Literature summary for 2.6.1.39 extracted from

Ouchi, T.; Tomita, T.; Miyagawa, T.; Kuzuyama, T.; Nishiyama, M.
Dual roles of a conserved pair, Arg23 and Ser20, in recognition of multiple substrates in alpha-aminoadipate aminotransferase from Thermus thermophilus (2009), Biochem. Biophys. Res. Commun., 388, 21-27.

Cloned(Commentary)

Cloned (Comment)	Organism
expressed in Escherichia coli BL21(DE3) cells	Thermus thermophilus

Crystallization (Commentary)

Crystallization (Comment)	Organism
complexed with N-(5'-phosphopyridoxyl)-L-glutamate, vapor diffusion method, using 16% (w/v) PEG3350, 100 mM HEPES, pH 7.0, and 200 mM calcium acetate, at 20°C	Thermus thermophilus

Protein Variants

Protein Variants	Comment	Organism
R23A	the mutation causes 4.1fold increase in the Km value for L-2-aminoadipate, the Km value for L-Glu increases 546fold, the Km value for L-Leu is not greatly affected	Thermus thermophilus
R23Q	the mutation causes 13.7fold increase in the Km value for L-2-aminoadipate, the Km value for L-Glu increases 134fold, the Km value for L-Leu is not greatly affected	Thermus thermophilus
S20E	the mutant shows decreased kcat values compared to the wild type enzyme	Thermus thermophilus

KM Value [mM]

KM Value [mM]	KM Value Maximum [mM]	Substrate	Comment	Organism
0.048	-	2-oxoisocaproate	mutant enzyme R23A, in 50 mM HEPESNaOH buffer, pH 8.0, containing 50 mM NH4Cl, 0.15 mM pyridoxal 5'-phosphate, at 45°C	Thermus thermophilus
0.05	-	2-oxoisocaproate	mutant enzyme S20E, in 50 mM HEPESNaOH buffer, pH 8.0, containing 50 mM NH4Cl, 0.15 mM pyridoxal 5'-phosphate, at 45°C	Thermus thermophilus
0.091	-	2-oxoisocaproate	mutant enzyme R23Q, in 50 mM HEPESNaOH buffer, pH 8.0, containing 50 mM NH4Cl, 0.15 mM pyridoxal 5'-phosphate, at 45°C	Thermus thermophilus
0.2	-	L-leucine	mutant enzyme S20E, in 50 mM HEPES-NaOH buffer, pH 8.0, 50 mM KCl, 0.15 mM pyridoxal 5'-phosphate, at 45°C	Thermus thermophilus
0.27	-	2-oxoglutarate	wild type enzyme, using L-leucine as cosubstrate, in 50 mM HEPES-NaOH buffer, pH 8.0, 50 mM KCl, 0.15 mM pyridoxal 5'-phosphate, at 45°C	Thermus thermophilus
0.3	-	2-oxoglutarate	wild type enzyme, using L-2-aminoadipate as cosubstrate, in 50 mM HEPES-NaOH buffer, pH 8.0, 50 mM KCl, 0.15 mM pyridoxal 5'-phosphate, at 45°C	Thermus thermophilus
0.39	-	L-leucine	mutant enzyme R23A, in 50 mM HEPES-NaOH buffer, pH 8.0, 50 mM KCl, 0.15 mM pyridoxal 5'-phosphate, at 45°C	Thermus thermophilus
0.4	-	2-oxoglutarate	mutant enzyme S20E, using L-leucine as cosubstrate, in 50 mM HEPES-NaOH buffer, pH 8.0, 50 mM KCl, 0.15 mM pyridoxal 5'-phosphate, at 45°C	Thermus thermophilus
