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Information on EC 6.1.2.1 - D-alanine-(R)-lactate ligase
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6.1.2.1 D-alanine-(R)-lactate ligaseIUBMB Comments
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.
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Word Map
- 6.1.2.1
- vancomycin
- glycopeptide
- peptidoglycan
-
vancomycin-resistant
- faecium
- ligases
- aureus
- d-alanyl-d-alanine
-
glycopeptide-resistant
-
enterococcal
-
subsite
-
hydrogen-bonding
- faecalis
- teicoplanins
-
omega-loop
- d-ala-d-lactate
- penicillin
-
d-ala:d-ala
-
resort
-
vand-type
-
d-alanine:d-alanine
- desulfitobacterium
-
amycolatopsis
- hafniense
-
teicoplanin-resistant
- mesenteroides
-
leuconostoc
-
d-ala2
- synthesis
The enzyme appears in viruses and cellular organisms
Synonyms
d-alanine-d-lactate ligase, depsipeptide ligase, d-ala:d-lac ligase, d-ala-d-lac ligase, vand4, d-alanyl-d-lactate ligase, vrsa-9 ddl, d-alanine:d-lactate ligase, 
more
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SYNONYM 
ORGANISM
UNIPROT
COMMENTARY 
LITERATURE
D-Ala-D-Lac ligase
D-Ala:D-Lac ligase
D-alanine-D-lactate ligase
D-alanyl-D-lactate ligase
VanA
VanB
VanD
VanD4
VanI
VRSA-9 Ddl
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REACTION
REACTION DIAGRAM
COMMENTARY
ORGANISM
UNIPROT
LITERATURE
D-alanine + (R)-lactate + ATP = D-alanyl-(R)-lactate + ADP + phosphate
-
-
-
-
D-alanine + (R)-lactate + ATP = D-alanyl-(R)-lactate + ADP + phosphate
Arg301 has a dual function in a sequential reaction mechanism, i.e. substrate orientation in subsite 2 aswell as stabilization of the transition state, overview
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PATHWAY SOURCE
PATHWAYS
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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.
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SUBSTRATE
PRODUCT
REACTION DIAGRAM
ORGANISM
UNIPROT
COMMENTARY
(Substrate)
(Substrate)
LITERATURE
(Substrate)
(Substrate)
COMMENTARY
(Product)
(Product)
LITERATURE
(Product)
(Product)
Reversibility
r=reversible
ir=irreversible
?=not specified
r=reversible
ir=irreversible
?=not specified
D-Ala + D-2-hydroxybutanoate + ATP
D-Ala-D-2-hydroxybutanoate + ADP + phosphate
-
-
-
-
?
D-Ala + D-2-hydroxybutanoate + ATP
D-Ala-D-2-hydroxybutanoate + ADP + phosphate
-
-
-
-
?
D-Ala + D-2-hydroxybutanoate + ATP
D-Ala-D-2-hydroxybutanoate + ADP + phosphate
-
-
-
-
?
D-Ala + D-2-hydroxybutanoate + ATP
D-Ala-D-2-hydroxybutanoate + ADP + phosphate
-
-
-
-
?
D-Ala + D-2-hydroxyvalerate + ATP
D-Ala-D-2-hydroxyvalerate + ADP + phosphate
-
-
-
-
?
D-Ala + D-2-hydroxyvalerate + ATP
D-Ala-D-2-hydroxyvalerate + ADP + phosphate
-
-
-
-
?
D-Ala + D-2-hydroxyvalerate + ATP
D-Ala-D-2-hydroxyvalerate + ADP + phosphate
-
-
-
-
?
D-Ala + D-lactate + ATP
D-Ala-D-lactate + ADP + phosphate
-
-
-
-
?
D-Ala + D-lactate + ATP
D-Ala-D-lactate + ADP + phosphate
-
-
-
-
?
D-alanine + (R)-lactate + ATP
D-alanyl-(R)-lactate + ADP + phosphate
-
-
-
-
?
D-alanine + (R)-lactate + ATP
D-alanyl-(R)-lactate + ADP + phosphate
-
-
-
-
?
D-alanine + (R)-lactate + ATP
D-alanyl-(R)-lactate + ADP + phosphate
-
-
-
-
?
D-alanine + (R)-lactate + ATP
D-alanyl-(R)-lactate + ADP + phosphate
-
-
-
-
?
D-alanine + (R)-lactate + ATP
D-alanyl-(R)-lactate + ADP + phosphate
-
-
-
-
?
D-alanine + (R)-lactate + ATP
D-alanyl-(R)-lactate + ADP + phosphate
-
-
-
-
?
D-alanine + (R)-lactate + ATP
D-alanyl-(R)-lactate + ADP + phosphate
-
-
-
-
?
D-alanine + (R)-lactate + ATP
D-alanyl-(R)-lactate + ADP + phosphate
-
-
-
-
?
D-alanine + (R)-lactate + ATP
D-alanyl-(R)-lactate + ADP + phosphate
-
-
-
-
?
D-alanine + (R)-lactate + ATP
D-alanyl-(R)-lactate + ADP + phosphate
-
-
-
?
