Literature summary for 5.1.1.1 extracted from

Asojo, O.A.; Nelson, S.K.; Mootien, S.; Lee, Y.; Rezende, W.C.; Hyman, D.A.; Matsumoto, M.M.; Reiling, S.; Kelleher, A.; Ledizet, M.; Koski, R.A.; Anthony, K.G.
Structural and biochemical analyses of alanine racemase from the multidrug-resistant Clostridium difficile strain 630 (2014), Acta Crystallogr. Sect. D, 70, 1922-1933 .

Application

Application	Comment	Organism
drug development	the enzyme is a potential target for development of antibiotic compounds	Clostridioides difficile

Cloned(Commentary)

Cloned (Comment)	Organism
gene alr, sequence comparisons, recombinant expression of codon-optimized wild-type and mutant enzymes in Escherichia coli strain Rosetta 2 (pLysS)DE3, recombinant expression of wild-type and mutant enzymes in Escherichia coli alr/dadX double-knockout mutant strain MB2159, the wild-type and K271T mutant CdAlr proteins are able to complement the D-alanine auxotrophy and restore its growth in D-alanine-free medium	Clostridioides difficile

Cloned (Comment)

Organism

gene alr, sequence comparisons, recombinant expression of codon-optimized wild-type and mutant enzymes in Escherichia coli strain Rosetta 2 (pLysS)DE3, recombinant expression of wild-type and mutant enzymes in Escherichia coli alr/dadX double-knockout mutant strain MB2159, the wild-type and K271T mutant CdAlr proteins are able to complement the D-alanine auxotrophy and restore its growth in D-alanine-free medium

Clostridioides difficile

Crystallization (Commentary)

Crystallization (Comment)	Organism
wild-type enzyme in complex with pyridoxal-5'-phosphate or with D-cycloserine, and enzyme mutant K271T in complex with pyridoxal 5'-phosphate, sitting drop vapor diffusion method, mixing of 0.002 ml of 32 m/ml protein in 10 mM PLP, 1 mM TCEP, and 50 mM Tris-HCl, pH 8.0, with 0.0015 ml of reservoir solution containing 0.2 M ammonium sulfate, 0.1 M Bis-Tris, pH 6.5, 25% w/v PEG 3350 for the wild-type/PLP complex, and additional 100 mM cycloserine, 200 mM sodium formate, 20% w/v PEG 3350 for the wild-type enzyme/inhibitor complex, or containing 0.17 M lithium sulfate, 0.085 M Tris-HCl, pH 8.5, 25.5% w/v PEG 4000, and 20% v/v glycerol for the enzyme mutant/PLP complex, X-ray diffraction structure determination and analysis at 2.1-2.6 A resolution	Clostridioides difficile

Crystallization (Comment)

Organism

wild-type enzyme in complex with pyridoxal-5'-phosphate or with D-cycloserine, and enzyme mutant K271T in complex with pyridoxal 5'-phosphate, sitting drop vapor diffusion method, mixing of 0.002 ml of 32 m/ml protein in 10 mM PLP, 1 mM TCEP, and 50 mM Tris-HCl, pH 8.0, with 0.0015 ml of reservoir solution containing 0.2 M ammonium sulfate, 0.1 M Bis-Tris, pH 6.5, 25% w/v PEG 3350 for the wild-type/PLP complex, and additional 100 mM cycloserine, 200 mM sodium formate, 20% w/v PEG 3350 for the wild-type enzyme/inhibitor complex, or containing 0.17 M lithium sulfate, 0.085 M Tris-HCl, pH 8.5, 25.5% w/v PEG 4000, and 20% v/v glycerol for the enzyme mutant/PLP complex, X-ray diffraction structure determination and analysis at 2.1-2.6 A resolution

Clostridioides difficile

Inhibitors

Inhibitors	Comment	Organism	Structure
D-cycloserine	active site bound inhibitor, binding structure, overview	Clostridioides difficile

KM Value [mM]