0.41	-	2-oxoglutarate	mutant enzyme S20E, using L-2-aminoadipate as cosubstrate, in 50 mM HEPES-NaOH buffer, pH 8.0, 50 mM KCl, 0.15 mM pyridoxal 5'-phosphate, at 45°C	Thermus thermophilus
0.42	-	2-oxoglutarate	mutant enzyme R23Q, using L-2-aminoadipate as cosubstrate, in 50 mM HEPES-NaOH buffer, pH 8.0, 50 mM KCl, 0.15 mM pyridoxal 5'-phosphate, at 45°C	Thermus thermophilus
0.43	-	2-oxoisocaproate	wild type enzyme, in 50 mM HEPESNaOH buffer, pH 8.0, containing 50 mM NH4Cl, 0.15 mM pyridoxal 5'-phosphate, at 45°C	Thermus thermophilus
0.46	-	L-glutamate	wild type enzyme, using 2-oxoisocaproate as cosubstrate, in 50 mM HEPESNaOH buffer, pH 8.0, containing 50 mM NH4Cl, 0.15 mM pyridoxal 5'-phosphate, at 45°C	Thermus thermophilus
0.54	-	2-oxoglutarate	mutant enzyme R23A, using L-2-aminoadipate as cosubstrate, in 50 mM HEPES-NaOH buffer, pH 8.0, 50 mM KCl, 0.15 mM pyridoxal 5'-phosphate, at 45°C	Thermus thermophilus
0.55	-	2-oxoglutarate	mutant enzyme R23Q, using L-leucine as cosubstrate, in 50 mM HEPES-NaOH buffer, pH 8.0, 50 mM KCl, 0.15 mM pyridoxal 5'-phosphate, at 45°C	Thermus thermophilus
0.61	-	2-oxoglutarate	mutant enzyme R23A, using L-leucine as cosubstrate, in 50 mM HEPES-NaOH buffer, pH 8.0, 50 mM KCl, 0.15 mM pyridoxal 5'-phosphate, at 45°C	Thermus thermophilus
0.81	-	L-2-aminoadipate	wild type enzyme, in 50 mM HEPES-NaOH buffer, pH 8.0, 50 mM KCl, 0.15 mM pyridoxal 5'-phosphate, at 45°C	Thermus thermophilus
0.88	-	L-leucine	mutant enzyme R23Q, in 50 mM HEPES-NaOH buffer, pH 8.0, 50 mM KCl, 0.15 mM pyridoxal 5'-phosphate, at 45°C	Thermus thermophilus
0.95	-	L-leucine	wild type enzyme, in 50 mM HEPES-NaOH buffer, pH 8.0, 50 mM KCl, 0.15 mM pyridoxal 5'-phosphate, at 45°C	Thermus thermophilus
3.3	-	L-2-aminoadipate	mutant enzyme R23A, in 50 mM HEPES-NaOH buffer, pH 8.0, 50 mM KCl, 0.15 mM pyridoxal 5'-phosphate, at 45°C	Thermus thermophilus
7	-	L-2-aminoadipate	mutant enzyme S20E, in 50 mM HEPES-NaOH buffer, pH 8.0, 50 mM KCl, 0.15 mM pyridoxal 5'-phosphate, at 45°C	Thermus thermophilus
11	-	L-2-aminoadipate	mutant enzyme R23Q, in 50 mM HEPES-NaOH buffer, pH 8.0, 50 mM KCl, 0.15 mM pyridoxal 5'-phosphate, at 45°C	Thermus thermophilus
62	-	L-glutamate	mutant enzyme R23Q, using 2-oxoisocaproate as cosubstrate, in 50 mM HEPESNaOH buffer, pH 8.0, containing 50 mM NH4Cl, 0.15 mM pyridoxal 5'-phosphate, at 45°C	Thermus thermophilus
82	-	L-glutamate	mutant enzyme S20E, using 2-oxoisocaproate as cosubstrate, in 50 mM HEPESNaOH buffer, pH 8.0, containing 50 mM NH4Cl, 0.15 mM pyridoxal 5'-phosphate, at 45°C	Thermus thermophilus
250	-	L-glutamate	mutant enzyme R23A, using 2-oxoisocaproate as cosubstrate, in 50 mM HEPESNaOH buffer, pH 8.0, containing 50 mM NH4Cl, 0.15 mM pyridoxal 5'-phosphate, at 45°C	Thermus thermophilus