D-alanine + (R)-lactate + ATP
D-alanyl-(R)-lactate + ADP + phosphate
-
-
-
-
?
D-alanine + (R)-lactate + ATP
D-alanyl-(R)-lactate + ADP + phosphate
-
-
-
?
D-alanine + (R)-lactate + ATP
D-alanyl-(R)-lactate + ADP + phosphate
-
-
-
-
?
D-alanine + (R)-lactate + ATP
D-alanyl-(R)-lactate + ADP + phosphate
-
-
-
-
?
D-alanine + (R)-lactate + ATP
D-alanyl-(R)-lactate + ADP + phosphate
-
-
-
-
?
D-alanine + (R)-lactate + ATP
D-alanyl-(R)-lactate + ADP + phosphate
-
-
-
-
?
D-alanine + (R)-lactate + ATP
D-alanyl-(R)-lactate + ADP + phosphate
-
-
-
-
?
D-alanine + (R)-lactate + ATP
D-alanyl-(R)-lactate + ADP + phosphate
-
-
-
-
?
D-alanine + (R)-lactate + ATP
D-alanyl-(R)-lactate + ADP + phosphate
-
-
-
-
?
D-alanine + (R)-lactate + ATP
D-alanyl-(R)-lactate + ADP + phosphate
-
-
-
-
?
D-alanine + (R)-lactate + ATP
D-alanyl-(R)-lactate + ADP + phosphate
-
-
-
-
?
D-alanine + (R)-lactate + ATP
D-alanyl-(R)-lactate + ADP + phosphate
-
-
-
-
?
D-alanine + (R)-lactate + ATP
D-alanyl-(R)-lactate + ADP + phosphate
-
-
-
-
?
D-alanine + (R)-lactate + ATP
D-alanyl-(R)-lactate + ADP + phosphate
-
-
-
-
?
D-alanine + (R)-lactate + ATP
D-alanyl-(R)-lactate + ADP + phosphate
-
-
-
-
?
D-alanine + (R)-lactate + ATP
D-alanyl-(R)-lactate + ADP + phosphate
-
-
-
-
?
D-alanine + (R)-lactate + ATP
D-alanyl-(R)-lactate + ADP + phosphate
-
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
-
-
?
D-alanine + (R)-lactate + ATP
D-alanyl-(R)-lactate + ADP + phosphate
-
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
-
-
?
additional information
?
-
-
reaction proceeds via rapid and reversible formation of the enzyme intermediate D-Ala-phosphate. Reversible cleavage of ATP to ADP and D-Ala-phosphate and reformation of ATP are detectable. D-Ala-phosphate is a common intermediate that serves as the electrophilic substrate in the condensation with D-Lac as nucleophilic cosubstrate
-
-
?
additional information
?
-
-
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
-
-
?
additional information
?
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ligand binding sites of VRSA-9 Ddl, overview
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-
?
additional information
?
-
-
ligand binding sites of VRSA-9 Ddl, overview
-
-
?
additional information
?
-
-
substrate specificity of recombinant D-alanine-D-alanine ligase mutant S137G/Y207F, overview
-
-
?
additional information
?
-
-
substrate specificity of recombinant D-alanine-D-alanine ligase mutant S137G/Y207F, overview
-
-
?
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NATURAL SUBSTRATE
NATURAL PRODUCT
REACTION DIAGRAM
ORGANISM
UNIPROT
COMMENTARY
(Substrate)
(Substrate)
LITERATURE
(Substrate)
(Substrate)
COMMENTARY
(Product)
(Product)
LITERATURE
(Product)
(Product)
REVERSIBILITY
r=reversible
ir=irreversible
?=not specified
r=reversible
ir=irreversible
?=not specified
additional information
?
-
-
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
-
-
?
D-alanine + (R)-lactate + ATP
D-alanyl-(R)-lactate + ADP + phosphate
-
-
-
-
?
D-alanine + (R)-lactate + ATP
D-alanyl-(R)-lactate + ADP + phosphate
-
-
-
-
?
D-alanine + (R)-lactate + ATP
D-alanyl-(R)-lactate + ADP + phosphate
-
-
-
-
?
D-alanine + (R)-lactate + ATP
D-alanyl-(R)-lactate + ADP + phosphate
-
-
-
-
?
D-alanine + (R)-lactate + ATP
D-alanyl-(R)-lactate + ADP + phosphate
-
-
-
-
?
D-alanine + (R)-lactate + ATP
D-alanyl-(R)-lactate + ADP + phosphate
-
-
-
-
?
D-alanine + (R)-lactate + ATP
D-alanyl-(R)-lactate + ADP + phosphate
-
-
-
-
?
D-alanine + (R)-lactate + ATP
D-alanyl-(R)-lactate + ADP + phosphate
-
-
-
-
?
D-alanine + (R)-lactate + ATP
D-alanyl-(R)-lactate + ADP + phosphate
-
-
-
-
?
D-alanine + (R)-lactate + ATP
D-alanyl-(R)-lactate + ADP + phosphate
-
-
-
?
D-alanine + (R)-lactate + ATP
D-alanyl-(R)-lactate + ADP + phosphate
-
-
-
-
?
