Information on EC 6.1.2.1 - D-alanine-(R)-lactate ligase

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The enzyme appears in viruses and cellular organisms

EC NUMBER
COMMENTARY hide
6.1.2.1
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RECOMMENDED NAME
GeneOntology No.
D-alanine-(R)-lactate ligase
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REACTION
REACTION DIAGRAM
COMMENTARY hide
ORGANISM
UNIPROT
LITERATURE
D-alanine + (R)-lactate + ATP = D-alanyl-(R)-lactate + ADP + phosphate
show the reaction diagram
PATHWAY
BRENDA Link
KEGG Link
MetaCyc Link
vancomycin resistance I
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SYSTEMATIC NAME
IUBMB Comments
D-alanine:(R)-lactate ligase (ADP-forming)
The product of this enzyme, the depsipeptide D-alanyl-(R)-lactate, can be incorporated into the peptidoglycan pentapeptide instead of the usual D-alanyl-D-alanine dipeptide, which is formed by EC 6.3.2.4, D-alanine---D-alanine ligase. The resulting peptidoglycan does not bind the glycopeptide antibiotics vancomycin and teicoplanin, conferring resistance on the bacteria.
ORGANISM
COMMENTARY hide
LITERATURE
UNIPROT
SEQUENCE DB
SOURCE
gene vanA
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Manually annotated by BRENDA team
gene vanA
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Manually annotated by BRENDA team
V583, isoform VanB
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Manually annotated by BRENDA team
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Manually annotated by BRENDA team
GENERAL INFORMATION
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
evolution
physiological function
additional information
SUBSTRATE
PRODUCT                       
REACTION DIAGRAM
ORGANISM
UNIPROT
COMMENTARY
(Substrate) hide
LITERATURE
(Substrate)
COMMENTARY
(Product) hide
LITERATURE
(Product)
Reversibility
r=reversible
ir=irreversible
?=not specified
D-Ala + D-2-hydroxybutanoate + ATP
D-Ala-D-2-hydroxybutanoate + ADP + phosphate
show the reaction diagram
D-Ala + D-2-hydroxyvalerate + ATP
D-Ala-D-2-hydroxyvalerate + ADP + phosphate
show the reaction diagram
D-Ala + D-Ala + ATP
D-Ala-D-Ala + ADP + phosphate
show the reaction diagram
D-Ala + D-lactate + ATP
D-Ala-D-lactate + ADP + phosphate
show the reaction diagram
D-alanine + (R)-lactate + ATP
D-alanyl-(R)-lactate + ADP + phosphate
show the reaction diagram
additional information
?
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NATURAL SUBSTRATES
NATURAL PRODUCTS
REACTION DIAGRAM
ORGANISM
UNIPROT
COMMENTARY
(Substrate) hide
LITERATURE
(Substrate)
COMMENTARY
(Product) hide
LITERATURE
(Product)
REVERSIBILITY
r=reversible
ir=irreversible
?=not specified
D-alanine + (R)-lactate + ATP
D-alanyl-(R)-lactate + ADP + phosphate
show the reaction diagram
additional information
?
