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Literature summary extracted from

  • Nakagawa, T.; Satake, R.; Sato, M.; Kino, K.
    Structure-based modification of D-alanine-D-alanine ligase from Thermotoga maritima ATCC 43589 for depsipeptide synthesis (2011), Biosci. Biotechnol. Biochem., 75, 700-704.
    View publication on PubMed

Application

EC Number Application Comment Organism
6.1.2.1 synthesis structure-based modification of D-alanine-D-alanine ligase from strain ATCC 43589 for D-alanyl-D-lactate and other depsipeptide synthesis by mutant S137G/Y207F Thermotoga maritima

Cloned(Commentary)

EC Number Cloned (Comment) Organism
6.1.2.1 expression of mutant enzymes in Escherichia coli strain BL21(DE3) as His6-tagged proteins Thermotoga maritima

Protein Variants

EC Number Protein Variants Comment Organism
6.1.2.1 additional information structure-based modification of D-alanine-D-alanine ligase from strain ATCC 43589 for depsipeptide synthesis, overview Thermotoga maritima
6.1.2.1 S137A site-directed mutagenesis, the mutant D-alanine-D-alanine ligase also shows formation of D-alanyl-D-lactate depsipeptide in contrast to the wild-type enzyme, EC 6.3.2.4, which is inactive with (R)-lactate Thermotoga maritima
6.1.2.1 S137A/Y207F site-directed mutagenesis, the mutant D-alanine-D-alanine ligase also shows formation of D-alanyl-D-lactate depsipeptide in contrast to the wild-type enzyme, EC 6.3.2.4, which is inactive with (R)-lactate Thermotoga maritima
6.1.2.1 S137F/Y207F site-directed mutagenesis, the mutant D-alanine-D-alanine ligase also shows formation of D-alanyl-D-lactate depsipeptide in contrast to the wild-type enzyme, EC 6.3.2.4, which is inactive with (R)-lactate Thermotoga maritima
6.1.2.1 S137G/Y207F site-directed mutagenesis, the mutant D-alanine-D-alanine ligase also shows formation of D-alanyl-D-lactate depsipeptide in contrast to the wild-type enzyme, EC 6.3.2.4, which is inactive with (R)-lactate Thermotoga maritima
6.1.2.1 S137T/Y207F site-directed mutagenesis, the mutant D-alanine-D-alanine ligase also shows formation of D-alanyl-D-lactate depsipeptide in contrast to the wild-type enzyme, EC 6.3.2.4, which is inactive with (R)-lactate Thermotoga maritima
6.1.2.1 Y201F/Y207F site-directed mutagenesis, the mutant D-alanine-D-alanine ligase also shows formation of D-alanyl-D-lactate depsipeptide in contrast to the wild-type enzyme, EC 6.3.2.4, which is inactive with (R)-lactate Thermotoga maritima
6.1.2.1 Y207F site-directed mutagenesis, the mutant D-alanine-D-alanine ligase also shows formation of D-alanyl-D-lactate depsipeptide in contrast to the wild-type enzyme, EC 6.3.2.4, which is inactive with (R)-lactate Thermotoga maritima
6.3.2.4 S137A/Y207F higher D-alanyl-D-lactate and lower D-alanyl-D-alanine synthase activity than mutant Y207F. Broad substrate specificity toward D-amino acids, accepts D-lactate and D,L-isoleucine as substrate Thermotoga maritima
6.3.2.4 S137F/Y207F about 20% of wild-type activity. Accepts D-lactate as substrate Thermotoga maritima
6.3.2.4 S137G/Y207F about 200% of wild-type activity. Accepts D-lactate as substrate Thermotoga maritima
6.3.2.4 S137T/Y207F about 40% of wild-type activity. Does not accept D-lactate as substrate Thermotoga maritima
6.3.2.4 Y201F increase in activity compared to wild-type, mutant is not able to synthesize D-alanyl-D-lactate Thermotoga maritima
6.3.2.4 Y207F mutant is able to synthesize D-alanyl-D-lactate Thermotoga maritima

Organism

EC Number Organism UniProt Comment Textmining
6.1.2.1 Thermotoga maritima
-
-
-
6.1.2.1 Thermotoga maritima ATCC 43589
-
-
-
6.3.2.4 Thermotoga maritima
-
-
-
6.3.2.4 Thermotoga maritima ATCC 43589
-
-
-

Purification (Commentary)

EC Number Purification (Comment) Organism
6.1.2.1 recombinant His6-tagged mutant enzymes from Escherichia coli strain BL21(DE3) by nickel affinity chromatography Thermotoga maritima

Specific Activity [micromol/min/mg]

