Literature summary for 5.1.1.1 extracted from

Sun, X.; He, G.; Wang, X.; Xu, S.; Ju, J.; Xu, X.
Crystal structure of a thermostable alanine racemase from Thermoanaerobacter tengcongensis MB4 reveals the role of Gln360 in substrate selection (2015), PLoS ONE, 10, e0133516 .

Cloned(Commentary)

Cloned (Comment)	Organism
gene alr1, recombinant expression of His-tagged wild-type and mutant enzymes in Escherichia coli strain BL21(DE3)	Caldanaerobacter subterraneus subsp. tengcongensis

Crystallization (Commentary)

Crystallization (Comment)	Organism
purified recombinant His-tagged wild-type enzyme in complex with L-alanine and pyridoxal 5'-phosphate, hanging drop vapor diffusion method, mixing of 0.001 ml of 10 mg/ml protein in 10 mM Tris-HCl, pH 8.0, L-Ala and PLP at 1:1.5:1.5 molar ratio, with 0.001 ml of reservoir solution containing 22% PEG 4000, and 0.1 M Bis-Tris, pH 7.0, and equilibration against 0.3 ml reservoir solution, 16°C, X-ray diffraction structure determination and analysis at 2.7 A resolution, molecular replacement using the alanine racemase from Bacillus stearothermophilus, PDB ID 1SFT, as a search model	Caldanaerobacter subterraneus subsp. tengcongensis

Crystallization (Comment)

Organism

purified recombinant His-tagged wild-type enzyme in complex with L-alanine and pyridoxal 5'-phosphate, hanging drop vapor diffusion method, mixing of 0.001 ml of 10 mg/ml protein in 10 mM Tris-HCl, pH 8.0, L-Ala and PLP at 1:1.5:1.5 molar ratio, with 0.001 ml of reservoir solution containing 22% PEG 4000, and 0.1 M Bis-Tris, pH 7.0, and equilibration against 0.3 ml reservoir solution, 16°C, X-ray diffraction structure determination and analysis at 2.7 A resolution, molecular replacement using the alanine racemase from Bacillus stearothermophilus, PDB ID 1SFT, as a search model

Caldanaerobacter subterraneus subsp. tengcongensis

Protein Variants

Protein Variants	Comment	Organism
additional information	enzyme residue Gln360 and conformational changes of active site residues disrupt the hydrogen bonding interactions necessary for proper pyridoxal 5'-phosphate immobilization, and decrease both the substrate affinity and turnover number of AlrTt. Introduction of hydrophobic amino acids at Gln360 increase the racemase activity of AlrTt	Caldanaerobacter subterraneus subsp. tengcongensis
Q360A	site-directed mutagenesis, the mutant shows about 1.2fold increased activity compared to the wild-type enzyme	Caldanaerobacter subterraneus subsp. tengcongensis
Q360C	site-directed mutagenesis, the mutant shows slightly reduced activity compared to the wild-type enzyme	Caldanaerobacter subterraneus subsp. tengcongensis
Q360D	site-directed mutagenesis, the mutant shows about 70% reduced activity compared to the wild-type enzyme	Caldanaerobacter subterraneus subsp. tengcongensis
Q360E	site-directed mutagenesis, the mutant shows slightly reduced activity compared to the wild-type enzyme	Caldanaerobacter subterraneus subsp. tengcongensis
Q360F	site-directed mutagenesis, the mutant shows about 1.5fold increased activity compared to the wild-type enzyme	Caldanaerobacter subterraneus subsp. tengcongensis
Q360G	site-directed mutagenesis, the mutant shows similar activity compared to the wild-type enzyme	Caldanaerobacter subterraneus subsp. tengcongensis
Q360H	site-directed mutagenesis, the mutant shows about 2.4fold increased activity compared to the wild-type enzyme	Caldanaerobacter subterraneus subsp. tengcongensis
Q360I	site-directed mutagenesis, the mutant shows 3fold increased activity compared to the wild-type enzyme	Caldanaerobacter subterraneus subsp. tengcongensis
Q360K	site-directed mutagenesis, the mutant shows about 70% reduced activity compared to the wild-type enzyme	Caldanaerobacter subterraneus subsp. tengcongensis
Q360L	site-directed mutagenesis, the mutant shows about 2.5fold increased activity compared to the wild-type enzyme	Caldanaerobacter subterraneus subsp. tengcongensis
Q360M	site-directed mutagenesis, the mutant shows similar activity compared to the wild-type enzyme	Caldanaerobacter subterraneus subsp. tengcongensis
Q360N	site-directed mutagenesis, the mutant shows about 2.3fold increased activity compared to the wild-type enzyme	Caldanaerobacter subterraneus subsp. tengcongensis
Q360P	site-directed mutagenesis, the mutant shows about 1.5fold increased activity compared to the wild-type enzyme	Caldanaerobacter subterraneus subsp. tengcongensis
Q360R	site-directed mutagenesis, the mutant shows similar activity compared to the wild-type enzyme	Caldanaerobacter subterraneus subsp. tengcongensis
Q360S	site-directed mutagenesis, the mutant shows similar activity compared to the wild-type enzyme	Caldanaerobacter subterraneus subsp. tengcongensis
Q360T	site-directed mutagenesis, the mutant shows about 1.8fold increased activity compared to the wild-type enzyme	Caldanaerobacter subterraneus subsp. tengcongensis
Q360V	site-directed mutagenesis, the mutant shows about 2.3fold increased activity compared to the wild-type enzyme	Caldanaerobacter subterraneus subsp. tengcongensis
Q360W	site-directed mutagenesis, the mutant shows 3fold increased activity compared to the wild-type enzyme	Caldanaerobacter subterraneus subsp. tengcongensis
Q360Y	site-directed mutagenesis, the mutant shows 3fold increased activity compared to the wild-type enzyme	Caldanaerobacter subterraneus subsp. tengcongensis
S173D	site-directed mutagenesis	Caldanaerobacter subterraneus subsp. tengcongensis