KM Value [mM]	KM Value Maximum [mM]	Substrate	Comment	Organism
additional information	-	additional information	Michaelis-Menten kinetics	Clostridioides difficile
5.4	-	D-alanine	pH 8.5, temperature not specified in the publication, recombinant mutant K271T	Clostridioides difficile
7	-	D-alanine	pH 8.5, temperature not specified in the publication, recombinant wild-type enzyme	Clostridioides difficile
13.8	-	L-alanine	pH 8.5, temperature not specified in the publication, recombinant mutant K271T	Clostridioides difficile
17.6	-	L-alanine	pH 8.5, temperature not specified in the publication, recombinant wild-type enzyme	Clostridioides difficile

Molecular Weight [Da]

Molecular Weight [Da]	Molecular Weight Maximum [Da]	Comment	Organism
80530	-	mutant enzyme, gel filtration	Clostridioides difficile
81090	-	wild-type enzyme, gel filtration	Clostridioides difficile
86400	-	mutant enzyme, about, sequence calculation	Clostridioides difficile
86600	-	wild-type enzyme, about, sequence calculation	Clostridioides difficile

Natural Substrates/ Products (Substrates)

Natural Substrates	Organism	Comment (Nat. Sub.)	Natural Products	Comment (Nat. Pro.)	Rev.	Reac.
L-alanine	Clostridioides difficile	-	D-alanine	-	r
L-alanine	Clostridioides difficile 630	-	D-alanine	-	r

Organism

Organism	UniProt	Comment	Textmining
Clostridioides difficile	Q180W0	strain 630 is virulent and multidrug-resistant	-
Clostridioides difficile 630	Q180W0	strain 630 is virulent and multidrug-resistant	-

Purification (Commentary)

Purification (Comment)	Organism
recombinant codon-optimized wild-type and mutant enzymes from Escherichia coli strain Rosetta 2 (pLysS) DE3 by anion exchange chromatography, gel filtration, and dialysis	Clostridioides difficile

Substrates and Products (Substrate)

Substrates	Comment Substrates	Organism	Products	Comment (Products)	Rev.
L-alanine	-	Clostridioides difficile	D-alanine	-	r
L-alanine	reversible racemization	Clostridioides difficile	D-alanine	-	r
L-alanine	-	Clostridioides difficile 630	D-alanine	-	r
L-alanine	reversible racemization	Clostridioides difficile 630	D-alanine	-	r

Subunits

Subunits	Comment	Organism
homodimer	2 x 43306, recombinant wild-type enzyme, mass spectrometry, 2 * 43279, recombinant mutant enzyme, mass spectrometry	Clostridioides difficile
More	the enzyme forms a homodimer with two active sites in which the cofactor pyridoxal 5'-phosphate is bound. Intermolecular interactions, overview	Clostridioides difficile

Synonyms

Synonyms	Comment	Organism
ALR	-	Clostridioides difficile
CdAlr	-	Clostridioides difficile

Turnover Number [1/s]

Turnover Number Minimum [1/s]	Turnover Number Maximum [1/s]	Substrate	Comment	Organism
19.25	-	D-alanine	pH 8.5, temperature not specified in the publication, recombinant wild-type enzyme	Clostridioides difficile
59.3	-	L-alanine	pH 8.5, temperature not specified in the publication, recombinant wild-type enzyme	Clostridioides difficile
1298.4	-	D-alanine	pH 8.5, temperature not specified in the publication, recombinant mutant K271T	Clostridioides difficile
3275.5	-	L-alanine	pH 8.5, temperature not specified in the publication, recombinant mutant K271T	Clostridioides difficile

pH Optimum

pH Optimum Minimum	pH Optimum Maximum	Comment	Organism
8.5	-	assay at	Clostridioides difficile

Cofactor

Cofactor	Comment	Organism	Structure
pyridoxal 5'-phosphate	-	Clostridioides difficile

General Information

General Information	Comment	Organism
additional information	analysis of the enzyme structure, active site structure, and intermolecular interactions, structure comparisons, overview. The N-terminal and C-terminal domains of all reported Alr structures are connected by a short hinge region, but the hinge angles between the N- and C-terminal of Alrs vary	Clostridioides difficile
physiological function	alanine racemase (Alr) is a pyridoxal 5'-phosphate (PLP)-dependent enzyme that catalyzes the reversible racemization of L- and D-alanine. D-alanine is an essential component of the bacterial cell-wall peptidoglycan	Clostridioides difficile