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Literature summary for 6.1.2.1 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

Application Comment Organism
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)

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

Protein Variants

Protein Variants Comment Organism
additional information structure-based modification of D-alanine-D-alanine ligase from strain ATCC 43589 for depsipeptide synthesis, overview Thermotoga maritima
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
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
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
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
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
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
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

Organism

Organism UniProt Comment Textmining
Thermotoga maritima
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Thermotoga maritima ATCC 43589
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-
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Purification (Commentary)

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

Specific Activity [micromol/min/mg]

Specific Activity Minimum [µmol/min/mg] Specific Activity Maximum [µmol/min/mg] Comment Organism
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
0.39
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D-alanyl-D-lactate depsipeptide formation activity, recombinant D-alanine-D-alanine ligase mutant Y207F, pH 7.5, 60°C Thermotoga maritima
0.42
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D-alanyl-D-lactate depsipeptide formation activity, recombinant D-alanine-D-alanine ligase mutant S137A/Y207F, pH 7.5, 60°C Thermotoga maritima
1.2
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D-alanyl-D-lactate depsipeptide formation activity, recombinant D-alanine-D-alanine ligase mutant S137G/Y207F, pH 7.5, 60°C Thermotoga maritima

Substrates and Products (Substrate)

Substrates Comment Substrates Organism Products Comment (Products) Rev. Reac.
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
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?
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
-
?
additional information substrate specificity of recombinant D-alanine-D-alanine ligase mutant S137G/Y207F, overview Thermotoga maritima ?
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?
additional information substrate specificity of recombinant D-alanine-D-alanine ligase mutant S137G/Y207F, overview Thermotoga maritima ATCC 43589 ?
-
?

Temperature Optimum [°C]

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

pH Optimum

pH Optimum Minimum pH Optimum Maximum Comment Organism
7.5
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assay at Thermotoga maritima

Cofactor

Cofactor Comment Organism Structure
ATP
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Thermotoga maritima