KM Value [mM]

KM Value [mM]	KM Value Maximum [mM]	Substrate	Comment	Organism
52	-	D-alanine	pH 10.0, 65°C, recombinant mutant Q360Y	Caldanaerobacter subterraneus subsp. tengcongensis
58.5	-	D-alanine	pH 10.0, 65°C, recombinant mutant S173D/Q360Y	Caldanaerobacter subterraneus subsp. tengcongensis
83.1	-	L-alanine	pH 10.0, 65°C, recombinant mutant S173D/Q360Y	Caldanaerobacter subterraneus subsp. tengcongensis
87.8	-	L-alanine	pH 10.0, 65°C, recombinant mutant Q360Y	Caldanaerobacter subterraneus subsp. tengcongensis
110.3	-	D-alanine	pH 10.0, 65°C, recombinant mutant Q360I	Caldanaerobacter subterraneus subsp. tengcongensis
136	-	L-alanine	pH 10.0, 65°C, recombinant mutant Q360I	Caldanaerobacter subterraneus subsp. tengcongensis
157.5	-	L-alanine	pH 10.0, 65°C, recombinant mutant S173D	Caldanaerobacter subterraneus subsp. tengcongensis
160.9	-	D-alanine	pH 10.0, 65°C, recombinant mutant S173D	Caldanaerobacter subterraneus subsp. tengcongensis
176.1	-	L-alanine	pH 10.0, 65°C, recombinant mutant Q360W	Caldanaerobacter subterraneus subsp. tengcongensis
216.1	-	D-alanine	pH 10.0, 65°C, recombinant mutant Q360W	Caldanaerobacter subterraneus subsp. tengcongensis
381.2	-	D-alanine	pH 10.0, 65°C, recombinant wild-type enzyme	Caldanaerobacter subterraneus subsp. tengcongensis
780.2	-	L-alanine	pH 10.0, 65°C, recombinant wild-type enzyme	Caldanaerobacter subterraneus subsp. tengcongensis

Natural Substrates/ Products (Substrates)

Natural Substrates	Organism	Comment (Nat. Sub.)	Natural Products	Comment (Nat. Pro.)	Rev.	Reac.
L-alanine	Caldanaerobacter subterraneus subsp. tengcongensis	-	D-alanine	-	r

Organism

Organism	UniProt	Comment	Textmining
Caldanaerobacter subterraneus subsp. tengcongensis	Q8RAK6	isolated from a hot spring in Tengchong, Yunnan province of China	-

Purification (Commentary)

Purification (Comment)	Organism
recombinant His-tagged wild-type and mutant enzymes from Escherichia coli strain BL21(DE3) by nickel affinity chromatography, gel filtration, dialysis, and ultrafiltration	Caldanaerobacter subterraneus subsp. tengcongensis

Source Tissue

Source Tissue	Comment	Organism	Textmining
additional information	optimal propagation occurs at 75°C and pH 7.0-7.5	Caldanaerobacter subterraneus subsp. tengcongensis	-

