 3-methyl-2-oxobutyrate Haliangium ochraceum
2.6.1.B21 0.135
-
purified enzyme, pH 10.0, 40°C, substrate 2-oxohexanoate Haliangium ochraceum
2.6.1.B21 0.135
-
purified recombinant enzyme, pH 10.0, 40°C, substrate (R)-alpha-methylbenzylamine and 2-oxohexanoate Haliangium ochraceum
2.6.1.B21 0.15
-
purified enzyme, pH 10.0, 40°C, substrate 4-methyl-2-oxovalerate Haliangium ochraceum
2.6.1.B21 0.15
-
purified recombinant enzyme, pH 10.0, 40°C, substrate (R)-alpha-methylbenzylamine and 4-methyl-2-oxovalerate Haliangium ochraceum
2.6.1.B21 0.19
-
purified enzyme, pH 10.0, 40°C, substrate 3-methyl-2-oxovalerate Haliangium ochraceum
2.6.1.B21 0.19
-
purified recombinant enzyme, pH 10.0, 40°C, substrate (R)-alpha-methylbenzylamine and 3-methyl-2-oxovalerate Haliangium ochraceum
2.6.1.B21 0.23
-
purified enzyme, pH 10.0, 40°C, substrate 2-oxovalerate Haliangium ochraceum
2.6.1.B21 0.23
-
purified recombinant enzyme, pH 10.0, 40°C, substrate (R)-alpha-methylbenzylamine and 2-oxovalerate Haliangium ochraceum
2.6.1.42 0.0042
-
purified enzyme, pH 10.0, 40°C, substrate 2-oxoglutarate Haliangium ochraceum
2.6.1.42 0.007
-
purified enzyme, pH 10.0, 40°C, substrate pyruvate Haliangium ochraceum
2.6.1.42 0.025
-
purified enzyme, pH 10.0, 40°C, substrate 2-oxobutyrate Haliangium ochraceum
2.6.1.42 0.12
-
purified enzyme, pH 10.0, 40°C, substrate 3-methyl-2-oxobutyrate Haliangium ochraceum
2.6.1.42 0.135
-
purified enzyme, pH 10.0, 40°C, substrate 2-oxohexanoate Haliangium ochraceum
2.6.1.42 0.15
-
purified enzyme, pH 10.0, 40°C, substrate 4-methyl-2-oxovalerate Haliangium ochraceum
2.6.1.42 0.19
-
purified enzyme, pH 10.0, 40°C, substrate 3-methyl-2-oxovalerate Haliangium ochraceum
2.6.1.42 0.23
-
purified enzyme, pH 10.0, 40°C, substrate 2-oxovalerate Haliangium ochraceum

Substrates and Products (Substrate)

