Literature summary for 5.1.1.21 extracted from

Hayashi, J.; Mutaguchi, Y.; Minemura, Y.; Nakagawa, N.; Yoneda, K.; Ohmori, T.; Ohshima, T.; Sakuraba, H.
Crystal structure of the novel amino-acid racemase isoleucine 2-epimerase from Lactobacillus buchneri (2017), Acta Crystallogr. Sect. D, 73, 428-437 .

Cloned(Commentary)

Cloned (Comment)	Organism
sequence comparisons, recombinant expression of His/ProS2-tagged wild-type and mutant enzymes	Lentilactobacillus buchneri

Crystallization (Commentary)

Crystallization (Comment)	Organism
purified recombinant detagged enzyme in apoform, in complex with pyridoxal 5'-phosphate, in complex with N-(5'-phosphopyridoxyl)-L-isoleucine, and in complex with N-(5'-phosphopyridoxyl)-D-allo-isoleucine. By sitting drop vapour diffusion method, for crystals of PLP-bound enzyme, mixing of 0.002 ml enzyme solution containing 0.05 mM PLP 33% 2-propanol, 0.2 M magnesium acetate, 0.1 M cacodylate buffer pH 6.5, for crystals of the apoenzyme mixing of 0.001 ml enzyme solution containing 2 mM D-tert-Leu and 0.1 mM PLP with 0.001 ml of 0.5 M NaCl, 10 mM MgCl2, 10mM hexadecyltrimethylammonium bromide, for crystals of the PLP-L-Ile-bound enzyme, mixing of 0.001 ml protein solution with 0.001 ml of PLP-L-Ile, 20% PEG 3000, 0.2 M NaCl, 0.1 M HEPES, pH 7.5, and for crystals of the PLP-D-allo-Ile-bound enzyme are grown in sitting drops composed of 0.001 ml enzyme solution containing 1 mM PLP-D-allo-Ile mixed with an equal volume of 50 mM cadmium sulfate hydrate, 0.76 M sodium acetate trihydrate, and 0.1 M HEPES, pH 7.5. In all cases, drops are equilibrated against 0.1 ml mother liquor at 20°C, X-ray diffraction structure determination and analysis at resolutions of 2.77 A, 1.94 A, 2.65 A, and 2.12 A, respectively, molecular replacement using with the tetrameric structure of GABA-AT from Sulfolobus tokodaii (PDB ID 2eo5)	Lentilactobacillus buchneri

Crystallization (Comment)

Organism

purified recombinant detagged enzyme in apoform, in complex with pyridoxal 5'-phosphate, in complex with N-(5'-phosphopyridoxyl)-L-isoleucine, and in complex with N-(5'-phosphopyridoxyl)-D-allo-isoleucine. By sitting drop vapour diffusion method, for crystals of PLP-bound enzyme, mixing of 0.002 ml enzyme solution containing 0.05 mM PLP 33% 2-propanol, 0.2 M magnesium acetate, 0.1 M cacodylate buffer pH 6.5, for crystals of the apoenzyme mixing of 0.001 ml enzyme solution containing 2 mM D-tert-Leu and 0.1 mM PLP with 0.001 ml of 0.5 M NaCl, 10 mM MgCl2, 10mM hexadecyltrimethylammonium bromide, for crystals of the PLP-L-Ile-bound enzyme, mixing of 0.001 ml protein solution with 0.001 ml of PLP-L-Ile, 20% PEG 3000, 0.2 M NaCl, 0.1 M HEPES, pH 7.5, and for crystals of the PLP-D-allo-Ile-bound enzyme are grown in sitting drops composed of 0.001 ml enzyme solution containing 1 mM PLP-D-allo-Ile mixed with an equal volume of 50 mM cadmium sulfate hydrate, 0.76 M sodium acetate trihydrate, and 0.1 M HEPES, pH 7.5. In all cases, drops are equilibrated against 0.1 ml mother liquor at 20°C, X-ray diffraction structure determination and analysis at resolutions of 2.77 A, 1.94 A, 2.65 A, and 2.12 A, respectively, molecular replacement using with the tetrameric structure of GABA-AT from Sulfolobus tokodaii (PDB ID 2eo5)

Lentilactobacillus buchneri

Protein Variants

Protein Variants	Comment	Organism
D222A	site-directed mutagenesis, the mutant shows reduced activity and altered kinetics compared to the wild-type enzyme	Lentilactobacillus buchneri
D222N	site-directed mutagenesis, the mutant shows reduced activity and altered kinetics compared to the wild-type enzyme	Lentilactobacillus buchneri
Y142F	site-directed mutagenesis, the mutant shows reduced activity and altered kinetics compared to the wild-type enzyme	Lentilactobacillus buchneri

KM Value [mM]

KM Value [mM]	KM Value Maximum [mM]	Substrate	Comment	Organism
4.46	-	L-isoleucine	recombinant mutant D222N, pH and temperature not specified in the publication	Lentilactobacillus buchneri
4.58	-	L-isoleucine	recombinant mutant D222A, pH and temperature not specified in the publication	Lentilactobacillus buchneri
5	-	L-isoleucine	recombinant wild-type enzyme, pH and temperature not specified in the publication	Lentilactobacillus buchneri
13.2	-	D-allo-isoleucine	recombinant wild-type enzyme, pH and temperature not specified in the publication	Lentilactobacillus buchneri
13.8	-	D-allo-isoleucine	recombinant mutant D222N, pH and temperature not specified in the publication	Lentilactobacillus buchneri
16	-	D-allo-isoleucine	recombinant mutant D222A, pH and temperature not specified in the publication	Lentilactobacillus buchneri
16	-	L-isoleucine	recombinant mutant Y142F, pH and temperature not specified in the publication	Lentilactobacillus buchneri
35	-	D-allo-isoleucine	recombinant mutant Y142F, pH and temperature not specified in the publication	Lentilactobacillus buchneri