Substrates and Products (Substrate)

Substrates	Comment Substrates	Organism	Products	Comment (Products)	Rev.
D-alanine	-	Caldanaerobacter subterraneus subsp. tengcongensis	L-alanine	-	r
L-alanine	-	Caldanaerobacter subterraneus subsp. tengcongensis	D-alanine	-	r
L-alanine	reversible racemization	Caldanaerobacter subterraneus subsp. tengcongensis	D-alanine	-	r
additional information	the enzyme is absolutely specific for L-alanine compared to other L-amino acids, overview	Caldanaerobacter subterraneus subsp. tengcongensis	?	-	?

Subunits

Subunits	Comment	Organism
homodimer	each monomer covers full-length AlrTt (Val1-Lys383), it contains an N-terminal eight-stranded alpha/beta barrel domain (residues 1-244) and a C-terminal extended beta-strand domain (residues 245-383). The active site is located in the center of alpha/beta barrel domain, it is surrounded by parallel beta strands. Two identical monomers associate at the C-terminal beta-strand domain and the alpha/beta barrel domain to form the functional dimer, enzyme structure determination and analysis, active site structure, detailed overview. A hydrophobic patch localizes underneath Gln360 and phosphate group of pyridoxal 5'-phosphate. This hydrophobic patch is conserved both in sequence and conformation among bacterial alanine racemase	Caldanaerobacter subterraneus subsp. tengcongensis

Synonyms

Synonyms	Comment	Organism
ALR1	-	Caldanaerobacter subterraneus subsp. tengcongensis
alrTt	-	Caldanaerobacter subterraneus subsp. tengcongensis
MBalr1	-	Caldanaerobacter subterraneus subsp. tengcongensis

Temperature Optimum [°C]

Temperature Optimum [°C]	Temperature Optimum Maximum [°C]	Comment	Organism
65	-	assay at	Caldanaerobacter subterraneus subsp. tengcongensis

Turnover Number [1/s]

Turnover Number Minimum [1/s]	Turnover Number Maximum [1/s]	Substrate	Comment	Organism
32.5	-	D-alanine	pH 10.0, 65°C, recombinant mutant S173D	Caldanaerobacter subterraneus subsp. tengcongensis
34.7	-	L-alanine	pH 10.0, 65°C, recombinant mutant S173D	Caldanaerobacter subterraneus subsp. tengcongensis
96.9	-	D-alanine	pH 10.0, 65°C, recombinant wild-type enzyme	Caldanaerobacter subterraneus subsp. tengcongensis
133.1	-	D-alanine	pH 10.0, 65°C, recombinant mutant S173D/Q360Y	Caldanaerobacter subterraneus subsp. tengcongensis
212.4	-	L-alanine	pH 10.0, 65°C, recombinant wild-type enzyme	Caldanaerobacter subterraneus subsp. tengcongensis
225	-	D-alanine	pH 10.0, 65°C, recombinant mutant Q360I	Caldanaerobacter subterraneus subsp. tengcongensis
246.5	-	L-alanine	pH 10.0, 65°C, recombinant mutant S173D/Q360Y	Caldanaerobacter subterraneus subsp. tengcongensis
251.2	-	D-alanine	pH 10.0, 65°C, recombinant mutant Q360Y	Caldanaerobacter subterraneus subsp. tengcongensis
286.6	-	L-alanine	pH 10.0, 65°C, recombinant mutant Q360I	Caldanaerobacter subterraneus subsp. tengcongensis
431.5	-	L-alanine	pH 10.0, 65°C, recombinant mutant Q360Y	Caldanaerobacter subterraneus subsp. tengcongensis
612.4	-	L-alanine	pH 10.0, 65°C, recombinant mutant Q360W	Caldanaerobacter subterraneus subsp. tengcongensis
633.5	-	D-alanine	pH 10.0, 65°C, recombinant mutant Q360W	Caldanaerobacter subterraneus subsp. tengcongensis

pH Optimum

pH Optimum Minimum	pH Optimum Maximum	Comment	Organism
10	-	assay at	Caldanaerobacter subterraneus subsp. tengcongensis

Cofactor

Cofactor	Comment	Organism	Structure
pyridoxal 5'-phosphate	PLP, the PLP content of the enzyme is determined by a spectroscopic method	Caldanaerobacter subterraneus subsp. tengcongensis