EC Number Substrates Comment Substrates Organism Products Comment (Products) Rev. Reac.
2.6.1.B21 (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
2.6.1.B21 (R)-alpha-ethylbenzylamine + pyruvate R-EtBA Haliangium ochraceum alpha-ethylbenzaldehyde + D-alanine
-
r
2.6.1.B21 (R)-alpha-methylbenzylamine + 2-oxobutyrate
-
Haliangium ochraceum acetophenone + L-alpha-aminobutyric acid
-
r
2.6.1.B21 (R)-alpha-methylbenzylamine + 2-oxoglutarate
-
Haliangium ochraceum acetophenone + L-glutamate
-
r
2.6.1.B21 (R)-alpha-methylbenzylamine + 2-oxoglutarate reaction of (R)-amine:pyruvate transaminase, EC 2.6.1.B21 Haliangium ochraceum acetophenone + L-glutamate
-
r
2.6.1.B21 (R)-alpha-methylbenzylamine + 2-oxohexanoate
-
Haliangium ochraceum acetophenone + L-norleucine
-
r
2.6.1.B21 (R)-alpha-methylbenzylamine + 2-oxovalerate
-
Haliangium ochraceum acetophenone + L-norvaline
-
r
2.6.1.B21 (R)-alpha-methylbenzylamine + 3-methyl-2-oxobutyrate
-
Haliangium ochraceum acetophenone + L-valine
-
r
2.6.1.B21 (R)-alpha-methylbenzylamine + 3-methyl-2-oxovalerate
-
Haliangium ochraceum acetophenone + D-isoleucine
-
r
2.6.1.B21 (R)-alpha-methylbenzylamine + 3-methyl-2-oxovalerate
-
Haliangium ochraceum acetophenone + L-isoleucine
-
r
2.6.1.B21 (R)-alpha-methylbenzylamine + 3-methyl-2-oxovalerate reaction of (R)-amine:pyruvate transaminase, EC 2.6.1.B21 Haliangium ochraceum acetophenone + L-isoleucine
-
r
2.6.1.B21 (R)-alpha-methylbenzylamine + 4-methyl-2-oxovalerate
-
Haliangium ochraceum acetophenone + L-leucine
-
r
2.6.1.B21 (R)-alpha-methylbenzylamine + pyruvate R-MBA, specific substrate for (R)-amine:pyruvate transaminases Haliangium ochraceum acetophenone + D-alanine
-
r
2.6.1.B21 2-oxohexanoate + L-glutamate reaction of branched-chain amino acid transaminase, EC 2.6.1.42 Haliangium ochraceum norleucine + 2-oxoglutarate
-
r
2.6.1.B21 L-alanine + 2-oxoglutarate reaction of branched-chain amino acid transaminase, EC 2.6.1.42 Haliangium ochraceum pyruvate + L-glutamate
-
r
2.6.1.B21 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
2.6.1.B21 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
2.6.1.B21 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
2.6.1.B21 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 ?
-
-
2.6.1.B21 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 ?
-
-
2.6.1.B21 norvaline + 2-oxoglutarate reaction of branched-chain amino acid transaminase, EC 2.6.1.42 Haliangium ochraceum 2-oxovalerate + L-glutamate
-
r
2.6.1.42 (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
2.6.1.42 (R)-alpha-methylbenzylamine + 3-methyl-2-oxovalerate reaction of (R)-amine:pyruvate transaminase, EC 2.6.1.B21 Haliangium ochraceum acetophenone + L-isoleucine
-
r
2.6.1.42 2-oxohexanoate + L-glutamate reaction of branched-chain amino acid transaminase, EC 2.6.1.42 Haliangium ochraceum L-norleucine + 2-oxoglutarate
-
r
2.6.1.42 L-alanine + 2-oxoglutarate reaction of branched-chain amino acid transaminase, EC 2.6.1.42 Haliangium ochraceum pyruvate + L-glutamate
-
r
2.6.1.42 L-alanine + 2-oxoglutarate reaction of branched-chain amino acid transaminase, EC 2.6.1.42 Haliangium ochraceum JCM 11303 pyruvate + L-glutamate
-
r
2.6.1.42 L-alanine + 2-oxoglutarate reaction of branched-chain amino acid transaminase, EC 2.6.1.42 Haliangium ochraceum DSM 14365 pyruvate + L-glutamate
-
r
2.6.1.42 L-alanine + 2-oxoglutarate reaction of branched-chain amino acid transaminase, EC 2.6.1.42 Haliangium ochraceum SMP-2 pyruvate + L-glutamate
-
r
2.6.1.42 L-isoleucine + 2-oxoglutarate
-
Haliangium ochraceum 3-methyl-2-oxopentanoate + L-glutamate
-
r
2.6.1.42 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
2.6.1.42 L-isoleucine + 2-oxoglutarate
-
Haliangium ochraceum JCM 11303 3-methyl-2-oxopentanoate + L-glutamate
-
r
2.6.1.42 L-isoleucine + 2-oxoglutarate
-
Haliangium ochraceum DSM 14365 3-methyl-2-oxopentanoate + L-glutamate
-
r
2.6.1.42 L-isoleucine + 2-oxoglutarate
-
Haliangium ochraceum SMP-2 3-methyl-2-oxopentanoate + L-glutamate
-
r
2.6.1.42 L-leucine + 2-oxoglutarate
-
Haliangium ochraceum 4-methyl-2-oxopentanoate + L-glutamate
-
r
2.6.1.42 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
2.6.1.42 L-leucine + 2-oxoglutarate
-
Haliangium ochraceum JCM 11303 4-methyl-2-oxopentanoate + L-glutamate
-
r
2.6.1.42 L-leucine + 2-oxoglutarate reaction of branched-chain amino acid transaminase, EC 2.6.1.42 Haliangium ochraceum JCM 11303 4-methyl-2-oxopentanoate + L-glutamate
-
r
2.6.1.42 L-leucine + 2-oxoglutarate
-
Haliangium ochraceum DSM 14365 4-methyl-2-oxopentanoate + L-glutamate
-
r
2.6.1.42 L-leucine + 2-oxoglutarate reaction of branched-chain amino acid transaminase, EC 2.6.1.42 Haliangium ochraceum DSM 14365 4-methyl-2-oxopentanoate + L-glutamate
-
r
2.6.1.42 L-leucine + 2-oxoglutarate
-
Haliangium ochraceum SMP-2 4-methyl-2-oxopentanoate + L-glutamate
-
r
2.6.1.42 L-leucine + 2-oxoglutarate reaction of branched-chain amino acid transaminase, EC 2.6.1.42 Haliangium ochraceum SMP-2 4-methyl-2-oxopentanoate + L-glutamate
-
r
2.6.1.42 L-norvaline + 2-oxoglutarate reaction of branched-chain amino acid transaminase, EC 2.6.1.42 Haliangium ochraceum 2-oxovalerate + L-glutamate
-
r
2.6.1.42 L-valine + 2-oxoglutarate
-
Haliangium ochraceum 3-methyl-2-oxobutanoate + L-glutamate
-
r
2.6.1.42 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
2.6.1.42 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 ?
-
-
2.6.1.42 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 JCM 11303 ?
-
-
2.6.1.42 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 DSM 14365 ?
-
-
2.6.1.42 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 SMP-2 ?
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Subunits