Natural Substrates/ Products (Substrates)

Natural Substrates	Organism	Comment (Nat. Sub.)	Natural Products	Comment (Nat. Pro.)	Rev.	Reac.
L-isoleucine	Lentilactobacillus buchneri	-	D-allo-isoleucine	-	r

Organism

Organism	UniProt	Comment	Textmining
Lentilactobacillus buchneri	M1GRN3	-	-

Purification (Commentary)

Purification (Comment)	Organism
recombinant His/ProS2-tagged wild-type and mutant enzymes, tag cleavage	Lentilactobacillus buchneri

Reaction

Reaction	Comment	Organism	Reaction ID
L-isoleucine = D-allo-isoleucine	L-isoleucine epimerization proceeds through abstraction of the alpha-hydrogen of the substrate by Lys280, while Asp222 serves as the catalytic residue adding an alpha-hydrogen to the quinonoid intermediate to form D-allo-isoleucine, structure-function relationship	Lentilactobacillus buchneri	a

Substrates and Products (Substrate)

Substrates	Comment Substrates	Organism	Products	Comment (Products)	Rev.	Reac.
L-isoleucine	-	Lentilactobacillus buchneri	D-allo-isoleucine	-	r

Subunits

Subunits	Comment	Organism
More	detailed enzyme structure analysis and structure comparisons, analysis of the structures bound with reaction-intermediate analogues (PLP-L-Ile and PLP-D-allo-Ile), overview	Lentilactobacillus buchneri
tetramer	the enzyme assembles as a tetramer, with each subunit being composed of N-terminal, C-terminal, and large PLP-binding domains	Lentilactobacillus buchneri

Synonyms

Synonyms	Comment	Organism
BCAA racemase	UniProt	Lentilactobacillus buchneri
branched-chain amino-acid racemase	-	Lentilactobacillus buchneri
ILEP	-	Lentilactobacillus buchneri
isoleucine 2-epimerase	-	Lentilactobacillus buchneri

Turnover Number [1/s]

Turnover Number Minimum [1/s]	Turnover Number Maximum [1/s]	Substrate	Comment	Organism
0.0238	-	L-isoleucine	recombinant mutant D222N, pH and temperature not specified in the publication	Lentilactobacillus buchneri
0.0461	-	D-allo-isoleucine	recombinant mutant D222N, pH and temperature not specified in the publication	Lentilactobacillus buchneri
1.65	-	L-isoleucine	recombinant mutant D222A, pH and temperature not specified in the publication	Lentilactobacillus buchneri
3.56	-	D-allo-isoleucine	recombinant mutant D222A, pH and temperature not specified in the publication	Lentilactobacillus buchneri
279	-	L-isoleucine	recombinant mutant Y142F, pH and temperature not specified in the publication	Lentilactobacillus buchneri
434	-	D-allo-isoleucine	recombinant mutant Y142F, pH and temperature not specified in the publication	Lentilactobacillus buchneri
502	-	L-isoleucine	recombinant wild-type enzyme, pH and temperature not specified in the publication	Lentilactobacillus buchneri
939	-	D-allo-isoleucine	recombinant wild-type enzyme, pH and temperature not specified in the publication	Lentilactobacillus buchneri

Cofactor

Cofactor	Comment	Organism	Structure
pyridoxal 5'-phosphate	PLP, a marked structural change occurs around the active site upon binding of pyridoxal 5'-phosphate that provides a solvent-inaccessible environment for the enzymatic reaction	Lentilactobacillus buchneri

General Information

General Information	Comment	Organism
evolution	the enzyme is a fold-type I racemase	Lentilactobacillus buchneri
additional information	the enzyme is a fold-type I racemase, similar to the alpha-amino-epsilon-caprolactam racemase (EC 5.1.1.15). The active-site cavity in the apoenzyme structure is much more solvent-accessible than that in the pyridoxal 5'-phosphate-bound structure. A marked structural change occurs around the active site upon binding of pyridoxal 5'-phosphate that provides a solvent-inaccessible environment for the enzymatic reaction. Detailed enzyme structure analysis and structure comparisons, active site and substrate/ligand-binding structre, structure-function relationship, overview	Lentilactobacillus buchneri

kcat/KM [mM/s]

kcat/KM Value [1/mMs^-1]	kcat/KM Value Maximum [1/mMs^-1]	Substrate	Comment	Organism
0.00334	-	D-allo-isoleucine	recombinant mutant D222N, pH and temperature not specified in the publication	Lentilactobacillus buchneri
0.00534	-	L-isoleucine	recombinant mutant D222N, pH and temperature not specified in the publication	Lentilactobacillus buchneri
0.223	-	D-allo-isoleucine	recombinant mutant D222A, pH and temperature not specified in the publication	Lentilactobacillus buchneri
0.361	-	L-isoleucine	recombinant mutant D222A, pH and temperature not specified in the publication	Lentilactobacillus buchneri
12.4	-	D-allo-isoleucine	recombinant mutant Y142F, pH and temperature not specified in the publication	Lentilactobacillus buchneri
17.5	-	L-isoleucine	recombinant mutant Y142F, pH and temperature not specified in the publication	Lentilactobacillus buchneri
71.4	-	D-allo-isoleucine	recombinant wild-type enzyme, pH and temperature not specified in the publication	Lentilactobacillus buchneri
101	-	L-isoleucine	recombinant wild-type enzyme, pH and temperature not specified in the publication	Lentilactobacillus buchneri