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Literature summary for 6.3.2.4 extracted from

  • Miki, Y.; Okazaki, S.; Asano, Y.
    Engineering an ATP-dependent D-Ala D-Ala ligase for synthesizing amino acid amides from amino acids (2017), J. Ind. Microbiol. Biotechnol., 44, 667-675 .
    View publication on PubMed

Cloned(Commentary)

Cloned (Comment) Organism
gene dll, recombinant expression of N-terminally His6-tagged enzyme in Escherichia coli strain BL21(DE3) Thermus thermophilus

Protein Variants

Protein Variants Comment Organism
additional information homology structure modeling analyses of wild-type enzyme and mutants S293D and S293E Thermus thermophilus
S293A site-directed mutagenesis, the mutant shows altered substrate specificity compared to wild-type enzyme with production of D-AlaNH2 from D-Ala and NH3 in contrast to the wild-type enzyme Thermus thermophilus
S293C site-directed mutagenesis, the mutant shows altered substrate specificity compared to wild-type enzyme with production of D-AlaNH2 from D-Ala and NH3 in contrast to the wild-type enzyme Thermus thermophilus
S293D site-directed mutagenesis, the mutant shows altered substrate specificity compared to wild-type enzyme with high production of D-AlaNH2 from D-Ala and NH3 in contrast to the wild-type enzyme Thermus thermophilus
S293E site-directed mutagenesis, the mutant shows altered substrate specificity compared to wild-type enzyme with high production of D-AlaNH2 from D-Ala and NH3 in contrast to the wild-type enzyme Thermus thermophilus
S293F site-directed mutagenesis, the mutant shows altered substrate specificity compared to wild-type enzyme with production of D-AlaNH2 from D-Ala and NH3 in contrast to the wild-type enzyme Thermus thermophilus
S293G site-directed mutagenesis, the mutant shows altered substrate specificity compared to wild-type enzyme with production of D-AlaNH2 from D-Ala and NH3 in contrast to the wild-type enzyme Thermus thermophilus
S293H site-directed mutagenesis, the mutant shows altered substrate specificity compared to wild-type enzyme with production of D-AlaNH2 from D-Ala and NH3 in contrast to the wild-type enzyme Thermus thermophilus
S293I site-directed mutagenesis, the mutant shows altered substrate specificity compared to wild-type enzyme with high production of D-AlaNH2 from D-Ala and NH3 in contrast to the wild-type enzyme Thermus thermophilus
S293K site-directed mutagenesis, the mutant shows altered substrate specificity compared to wild-type enzyme with production of D-AlaNH2 from D-Ala and NH3 in contrast to the wild-type enzyme Thermus thermophilus
S293L site-directed mutagenesis, the mutant shows altered substrate specificity compared to wild-type enzyme with production of D-AlaNH2 from D-Ala and NH3 in contrast to the wild-type enzyme Thermus thermophilus
S293M site-directed mutagenesis, the mutant shows altered substrate specificity compared to wild-type enzyme with high production of D-AlaNH2 from D-Ala and NH3 in contrast to the wild-type enzyme Thermus thermophilus
S293N site-directed mutagenesis, the mutant shows altered substrate specificity compared to wild-type enzyme with production of D-AlaNH2 from D-Ala and NH3 in contrast to the wild-type enzyme Thermus thermophilus
S293P site-directed mutagenesis, the mutant shows altered substrate specificity compared to wild-type enzyme, it catalyzes the synthesis of both D-AlaNH2 and D-Ala-dDAla Thermus thermophilus
S293Q site-directed mutagenesis, the mutant shows altered substrate specificity compared to wild-type enzyme with production of D-AlaNH2 from D-Ala and NH3 in contrast to the wild-type enzyme Thermus thermophilus
S293R site-directed mutagenesis, the mutant shows altered substrate specificity compared to wild-type enzyme, it catalyzes the synthesis of both D-AlaNH2 and D-Ala-D-Ala Thermus thermophilus
S293S site-directed mutagenesis, the mutant shows altered substrate specificity compared to wild-type enzyme with production of D-AlaNH2 from D-Ala and NH3 in contrast to the wild-type enzyme Thermus thermophilus
S293T site-directed mutagenesis, the mutant shows altered substrate specificity compared to wild-type enzyme with production of D-AlaNH2 from D-Ala and NH3 in contrast to the wild-type enzyme Thermus thermophilus
S293V site-directed mutagenesis, the mutant shows altered substrate specificity compared to wild-type enzyme with high production of D-AlaNH2 from D-Ala and NH3 in contrast to the wild-type enzyme Thermus thermophilus
S293W site-directed mutagenesis, the mutant shows altered substrate specificity compared to wild-type enzyme with production of D-AlaNH2 from D-Ala and NH3 in contrast to the wild-type enzyme Thermus thermophilus
S293Y site-directed mutagenesis, the mutant shows altered substrate specificity compared to wild-type enzyme with production of D-AlaNH2 from D-Ala and NH3 in contrast to the wild-type enzyme Thermus thermophilus

KM Value [mM]

