Literature summary for 6.3.2.4 extracted from

Park, I.-S.; Lin, C.-H.; Walsh, C.T.
Gain of D-alanyl-D-lactate or D-lactyl-D-alanine synthetase activities in three active-site mutants of the Escherichia coli D-alanyl-D-alanine ligase B (1996), Biochemistry, 35, 10464-10471.

Protein Variants

Protein Variants	Comment	Organism
E15Q	mutant enzyme with negligible depsipeptide synthetase activity. The mutant uniquely activates D-Lac as the electrophilic rather than the nucleophilic partner for condensation with D-Ala to make a regioisomeric D-Lac-D-Ala, an amide rather than an ester product	Escherichia coli
S150A	mutant has gained depsipeptide (D-Ala-D-Lac, D-Ala-D-hydroxybutyrate) ligase activity with dipeptide/depsipeptide partition ratios that mimic the pH behavior of VanA (D-alanine(R)-lactate ligase)	Escherichia coli
Y216F	mutant has gained depsipeptide (D-Ala-D-Lac, D-Ala-D-hydroxybutyrate) ligase activity with dipeptide/depsipeptide partition ratios that mimic the pH behavior of VanA (D-alanine(R)-lactate ligase)	Escherichia coli

KM Value [mM]

KM Value [mM]	KM Value Maximum [mM]	Substrate	Comment	Organism
0.003	-	D-alanine	37°C, KM-value for D-alanine1 (the first molecule of substrate that is activated by the enzyme molecule) , wild-type enzyme, pH 7.5	Escherichia coli
0.004	-	D-alanine	37°C, KM-value for D-alanine1 (the first molecule of substrate that is activated by the enzyme molecule), wild-type enzyme, pH 9.2	Escherichia coli
0.11	-	D-alanine	37°C, KM-value for D-alanine1 (the first molecule of substrate that is activated by the enzyme molecule), wild-type enzyme, pH 6.0	Escherichia coli
0.51	-	D-alanine	37°C, KM-value for D-alanine1 (the first molecule of substrate that is activated by the enzyme molecule), mutant enzyme E15Q, pH 7.5	Escherichia coli
0.51	-	D-alanine	37°C, KM-value for D-alanine1 (the first molecule of substrate that is activated by the enzyme molecule), mutant enzyme E15Q, pH 9.2	Escherichia coli
0.85	-	D-alanine	37°C, KM-value for D-alanine1 (the first molecule of substrate that is activated by the enzyme molecule), mutant enzyme S150A, pH 7.5	Escherichia coli
1.1	-	D-alanine	37°C, KM-value for D-alanine2 (the second molecule of substrate that is activated by the enzyme molecule), wild-type enzyme, pH 9.2	Escherichia coli
2	-	D-alanine	37°C, KM-value for D-alanine2 (the second molecule of substrate that is activated by the enzyme molecule), wild-type enzyme, pH 7.5	Escherichia coli
4.8	-	D-alanine	37°C, KM-value for D-alanine1 (the first molecule of substrate that is activated by the enzyme molecule), mutant enzyme Y216F, pH 9.2	Escherichia coli
6.2	-	D-alanine	37°C, KM-value for D-alanine1 (the first molecule of substrate that is activated by the enzyme molecule), mutant enzyme S150A, pH 6.0	Escherichia coli
8	-	D-alanine	37°C, KM-value for D-alanine2 (the second molecule of substrate that is activated by the enzyme molecule), wild-type enzyme, pH 6.0	Escherichia coli
11	-	D-alanine	37°C, KM-value for D-alanine1 (the first molecule of substrate that is activated by the enzyme molecule), mutant enzyme E15Q, pH 6.0	Escherichia coli
16	-	D-alanine	37°C, KM-value for D-alanine1 (the first molecule of substrate that is activated by the enzyme molecule), mutant enzyme Y216F, pH 7.5	Escherichia coli
44	-	D-alanine	37°C, KM-value for D-alanine1 (the first molecule of substrate that is activated by the enzyme molecule) , mutant enzyme S150A, pH 9.2	Escherichia coli
65	-	D-alanine	37°C, KM-value for D-alanine2 (the second molecule of substrate that is activated by the enzyme molecule), mutant enzyme E15Q, pH 9.2	Escherichia coli
74	-	D-alanine	37°C, KM-value for D-alanine2 (the second molecule of substrate that is activated by the enzyme molecule), mutant enzyme E15Q, pH 7.5	Escherichia coli
80	-	D-alanine	37°C, KM-value for D-alanine2 (the second molecule of substrate that is activated by the enzyme molecule), mutant enzyme S150A, pH 7.5	Escherichia coli
82	-	D-alanine	37°C, KM-value for D-alanine2 (the second molecule of substrate that is activated by the enzyme molecule), mutant enzyme E15Q, pH 6.0	Escherichia coli
140	-	D-alanine	37°C, KM-value for D-alanine2 (the second molecule of substrate that is activated by the enzyme molecule), mutant enzyme S150A, pH 6.0	Escherichia coli
150	-	D-alanine	37°C, KM-value for D-alanine1 (the first molecule of substrate that is activated by the enzyme molecule) , mutant enzyme Y216F, pH 6.0	Escherichia coli
450	-	D-alanine	37°C, KM-value for D-alanine2 (the second molecule of substrate that is activated by the enzyme molecule), mutant enzyme Y216F, pH 6.0	Escherichia coli
630	-	D-alanine	37°C, KM-value for D-alanine2 (the second molecule of substrate that is activated by the enzyme molecule), mutant enzyme Y216F, pH 7.5	Escherichia coli
1100	-	D-alanine	37°C, KM-value for D-alanine2 (the second molecule of substrate that is activated by the enzyme molecule), mutant enzyme S150A, pH 9.2	Escherichia coli
1200	-	D-alanine	37°C, KM-value for D-alanine2 (the second molecule of substrate that is activated by the enzyme molecule), mutant enzyme Y216F, pH 9.2	Escherichia coli