General Information

General Information	Comment	Organism
evolution	in bacteria, two types of alanine racemase are encoded independently by two genes named dadX and alr. The dadX gene encodes a catabolic alanine racemase DadX, which catalyzes direct conversion of L-Ala to D-Ala. Its expression is induced by L- or D-Ala. The alr gene encodes an anabolic alanine racemase Alr, it is expressed constitutively at low level and essential for providing abundant D-Ala for peptidoglycan biosynthesis. Some bacteria only contain one type alanine racemase gene, whereas others have two of them. Thermoanaerobacter tengcongensis strain MB4 contains two annotated alanine racemase genes MBalr1 and MBalr2. Both genes encode 388 amino acids long alanine racemase, sharing a 58.3% amino acid sequence identity. Compared with MBAlr2, MBAlr1 shows very low catalytic efficiency and limited substrate spectrum. It is probable that MBAlr1 serves as an anabolic and MBAlr2 as the catabolic alanine racemase in Thermoanaerobacter tengcongensis strain MB4	Caldanaerobacter subterraneus subsp. tengcongensis
malfunction	Mycobacterium smegmatis strains with a deletion of the alr gene require D-Ala for growth, indicating the essential role of Alr in D-Ala production. Gln360 and conformational changes of active site residues disrupt the hydrogen bonding interactions necessary for proper pyridoxal 5'-phosphate immobilization, and decrease both the substrate affinity and turnover number of AlrTt. Introduction of hydrophobic amino acids at Gln360 increase the racemase activity of AlrTt	Caldanaerobacter subterraneus subsp. tengcongensis
additional information	Residue Gln360 plays an essential role in substrate selection and has a preference for hydrophobic amino acids, especially Tyr, in bacterial alanine racemization. Enzyme structure determination and analysis, active site structure, detailed overview	Caldanaerobacter subterraneus subsp. tengcongensis
physiological function	in bacteria, two types of alanine racemase are encoded independently by two genes named dadX and alr. The dadX gene encodes a catabolic alanine racemase DadX, which catalyzes direct conversion of L-Ala to D-Ala. Its expression is induced by L- or D-Ala. The alr gene encodes an anabolic alanine racemase Alr, it is expressed constitutively at low level and essential for providing abundant D-Ala for peptidoglycan biosynthesis. The catabolic alanine racemase DadX usually shows much higher catalytic efficiency than the anabolic enzyme Alr. Essential role of Alr in D-Ala production	Caldanaerobacter subterraneus subsp. tengcongensis

kcat/KM [mM/s]

kcat/KM Value [1/mMs^-1]	kcat/KM Value Maximum [1/mMs^-1]	Substrate	Comment	Organism
0.2	-	D-alanine	pH 10.0, 65°C, recombinant mutant S173D	Caldanaerobacter subterraneus subsp. tengcongensis
0.22	-	L-alanine	pH 10.0, 65°C, recombinant mutant S173D	Caldanaerobacter subterraneus subsp. tengcongensis
0.25	-	D-alanine	pH 10.0, 65°C, recombinant wild-type enzyme	Caldanaerobacter subterraneus subsp. tengcongensis
0.27	-	L-alanine	pH 10.0, 65°C, recombinant wild-type enzyme	Caldanaerobacter subterraneus subsp. tengcongensis
2.04	-	D-alanine	pH 10.0, 65°C, recombinant mutant Q360I	Caldanaerobacter subterraneus subsp. tengcongensis
2.11	-	L-alanine	pH 10.0, 65°C, recombinant mutant Q360I	Caldanaerobacter subterraneus subsp. tengcongensis
2.27	-	D-alanine	pH 10.0, 65°C, recombinant mutant S173D/Q360Y	Caldanaerobacter subterraneus subsp. tengcongensis
2.93	-	D-alanine	pH 10.0, 65°C, recombinant mutant Q360W	Caldanaerobacter subterraneus subsp. tengcongensis
2.97	-	L-alanine	pH 10.0, 65°C, recombinant mutant S173D/Q360Y	Caldanaerobacter subterraneus subsp. tengcongensis
3.48	-	L-alanine	pH 10.0, 65°C, recombinant mutant Q360W	Caldanaerobacter subterraneus subsp. tengcongensis
4.83	-	D-alanine	pH 10.0, 65°C, recombinant mutant Q360Y	Caldanaerobacter subterraneus subsp. tengcongensis
4.91	-	L-alanine	pH 10.0, 65°C, recombinant mutant Q360Y	Caldanaerobacter subterraneus subsp. tengcongensis