KM Value [mM] KM Value Maximum [mM] Substrate Comment Organism Structure
additional information
-
additional information Michaelis-Menten steady-state kinetics Thermus thermophilus
7.35
-
D-alanine pH 9.0, 40°C, mutant S293E Thermus thermophilus
8.86
-
D-alanine pH 9.0, 40°C, mutant S293D Thermus thermophilus
1530
-
NH3 pH 9.0, 40°C, mutant S293D Thermus thermophilus
1580
-
NH3 pH 9.0, 40°C, mutant S293E Thermus thermophilus

Metals/Ions

Metals/Ions Comment Organism Structure
Mg2+ required Thermus thermophilus

Natural Substrates/ Products (Substrates)

Natural Substrates Organism Comment (Nat. Sub.) Natural Products Comment (Nat. Pro.) Rev. Reac.
ATP + 2 D-alanine Thermus thermophilus
-
ADP + phosphate + D-alanyl-D-alanine
-
?
ATP + 2 D-alanine Thermus thermophilus HB8 / ATCC 27634 / DSM 579
-
ADP + phosphate + D-alanyl-D-alanine
-
?

Organism

Organism UniProt Comment Textmining
Thermus thermophilus Q5SHZ3
-
-
Thermus thermophilus HB8 / ATCC 27634 / DSM 579 Q5SHZ3
-
-

Purification (Commentary)

Purification (Comment) Organism
recombinant N-terminally His6-tagged enzyme from Escherichia coli strain BL21(DE3) by nickel affinity and anion exchange chromatography, followed by dialysis Thermus thermophilus

Substrates and Products (Substrate)

Substrates Comment Substrates Organism Products Comment (Products) Rev. Reac.
ATP + 2 D-alanine
-
Thermus thermophilus ADP + phosphate + D-alanyl-D-alanine
-
?
ATP + 2 D-alanine
-
Thermus thermophilus HB8 / ATCC 27634 / DSM 579 ADP + phosphate + D-alanyl-D-alanine
-
?
ATP + D-Ala + NH3 the D-AlaNH2 production of enzyme S293X mutants is optimized, the S293E variant, which is selected as the best enzyme for D-AlaNH2 production, exhibits an optimal activity at pH 9.0 and 40°C for D-AlaNH2 production. The S293E variant catalyzes the synthesis of 9.3 and 35.7 mM of D-AlaNH2 from 10 and 50 mM D-Ala and 3 M NH4Cl with conversion yields of 93% and 71.4%, respectively. The S293E variant exhibits higher reaction specificity to D-AlaNH2 production compared with the S293D variant and the other variants Thermus thermophilus ADP + phosphate + D-AlaNH2
-
?
ATP + D-Ala + NH3 the D-AlaNH2 production of enzyme S293X mutants is optimized, the S293E variant, which is selected as the best enzyme for D-AlaNH2 production, exhibits an optimal activity at pH 9.0 and 40°C for D-AlaNH2 production. The S293E variant catalyzes the synthesis of 9.3 and 35.7 mM of D-AlaNH2 from 10 and 50 mM D-Ala and 3 M NH4Cl with conversion yields of 93% and 71.4%, respectively. The S293E variant exhibits higher reaction specificity to D-AlaNH2 production compared with the S293D variant and the other variants Thermus thermophilus HB8 / ATCC 27634 / DSM 579 ADP + phosphate + D-AlaNH2
-
?

Synonyms

Synonyms Comment Organism
ATP-dependent D-Ala:D-Ala ligase
-
Thermus thermophilus
Ddl
-
Thermus thermophilus
TtDdL
-
Thermus thermophilus
TTHA1587
-
Thermus thermophilus

Temperature Optimum [°C]

Temperature Optimum [°C] Temperature Optimum Maximum [°C] Comment Organism
40
-
assay at Thermus thermophilus

Turnover Number [1/s]

Turnover Number Minimum [1/s] Turnover Number Maximum [1/s] Substrate Comment Organism Structure
0.076
-
D-alanine pH 9.0, 40°C, mutant S293E Thermus thermophilus
0.092
-
D-alanine pH 9.0, 40°C, mutant S293D Thermus thermophilus

pH Optimum

pH Optimum Minimum pH Optimum Maximum Comment Organism
9
-
assay at Thermus thermophilus

Cofactor

Cofactor Comment Organism Structure
ATP
-
Thermus thermophilus

General Information

General Information Comment Organism
additional information homology structure modeling analyses of wild-type enzyme and mutants S293D and S293E. Both amino acids Arg268 and Ser293 play an important role in the synthesis of D-Ala-D-Ala, residue Ser293 recognizes the carboxylate group of D-Ala2, but is not involved in the ATP hydrolysis, while the Arg268 residue recognizes the carboxylate group of D-Ala1 and is involved in ATP hydrolysis to form the activated acyl-phosphate intermediate. Ligand docking simulations Thermus thermophilus

kcat/KM [mM/s]

kcat/KM Value [1/mMs-1] kcat/KM Value Maximum [1/mMs-1] Substrate Comment Organism Structure
0.0103
-
D-alanine pH 9.0, 40°C, mutant S293E Thermus thermophilus
0.0104
-
D-alanine pH 9.0, 40°C, mutant S293D Thermus thermophilus