EC Number Subunits Comment Organism
2.6.1.B21 homodimer
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Haliangium ochraceum
2.6.1.42 homodimer
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Haliangium ochraceum

Synonyms

EC Number Synonyms Comment Organism
2.6.1.B21 (R)-amine:pyruvate transaminase
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Haliangium ochraceum
2.6.1.B21 halotolerant R-TA
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Haliangium ochraceum
2.6.1.B21 Hoch3033
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Haliangium ochraceum
2.6.1.B21 IlvE
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Haliangium ochraceum
2.6.1.B21 More see also branched-chain amino acid transaminase, EC 2.6.1.42 Haliangium ochraceum
2.6.1.B21 R-TA
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Haliangium ochraceum
2.6.1.42 BcaT
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Haliangium ochraceum
2.6.1.42 branched-chain amino acid transaminase
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Haliangium ochraceum
2.6.1.42 Hoch3033
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Haliangium ochraceum
2.6.1.42 IlvE
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Haliangium ochraceum
2.6.1.42 More see also (R)-amine:pyruvate transaminase, EC 2.6.1.B21 Haliangium ochraceum

Temperature Optimum [°C]

EC Number Temperature Optimum [°C] Temperature Optimum Maximum [°C] Comment Organism
2.6.1.B21 40 45
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Haliangium ochraceum
2.6.1.B21 45
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Haliangium ochraceum
2.6.1.42 40 45
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Haliangium ochraceum

Temperature Range [°C]

EC Number Temperature Minimum [°C] Temperature Maximum [°C] Comment Organism
2.6.1.B21 40 60 maximal activity at 40-45°C, 40% of maximal activity at 60°C Haliangium ochraceum
2.6.1.42 40 60 maximal activity at 40-45°C, 40% of maximal activity at 60°C Haliangium ochraceum

Temperature Stability [°C]

EC Number Temperature Stability Minimum [°C] Temperature Stability Maximum [°C] Comment Organism
2.6.1.B21 60
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the purified recombinant enzyme retains about 40% of the maximum activity at pH 11.0 Haliangium ochraceum

Turnover Number [1/s]