Organism

Organism	UniProt	Comment	Textmining
Thermus thermophilus	Q72LL6	-	-

Purification (Commentary)

Purification (Comment)	Organism
ammonium sulfate precipitation and Superdex 200 gel filtration	Thermus thermophilus

Substrates and Products (Substrate)

Substrates	Comment Substrates	Organism	Products	Comment (Products)	Rev.
2-oxoisocaproate + L-glutamate	-	Thermus thermophilus	?	-	?
L-2-aminoadipate + 2-oxoglutarate	-	Thermus thermophilus	2-oxoadipate + L-glutamate	-	?
L-leucine + 2-oxoglutarate	-	Thermus thermophilus	4-methyl-2-oxopentanoate + L-glutamate	-	?
additional information	negligible activity is observed for L-Asp	Thermus thermophilus	?	-	?

Synonyms

Synonyms	Comment	Organism
AAA-AT	-	Thermus thermophilus
alpha-aminoadipate aminotransferase	-	Thermus thermophilus

Turnover Number [1/s]

Turnover Number Minimum [1/s]	Turnover Number Maximum [1/s]	Substrate	Comment	Organism
0.009	-	L-leucine	mutant enzyme R23A, in 50 mM HEPES-NaOH buffer, pH 8.0, 50 mM KCl, 0.15 mM pyridoxal 5'-phosphate, at 45°C	Thermus thermophilus
0.011	-	L-2-aminoadipate	mutant enzyme R23A, in 50 mM HEPES-NaOH buffer, pH 8.0, 50 mM KCl, 0.15 mM pyridoxal 5'-phosphate, at 45°C	Thermus thermophilus
0.023	-	L-leucine	mutant enzyme S20E, in 50 mM HEPES-NaOH buffer, pH 8.0, 50 mM KCl, 0.15 mM pyridoxal 5'-phosphate, at 45°C	Thermus thermophilus
0.04	-	L-2-aminoadipate	mutant enzyme S20E, in 50 mM HEPES-NaOH buffer, pH 8.0, 50 mM KCl, 0.15 mM pyridoxal 5'-phosphate, at 45°C	Thermus thermophilus
0.05	-	L-leucine	mutant enzyme R23Q, in 50 mM HEPES-NaOH buffer, pH 8.0, 50 mM KCl, 0.15 mM pyridoxal 5'-phosphate, at 45°C	Thermus thermophilus
0.083	-	L-2-aminoadipate	mutant enzyme R23Q, in 50 mM HEPES-NaOH buffer, pH 8.0, 50 mM KCl, 0.15 mM pyridoxal 5'-phosphate, at 45°C	Thermus thermophilus
0.23	-	L-glutamate	mutant enzyme R23A, using 2-oxoisocaproate as cosubstrate, in 50 mM HEPESNaOH buffer, pH 8.0, containing 50 mM NH4Cl, 0.15 mM pyridoxal 5'-phosphate, at 45°C	Thermus thermophilus
0.5	-	L-glutamate	mutant enzyme R23Q, using 2-oxoisocaproate as cosubstrate, in 50 mM HEPES-NaOH buffer, pH 8.0, containing 50 mM NH4Cl, 0.15 mM pyridoxal 5'-phosphate, at 45°C	Thermus thermophilus
1.2	-	L-glutamate	mutant enzyme S20E, using 2-oxoisocaproate as cosubstrate, in 50 mM HEPESNaOH buffer, pH 8.0, containing 50 mM NH4Cl, 0.15 mM pyridoxal 5'-phosphate, at 45°C	Thermus thermophilus
5.1	-	L-leucine	wild type enzyme, in 50 mM HEPES-NaOH buffer, pH 8.0, 50 mM KCl, 0.15 mM pyridoxal 5'-phosphate, at 45°C	Thermus thermophilus
6.7	-	L-2-aminoadipate	wild type enzyme, in 50 mM HEPES-NaOH buffer, pH 8.0, 50 mM KCl, 0.15 mM pyridoxal 5'-phosphate, at 45°C	Thermus thermophilus
16	-	L-glutamate	wild type enzyme, using 2-oxoisocaproate as cosubstrate, in 50 mM HEPES-NaOH buffer, pH 8.0, containing 50 mM NH4Cl, 0.15 mM pyridoxal 5'-phosphate, at 45°C	Thermus thermophilus

Cofactor

Cofactor	Comment	Organism	Structure
pyridoxal 5'-phosphate	-	Thermus thermophilus

kcat/KM [mM/s]