EC Number Specific Activity Minimum [µmol/min/mg] Specific Activity Maximum [µmol/min/mg] Comment Organism
6.1.2.1 0.19
-
 D-alanyl-D-lactate depsipeptide formation activity, recombinant D-alanine-D-alanine ligase mutant S137F/Y207F, pH 7.5, 60°C Thermotoga maritima
6.1.2.1 0.39
-
 D-alanyl-D-lactate depsipeptide formation activity, recombinant D-alanine-D-alanine ligase mutant Y207F, pH 7.5, 60°C Thermotoga maritima
6.1.2.1 0.42
-
 D-alanyl-D-lactate depsipeptide formation activity, recombinant D-alanine-D-alanine ligase mutant S137A/Y207F, pH 7.5, 60°C Thermotoga maritima
6.1.2.1 1.2
-
 D-alanyl-D-lactate depsipeptide formation activity, recombinant D-alanine-D-alanine ligase mutant S137G/Y207F, pH 7.5, 60°C Thermotoga maritima
6.3.2.4 0.19
-
 mutant S137F/Y207F, substrates D-Ala + D-lactate, 60°C, pH 7.5 Thermotoga maritima
6.3.2.4 0.39
-
 mutant Y207F, substrates D-Ala + D-lactate, 60°C, pH 7.5 Thermotoga maritima
6.3.2.4 0.42
-
 mutant S137A/Y207F, substrates D-Ala + D-lactate, 60°C, pH 7.5 Thermotoga maritima
6.3.2.4 0.95
-
 mutant S137F/Y207F, substrate D-Ala, 60°C, pH 7.5 Thermotoga maritima
6.3.2.4 1.2
-
 mutant S137G/Y207F, substrates D-Ala + D-lactate, 60°C, pH 7.5 Thermotoga maritima
6.3.2.4 1.75
-
 mutant S137T/Y207F, substrate D-Ala, 60°C, pH 7.5 Thermotoga maritima
6.3.2.4 4.22
-
 wild-type, substrate D-Ala, 60°C, pH 7.5 Thermotoga maritima
6.3.2.4 6.489
-
 mutant S137A/Y207F, substrate D-Ala, 60°C, pH 7.5 Thermotoga maritima
6.3.2.4 9.66
-
 mutant S137G/Y207F, substrate D-Ala, 60°C, pH 7.5 Thermotoga maritima
6.3.2.4 17.22
-
 mutant Y207F, substrate D-Ala, 60°C, pH 7.5 Thermotoga maritima

Substrates and Products (Substrate)

EC Number Substrates Comment Substrates Organism Products Comment (Products) Rev. Reac.
6.1.2.1 D-alanine + (R)-lactate + ATP mutants variants Y207F, S137G/Y207F, S137F/Y207F, S137T/Y207F, and S137A/Y207F from D-alanine-D-alanine ligase, EC 6.3.2.4. The wild-type D-alanine-D-alanine ligase, EC 6.3.2.4 does not show activity with (R)-lactate Thermotoga maritima D-alanyl-(R)-lactate + ADP + phosphate
-
 ?
6.1.2.1 D-alanine + (R)-lactate + ATP mutants variants Y207F, S137G/Y207F, S137F/Y207F, S137T/Y207F, and S137A/Y207F from D-alanine-D-alanine ligase, EC 6.3.2.4. The wild-type D-alanine-D-alanine ligase, EC 6.3.2.4 does not show activity with (R)-lactate Thermotoga maritima ATCC 43589 D-alanyl-(R)-lactate + ADP + phosphate
-
 ?
6.1.2.1 additional information substrate specificity of recombinant D-alanine-D-alanine ligase mutant S137G/Y207F, overview Thermotoga maritima ?
-
 ?
6.1.2.1 additional information substrate specificity of recombinant D-alanine-D-alanine ligase mutant S137G/Y207F, overview Thermotoga maritima ATCC 43589 ?
-
 ?
6.3.2.4 ATP + D-Ala + D-Ala
-
 Thermotoga maritima ADP + phosphate + D-Ala-D-Ala
-
 ?
6.3.2.4 ATP + D-Ala + D-Ala
-
 Thermotoga maritima ATCC 43589 ADP + phosphate + D-Ala-D-Ala
-
 ?
6.3.2.4 ATP + D-Ala + D-lactate D-lactate is not a substrate for wild-type, but for mutants S137A/Y207F, S137F/Y207F, S137T/Y207F, S137G/Y207F, Y207F Thermotoga maritima ADP + phosphate + D-Ala-D-lactate
-
 ?
6.3.2.4 ATP + D-Ala + D-lactate D-lactate is not a substrate for wild-type, but for mutants S137A/Y207F, S137F/Y207F, S137T/Y207F, S137G/Y207F, Y207F Thermotoga maritima ATCC 43589 ADP + phosphate + D-Ala-D-lactate
-
 ?

Temperature Optimum [°C]

EC Number Temperature Optimum [°C] Temperature Optimum Maximum [°C] Comment Organism
6.1.2.1 60
-
 assay at Thermotoga maritima

pH Optimum

EC Number pH Optimum Minimum pH Optimum Maximum Comment Organism
6.1.2.1 7.5
-
 assay at Thermotoga maritima

Cofactor

EC Number Cofactor Comment Organism Structure
6.1.2.1 ATP
-
 Thermotoga maritima