Organism

Organism	UniProt	Comment	Textmining
Escherichia coli	P07862	-	-

Substrates and Products (Substrate)

Substrates	Comment Substrates	Organism	Products	Comment (Products)	Rev.	Reac.
ATP + 2 D-alanine	Y216, S150, and E15 form a hydrogen-bonding triad that orients an omega-loop to close over the active site and also to orient substrate D-Ala1 (the first molecule of substrate that is activated by the enzyme protein). The bifunctional enzyme also catalyzes the formation of D-alanyl-D-lactate (D-alanine-(R)-lactate ligase)	Escherichia coli	ADP + phosphate + D-alanyl-D-alanine	-	?

Synonyms

Synonyms	Comment	Organism
D-alanyl-D-alanine ligase B	-	Escherichia coli
DdlB	-	Escherichia coli

Turnover Number [1/s]

Turnover Number Minimum [1/s]	Turnover Number Maximum [1/s]	Substrate	Comment	Organism
3.3	-	D-alanine	37°C, mutant enzyme E15Q, pH 6.0	Escherichia coli
3.3	-	D-alanine	37°C, mutant enzyme S150A, pH 6.0	Escherichia coli
7.5	-	D-alanine	37°C, mutant enzyme Y216F, pH 6.0	Escherichia coli
10.5	-	D-alanine	37°C, mutant enzyme Y216F, pH 7.5	Escherichia coli
11.83	-	D-alanine	37°C, wild-type enzyme, pH 6.0	Escherichia coli
13.7	-	D-alanine	37°C, mutant enzyme E15Q, pH 7.5	Escherichia coli
14.7	-	D-alanine	37°C, wild-type enzyme, pH 7.5	Escherichia coli
16.3	-	D-alanine	37°C, mutant enzyme S150A, pH 7.5	Escherichia coli
18.3	-	D-alanine	37°C, mutant enzyme E15Q, pH 9.2	Escherichia coli
18.3	-	D-alanine	37°C, mutant enzyme S150A, pH 9.2	Escherichia coli
20	-	D-alanine	37°C, mutant enzyme Y216F, pH 9.2	Escherichia coli
26.7	-	D-alanine	37°C, wild-type enzyme, pH 9.2	Escherichia coli

kcat/KM [mM/s]

kcat/KM Value [1/mMs^-1]	kcat/KM Value Maximum [1/mMs^-1]	Substrate	Comment	Organism
1.4	-	D-alanine	37°C, mutant enzyme S150A, kcat/Km(D-alanine2), pH 6.0. D-Alanine2 is the second molecule of substrate that is activated by the enzyme molecule	Escherichia coli
2.4	-	D-alanine	37°C, mutant enzyme E15Q, kcat/Km(D-alanine2), pH 6.0. D-Alanine2 is the second molecule of substrate that is activated by the enzyme molecule	Escherichia coli
3	-	D-alanine	37°C, mutant enzyme Y216F, kcat/Km(D-alanine2), pH 6.0. D-Alanine2 is the second molecule of substrate that is activated by the enzyme molecule	Escherichia coli
11	-	D-alanine	37°C, mutant enzyme E15Q, kcat/Km(D-alanine2), pH 7.5. D-Alanine2 is the second molecule of substrate that is activated by the enzyme molecule	Escherichia coli
12	-	D-alanine	37°C, mutant enzyme S150A, kcat/Km(D-alanine2), pH 7.5. D-Alanine2 is the second molecule of substrate that is activated by the enzyme molecule	Escherichia coli
17	-	D-alanine	37°C, mutant enzyme E15Q, kcat/Km(D-alanine2), pH 9.2. D-Alanine2 is the second molecule of substrate that is activated by the enzyme molecule	Escherichia coli
26	-	D-alanine	37°C, mutant enzyme S150A, kcat/Km(D-alanine2), pH 9.2. D-Alanine2 is the second molecule of substrate that is activated by the enzyme molecule	Escherichia coli
39	-	D-alanine	37°C, mutant enzyme Y216F, kcat/Km(D-alanine2), pH 7.5. D-Alanine2 is the second molecule of substrate that is activated by the enzyme molecule	Escherichia coli
89	-	D-alanine	37°C, wild-type enzyme, kcat/Km(D-alanine2), pH 6.0. D-Alanine2 is the second molecule of substrate that is activated by the enzyme molecule	Escherichia coli
260	-	D-alanine	37°C, mutant enzyme Y216F, kcat/Km(D-alanine2), pH 9.2. D-Alanine2 is the second molecule of substrate that is activated by the enzyme molecule	Escherichia coli
440	-	D-alanine	37°C, wild-type enzyme, kcat/Km(D-alanine2), pH 7.5. D-Alanine2 is the second molecule of substrate that is activated by the enzyme molecule	Escherichia coli
1500	-	D-alanine	37°C, wild-type enzyme, kcat/Km(D-alanine2), pH 9.2. D-Alanine2 is the second molecule of substrate that is activated by the enzyme molecule	Escherichia coli