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D-alanine:D-alanine (D-lactate) ligase (ADP) from Leuconostoc mesenteroides synthesizes the depsipeptide, D-alanyl-D-lactate, in addition to D-alanyl-D-alanine, when D-alanine and D-lactate are incubated simultaneously. Structure of bound D-alanine and D-lactate at the active subsites and substrate orientations, overview. With D-lactate a bifurcated H-bond from Arg301 to the R-OH of D-lactate may account for its orientation and nucleophile activation. This orientation is observed when the guanidino side chain of this residue is flexible. D-Alanine adopts an orientation that utilizes H-bonding to water 2882 and the D-alanyl phosphate in subsite 1. Both of these orientations provide mechanisms of deprotonation and place the nucleophile within 3.2 A of the electrophilic carbonyl of the D-alanyl phosphate intermediate for formation of the transition state, molecular docking and chiral specificity of lactate and alanine dockings, detailed overview
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COFACTOR
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
METALS and IONS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
INHIBITORS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
1-[[(4-fluorophenyl)sulfonyl]amino]-3-(morpholin-4-yl)propan-2-yl dihydrogen phosphate
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75% inhibition at 0.5 mM
1-[[(4-fluorophenyl)sulfonyl]amino]-3-(morpholin-4-yl)propan-2-yl phenyl hydrogen phosphate
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65% inhibition at 0.5 mM
1-[[(4-methoxyphenyl)sulfonyl]amino]-3-(morpholin-4-yl)propan-2-yl dihydrogen phosphate
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83% inhibition at 0.5 mM
Oxacillin
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VRSA-9 Ddl is less sensitive than VRSA-6 Ddl
Vancomycin
KM VALUE [mM]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
0.6 - 3
D-2-hydroxybutanoate
3.2 - 8.3
D-2-hydroxyvalerate
1.2 - 150
D-Ala
0.88 - 27
D-lactate
TURNOVER NUMBER [1/s]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
0.25 - 1.8
D-2-hydroxybutanoate
0.42 - 2.6
D-2-hydroxyvalerate
3.3 - 16.3
D-Ala
0.023 - 1.57
D-lactate
kcat/KM VALUE [1/mMs-1]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
0.084 - 3
D-2-hydroxybutanoate
16073
0.05 - 0.81
D-2-hydroxyvalerate
11120
0.024 - 0.65
D-Ala
291
0.014 - 0.74
D-lactate
600
SPECIFIC ACTIVITY [µmol/min/mg]
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
0.19
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D-alanyl-D-lactate depsipeptide formation activity, recombinant D-alanine-D-alanine ligase mutant S137F/Y207F, pH 7.5, 60C
0.39
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D-alanyl-D-lactate depsipeptide formation activity, recombinant D-alanine-D-alanine ligase mutant Y207F, pH 7.5, 60C
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, 60C
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, 60C
pH OPTIMUM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
7.5
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assay at
TEMPERATURE OPTIMUM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
60
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assay at
MOLECULAR WEIGHT
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
38000
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x * 38000, SDS-PAGE
41000
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2 * 41000, recombinant C-terminally His6-tagged, SDS-PAGE
82000
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recombinant C-terminally His6-tagged, gel filtration
SUBUNITS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
?
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x * 38000, SDS-PAGE
Crystallization/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
VanA crystallizes only in the presence of a phosphinate inhibitor analogue of D-alanine-D-alanine. The crystals diffract to at least 2.5 A resolution and are in the centred orthorhombic space group C2221
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VRSA-9 Ddl X-ray diffraction crystal structure determination and analysis
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Purification/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
recombinant His6-tagged mutant enzymes from Escherichia coli strain BL21(DE3) by nickel affinity chromatography
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Cloned/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
epxression in Escherichia coli
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expression in Escherichia coli
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expression of C-terminally His6-tagged VRSA-9 Ddl
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expression of mutant enzymes in Escherichia coli strain BL21(DE3) as His6-tagged proteins
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gene vanA, the gene is located on a polycistronic van operon
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ENGINEERING
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
S150A
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mutant of D-Ala-D-Ala ligase Ddl, mutant has gained depsipeptide ligase activity, i.e. formation of D-Ala-D-Lac, D-Ala-D-hydroxybutyrate, with dipeptide/depsipeptide partition ratios that mimic the pH behaviour of D-Ala-D-lactate ligase VanA. Mutant displays a clear pH-dependent partitioning between the preferred depsipeptide product at low pH and the dipeptide product at high pH
Y216F
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mutant of D-Ala-D-Ala ligase Ddl, mutant has gained depsipeptide ligase activity, i.e. formation of D-Ala-D-Lac, D-Ala-D-hydroxybutyrate, with dipeptide/depsipeptide partition ratios that mimic the pH behaviour of D-Ala-D-lactate ligase VanA. Mutant displays a clear pH-dependent partitioning between the preferred depsipeptide product at low pH and the dipeptide product at high pH
F261Y
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the mutant shows a complete loss of the ability to make D-alanyl-(R)-lactate
Q260K/A283E
S137A
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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
S137A/Y207F
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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
S137F/Y207F
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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
S137G/Y207F
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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
S137T/Y207F
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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
Y201F/Y207F
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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
Y207F
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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
additional information
APPLICATION
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
synthesis