Literature summary for 2.6.1.42 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 .

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

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

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

Protein Variants

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

Inhibitors

Inhibitors	Comment	Organism
acetonitril	over 90% inhibition at 20-30%	Haliangium ochraceum
DMSO	40% inhibition at 20%, 70% inhibition at 30%	Haliangium ochraceum
methanol	65% inhibition at 20%, 85% inhibition at 30%	Haliangium ochraceum

KM Value [mM]

KM Value [mM]	KM Value Maximum [mM]	Substrate	Comment	Organism
additional information	-	additional information	Michaelis-Menten steady-state kinetics of the first half-reaction	Haliangium ochraceum
0.32	-	L-norvaline	pH 10.0, 40°C	Haliangium ochraceum
0.58	-	L-isoleucine	pH 10.0, 40°C	Haliangium ochraceum
0.65	-	L-leucine	pH 10.0, 40°C	Haliangium ochraceum
6.5	-	L-valine	pH 10.0, 40°C	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 results in a slight increase in the activity of enzyme Hoch3033	Haliangium ochraceum

Molecular Weight [Da]

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

Natural Substrates/ Products (Substrates)

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

Organism

Organism	UniProt	Comment	Textmining
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

Reaction

Reaction	Comment	Organism	Reaction ID
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	a

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
0.007	-	purified enzyme, pH 10.0, 40°C, substrate pyruvate	Haliangium ochraceum
0.025	-	purified enzyme, pH 10.0, 40°C, substrate 2-oxobutyrate	Haliangium ochraceum
0.12	-	purified enzyme, pH 10.0, 40°C, substrate 3-methyl-2-oxobutyrate	Haliangium ochraceum
0.135	-	purified enzyme, pH 10.0, 40°C, substrate 2-oxohexanoate	Haliangium ochraceum
0.15	-	purified enzyme, pH 10.0, 40°C, substrate 4-methyl-2-oxovalerate	Haliangium ochraceum
0.19	-	purified enzyme, pH 10.0, 40°C, substrate 3-methyl-2-oxovalerate	Haliangium ochraceum
0.23	-	purified enzyme, pH 10.0, 40°C, substrate 2-oxovalerate	Haliangium ochraceum

Substrates and Products (Substrate)

Substrates	Comment Substrates	Organism	Products	Comment (Products)	Rev.
(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-methylbenzylamine + 3-methyl-2-oxovalerate	reaction of (R)-amine:pyruvate transaminase, EC 2.6.1.B21	Haliangium ochraceum	acetophenone + L-isoleucine	-	r
2-oxohexanoate + L-glutamate	reaction of branched-chain amino acid transaminase, EC 2.6.1.42	Haliangium ochraceum	L-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-alanine + 2-oxoglutarate	reaction of branched-chain amino acid transaminase, EC 2.6.1.42	Haliangium ochraceum JCM 11303	pyruvate + L-glutamate	-	r
L-alanine + 2-oxoglutarate	reaction of branched-chain amino acid transaminase, EC 2.6.1.42	Haliangium ochraceum DSM 14365	pyruvate + L-glutamate	-	r
L-alanine + 2-oxoglutarate	reaction of branched-chain amino acid transaminase, EC 2.6.1.42	Haliangium ochraceum SMP-2	pyruvate + L-glutamate	-	r
L-isoleucine + 2-oxoglutarate	-	Haliangium ochraceum	3-methyl-2-oxopentanoate + 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-isoleucine + 2-oxoglutarate	-	Haliangium ochraceum JCM 11303	3-methyl-2-oxopentanoate + L-glutamate	-	r
L-isoleucine + 2-oxoglutarate	-	Haliangium ochraceum DSM 14365	3-methyl-2-oxopentanoate + L-glutamate	-	r
L-isoleucine + 2-oxoglutarate	-	Haliangium ochraceum SMP-2	3-methyl-2-oxopentanoate + L-glutamate	-	r
L-leucine + 2-oxoglutarate	-	Haliangium ochraceum	4-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-leucine + 2-oxoglutarate	-	Haliangium ochraceum JCM 11303	4-methyl-2-oxopentanoate + L-glutamate	-	r
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
L-leucine + 2-oxoglutarate	-	Haliangium ochraceum DSM 14365	4-methyl-2-oxopentanoate + L-glutamate	-	r
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
L-leucine + 2-oxoglutarate	-	Haliangium ochraceum SMP-2	4-methyl-2-oxopentanoate + L-glutamate	-	r
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
L-norvaline + 2-oxoglutarate	reaction of branched-chain amino acid transaminase, EC 2.6.1.42	Haliangium ochraceum	2-oxovalerate + L-glutamate	-	r
L-valine + 2-oxoglutarate	-	Haliangium ochraceum	3-methyl-2-oxobutanoate + 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	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	?	-	-
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	?	-	-
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	?	-	-
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	?	-	-

Subunits

Subunits	Comment	Organism
homodimer	-	Haliangium ochraceum

Synonyms

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

Temperature Optimum [°C]

Temperature Optimum [°C]	Temperature Optimum Maximum [°C]	Comment	Organism
40	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

Turnover Number [1/s]

Turnover Number Minimum [1/s]	Turnover Number Maximum [1/s]	Substrate	Comment	Organism
0.19	-	L-norvaline	pH 10.0, 40°C	Haliangium ochraceum
0.24	-	L-isoleucine	pH 10.0, 40°C	Haliangium ochraceum
0.29	-	L-valine	pH 10.0, 40°C	Haliangium ochraceum
0.95	-	L-leucine	pH 10.0, 40°C	Haliangium ochraceum

pH Optimum

pH Optimum Minimum	pH Optimum Maximum	Comment	Organism
10	-	-	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

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
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]

kcat/KM Value [1/mMs^-1]	kcat/KM Value Maximum [1/mMs^-1]	Substrate	Comment	Organism
0.045	-	L-valine	pH 10.0, 40°C	Haliangium ochraceum
0.41	-	L-isoleucine	pH 10.0, 40°C	Haliangium ochraceum
0.59	-	L-norvaline	pH 10.0, 40°C	Haliangium ochraceum
1.46	-	L-leucine	pH 10.0, 40°C	Haliangium ochraceum