kcat/KM Value [1/mMs^-1]	kcat/KM Value Maximum [1/mMs^-1]	Substrate	Comment	Organism
0.0009	-	L-glutamate	mutant enzyme R23A, using 2-oxoisocaproate as cosubstrate, in 50 mM HEPESNaOH buffer, pH 8.0, containing 50 mM NH4Cl, 0.15 mM pyridoxal 5'-phosphate, at 45°C	Thermus thermophilus
0.0033	-	L-2-aminoadipate	mutant enzyme R23A, in 50 mM HEPES-NaOH buffer, pH 8.0, 50 mM KCl, 0.15 mM pyridoxal 5'-phosphate, at 45°C	Thermus thermophilus
0.0057	-	L-2-aminoadipate	mutant enzyme S20E, in 50 mM HEPES-NaOH buffer, pH 8.0, 50 mM KCl, 0.15 mM pyridoxal 5'-phosphate, at 45°C	Thermus thermophilus
0.0075	-	L-2-aminoadipate	mutant enzyme R23Q, in 50 mM HEPES-NaOH buffer, pH 8.0, 50 mM KCl, 0.15 mM pyridoxal 5'-phosphate, at 45°C	Thermus thermophilus
0.0081	-	L-glutamate	mutant enzyme R23Q, using 2-oxoisocaproate as cosubstrate, in 50 mM HEPESNaOH buffer, pH 8.0, containing 50 mM NH4Cl, 0.15 mM pyridoxal 5'-phosphate, at 45°C	Thermus thermophilus
0.014	-	L-glutamate	mutant enzyme S20E, using 2-oxoisocaproate as cosubstrate, in 50 mM HEPESNaOH buffer, pH 8.0, containing 50 mM NH4Cl, 0.15 mM pyridoxal 5'-phosphate, at 45°C	Thermus thermophilus
0.015	-	2-oxoglutarate	mutant enzyme R23A, using L-leucine as cosubstrate, in 50 mM HEPES-NaOH buffer, pH 8.0, 50 mM KCl, 0.15 mM pyridoxal 5'-phosphate, at 45°C	Thermus thermophilus
0.021	-	2-oxoglutarate	mutant enzyme R23A, using L-2-aminoadipate as cosubstrate, in 50 mM HEPES-NaOH buffer, pH 8.0, 50 mM KCl, 0.15 mM pyridoxal 5'-phosphate, at 45°C	Thermus thermophilus
0.023	-	L-leucine	mutant enzyme R23A, in 50 mM HEPES-NaOH buffer, pH 8.0, 50 mM KCl, 0.15 mM pyridoxal 5'-phosphate, at 45°C	Thermus thermophilus
0.057	-	L-leucine	mutant enzyme R23Q, in 50 mM HEPES-NaOH buffer, pH 8.0, 50 mM KCl, 0.15 mM pyridoxal 5'-phosphate, at 45°C	Thermus thermophilus
0.06	-	2-oxoglutarate	mutant enzyme S20E, using L-leucine as cosubstrate, in 50 mM HEPES-NaOH buffer, pH 8.0, 50 mM KCl, 0.15 mM pyridoxal 5'-phosphate, at 45°C	Thermus thermophilus
0.091	-	2-oxoglutarate	mutant enzyme R23Q, using L-leucine as cosubstrate, in 50 mM HEPES-NaOH buffer, pH 8.0, 50 mM KCl, 0.15 mM pyridoxal 5'-phosphate, at 45°C	Thermus thermophilus
0.097	-	2-oxoglutarate	mutant enzyme S20E, using L-2-aminoadipate as cosubstrate, in 50 mM HEPES-NaOH buffer, pH 8.0, 50 mM KCl, 0.15 mM pyridoxal 5'-phosphate, at 45°C	Thermus thermophilus
0.12	-	L-leucine	mutant enzyme S20E, in 50 mM HEPES-NaOH buffer, pH 8.0, 50 mM KCl, 0.15 mM pyridoxal 5'-phosphate, at 45°C	Thermus thermophilus
0.2	-	2-oxoglutarate	mutant enzyme R23Q, using L-2-aminoadipate as cosubstrate, in 50 mM HEPES-NaOH buffer, pH 8.0, 50 mM KCl, 0.15 mM pyridoxal 5'-phosphate, at 45°C	Thermus thermophilus
4.8	-	2-oxoisocaproate	mutant enzyme R23A, in 50 mM HEPESNaOH buffer, pH 8.0, containing 50 mM NH4Cl, 0.15 mM pyridoxal 5'-phosphate, at 45°C	Thermus thermophilus
5.4	-	L-leucine	wild type enzyme, in 50 mM HEPES-NaOH buffer, pH 8.0, 50 mM KCl, 0.15 mM pyridoxal 5'-phosphate, at 45°C	Thermus thermophilus
5.5	-	2-oxoisocaproate	mutant enzyme R23Q, in 50 mM HEPESNaOH buffer, pH 8.0, containing 50 mM NH4Cl, 0.15 mM pyridoxal 5'-phosphate, at 45°C	Thermus thermophilus
8.2	-	L-2-aminoadipate	wild type enzyme, in 50 mM HEPES-NaOH buffer, pH 8.0, 50 mM KCl, 0.15 mM pyridoxal 5'-phosphate, at 45°C	Thermus thermophilus
19	-	2-oxoglutarate	wild type enzyme, using L-leucine as cosubstrate, in 50 mM HEPES-NaOH buffer, pH 8.0, 50 mM KCl, 0.15 mM pyridoxal 5'-phosphate, at 45°C	Thermus thermophilus
22	-	2-oxoglutarate	wild type enzyme, using L-2-aminoadipate as cosubstrate, in 50 mM HEPES-NaOH buffer, pH 8.0, 50 mM KCl, 0.15 mM pyridoxal 5'-phosphate, at 45°C	Thermus thermophilus
24	-	2-oxoisocaproate	mutant enzyme S20E, in 50 mM HEPESNaOH buffer, pH 8.0, containing 50 mM NH4Cl, 0.15 mM pyridoxal 5'-phosphate, at 45°C	Thermus thermophilus
34	-	2-oxoisocaproate	wild type enzyme, in 50 mM HEPESNaOH buffer, pH 8.0, containing 50 mM NH4Cl, 0.15 mM pyridoxal 5'-phosphate, at 45°C	Thermus thermophilus
37	-	L-glutamate	wild type enzyme, using 2-oxoisocaproate as cosubstrate, in 50 mM HEPESNaOH buffer, pH 8.0, containing 50 mM NH4Cl, 0.15 mM pyridoxal 5'-phosphate, at 45°C	Thermus thermophilus