EC Number Turnover Number Minimum [1/s] Turnover Number Maximum [1/s] Substrate Comment Organism Structure
2.6.1.B21 0.0113
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(R)-alpha-ethylbenzylamine pH 10.0, 40°C, with 2-oxoglutarate Haliangium ochraceum
2.6.1.B21 0.15
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3-methyl-2-oxovalerate pH 10.0, 40°C Haliangium ochraceum
2.6.1.B21 0.15
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3-methyl-2-oxovalerate recombinant enzyme, pH 10.0, 40°C, with (R)-alpha-methylbenzylamine Haliangium ochraceum
2.6.1.B21 0.19
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(R)-alpha-methylbenzylamine pH 10.0, 40°C, with 1.5 mM 3-methyl-2-oxovalerate Haliangium ochraceum
2.6.1.B21 0.19
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(R)-alpha-methylbenzylamine recombinant enzyme, pH 9.0, 40°C, with 3-methyl-2-oxovalerate Haliangium ochraceum
2.6.1.B21 0.22
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(R)-alpha-methylbenzylamine pH 10.0, 40°C, with 5 mM 3-methyl-2-oxovalerate Haliangium ochraceum
2.6.1.B21 0.22
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(R)-alpha-methylbenzylamine recombinant enzyme, pH 10.0, 40°C, with 3-methyl-2-oxovalerate Haliangium ochraceum
2.6.1.B21 0.23
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(R)-alpha-methylbenzylamine pH 10.0, 40°C, with 2-oxoglutarate Haliangium ochraceum
2.6.1.42 0.19
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L-norvaline pH 10.0, 40°C Haliangium ochraceum
2.6.1.42 0.24
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L-isoleucine pH 10.0, 40°C Haliangium ochraceum
2.6.1.42 0.29
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L-valine pH 10.0, 40°C Haliangium ochraceum
2.6.1.42 0.95
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L-leucine pH 10.0, 40°C Haliangium ochraceum

pH Optimum

EC Number pH Optimum Minimum pH Optimum Maximum Comment Organism
2.6.1.B21 10
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Haliangium ochraceum
2.6.1.B21 10 10.5
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Haliangium ochraceum
2.6.1.42 10
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Haliangium ochraceum

pH Range

EC Number pH Minimum pH Maximum Comment Organism
2.6.1.B21 10 11 maximal activity at pH 10.0, 70% of maximal activity at pH 11.0 Haliangium ochraceum
2.6.1.42 10 11 maximal activity at pH 10.0, 70% of maximal activity at pH 11.0 Haliangium ochraceum

pH Stability

EC Number pH Stability pH Stability Maximum Comment Organism
2.6.1.B21 11
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the purified recombinant enzyme retains more than 70% of the maximum activity at pH 11.0 Haliangium ochraceum

Cofactor

EC Number Cofactor Comment Organism Structure
2.6.1.B21 pyridoxal 5'-phosphate PLP, dependent on Haliangium ochraceum
2.6.1.42 pyridoxal 5'-phosphate PLP, dependent on Haliangium ochraceum

General Information

EC Number General Information Comment Organism
2.6.1.B21 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
2.6.1.B21 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
2.6.1.B21 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
2.6.1.B21 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
2.6.1.B21 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
2.6.1.42 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
2.6.1.42 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 site structure analysis Haliangium ochraceum

kcat/KM [mM/s]

EC Number kcat/KM Value [1/mMs-1] kcat/KM Value Maximum [1/mMs-1] Substrate Comment Organism Structure
2.6.1.B21 0.0011
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(R)-alpha-ethylbenzylamine pH 10.0, 40°C, with 2-oxoglutarate Haliangium ochraceum
2.6.1.B21 0.01
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(R)-alpha-methylbenzylamine pH 10.0, 40°C, with 2-oxoglutarate Haliangium ochraceum
2.6.1.B21 0.016
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(R)-alpha-methylbenzylamine pH 10.0, 40°C, with 1.5 mM 3-methyl-2-oxovalerate Haliangium ochraceum
2.6.1.B21 0.016
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(R)-alpha-methylbenzylamine recombinant enzyme, pH 9.0, 40°C, with 3-methyl-2-oxovalerate Haliangium ochraceum
2.6.1.B21 0.037
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(R)-alpha-methylbenzylamine pH 10.0, 40°C, with 5 mM 3-methyl-2-oxovalerate Haliangium ochraceum
2.6.1.B21 0.037
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(R)-alpha-methylbenzylamine recombinant enzyme, pH 10.0, 40°C, with 3-methyl-2-oxovalerate Haliangium ochraceum
2.6.1.B21 0.27
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3-methyl-2-oxovalerate pH 10.0, 40°C Haliangium ochraceum
2.6.1.B21 0.27
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3-methyl-2-oxovalerate recombinant enzyme, pH 10.0, 40°C, with (R)-alpha-methylbenzylamine Haliangium ochraceum
2.6.1.42 0.045
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L-valine pH 10.0, 40°C Haliangium ochraceum
2.6.1.42 0.41
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L-isoleucine pH 10.0, 40°C Haliangium ochraceum
2.6.1.42 0.59
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L-norvaline pH 10.0, 40°C Haliangium ochraceum
2.6.1.42 1.46
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L-leucine pH 10.0, 40°C Haliangium ochraceum