D-alanine + (R)-lactate + ATP
D-alanyl-(R)-lactate + ADP + phosphate
-
-
-
?
D-alanine + (R)-lactate + ATP
D-alanyl-(R)-lactate + ADP + phosphate
-
-
-
-
?
D-alanine + (R)-lactate + ATP
D-alanyl-(R)-lactate + ADP + phosphate
-
-
-
-
?
D-alanine + (R)-lactate + ATP
D-alanyl-(R)-lactate + ADP + phosphate
-
-
-
-
?
D-alanine + (R)-lactate + ATP
D-alanyl-(R)-lactate + ADP + phosphate
-
-
-
-
?
D-alanine + (R)-lactate + ATP
D-alanyl-(R)-lactate + ADP + phosphate
-
-
-
-
?
D-alanine + (R)-lactate + ATP
D-alanyl-(R)-lactate + ADP + phosphate
-
-
-
-
?
D-alanine + (R)-lactate + ATP
D-alanyl-(R)-lactate + ADP + phosphate
-
-
-
-
?
D-alanine + (R)-lactate + ATP
D-alanyl-(R)-lactate + ADP + phosphate
-
-
-
-
?
D-alanine + (R)-lactate + ATP
D-alanyl-(R)-lactate + ADP + phosphate
-
-
-
-
?
D-alanine + (R)-lactate + ATP
D-alanyl-(R)-lactate + ADP + phosphate
-
-
-
-
?
D-alanine + (R)-lactate + ATP
D-alanyl-(R)-lactate + ADP + phosphate
-
-
-
-
?
D-alanine + (R)-lactate + ATP
D-alanyl-(R)-lactate + ADP + phosphate
-
-
-
-
?
D-alanine + (R)-lactate + ATP
D-alanyl-(R)-lactate + ADP + phosphate
-
-
-
-
?
D-alanine + (R)-lactate + ATP
D-alanyl-(R)-lactate + ADP + phosphate
-
-
-
-
?
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COFACTOR
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
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METALS and IONS
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
Mg2+
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INHIBITOR
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
1-[[(4-fluorophenyl)sulfonyl]amino]-3-(morpholin-4-yl)propan-2-yl dihydrogen phosphate
-
75% inhibition at 0.5 mM
1-[[(4-fluorophenyl)sulfonyl]amino]-3-(morpholin-4-yl)propan-2-yl phenyl hydrogen phosphate
-
65% inhibition at 0.5 mM
1-[[(4-methoxyphenyl)sulfonyl]amino]-3-(morpholin-4-yl)propan-2-yl dihydrogen phosphate
-
83% inhibition at 0.5 mM
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KM VALUE [mM]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
3
D-2-hydroxybutanoate
-
pH not specified in the publication, temperature not specified in the publication
8.3
D-2-hydroxyvalerate
-
pH not specified in the publication, temperature not specified in the publication
1.2
D-Ala
-
cosubstrate D-Ala, binding of N-terminal Ala residue. pH not specified in the publication, temperature not specified in the publication
34
D-Ala
-
cosubstrate D-Ala, binding of C-terminal Ala residue. pH not specified in the publication, temperature not specified in the publication
11.4
D-lactate
-
pH not specified in the publication, temperature not specified in the publication
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TURNOVER NUMBER [1/s]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
0.25
D-2-hydroxybutanoate
-
pH not specified in the publication, temperature not specified in the publication
0.42
D-2-hydroxyvalerate
-
pH not specified in the publication, temperature not specified in the publication
4.1
D-Ala
-
pH not specified in the publication, temperature not specified in the publication
0.47
D-lactate
-
pH not specified in the publication, temperature not specified in the publication
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kcat/KM VALUE [1/mMs-1]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
0.084
D-2-hydroxybutanoate
-
pH not specified in the publication, temperature not specified in the publication
0.05
D-2-hydroxyvalerate
-
pH not specified in the publication, temperature not specified in the publication
0.12
D-Ala
-
pH not specified in the publication, temperature not specified in the publication
0.0414
D-lactate
-
pH not specified in the publication, temperature not specified in the publication
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SPECIFIC ACTIVITY [µmol/min/mg]
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
0.19
-
D-alanyl-D-lactate depsipeptide formation activity, recombinant D-alanine-D-alanine ligase mutant S137F/Y207F, pH 7.5, 60°C
0.39
-
D-alanyl-D-lactate depsipeptide formation activity, recombinant D-alanine-D-alanine ligase mutant Y207F, pH 7.5, 60°C
0.42
-
D-alanyl-D-lactate depsipeptide formation activity, recombinant D-alanine-D-alanine ligase mutant S137A/Y207F, pH 7.5, 60°C
1.2
-
D-alanyl-D-lactate depsipeptide formation activity, recombinant D-alanine-D-alanine ligase mutant S137G/Y207F, pH 7.5, 60°C
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pH OPTIMUM
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
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TEMPERATURE OPTIMUM
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
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ORGANISM
COMMENTARY
LITERATURE
UNIPROT
SEQUENCE DB
SOURCE
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GENERAL INFORMATION
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
evolution
physiological function
additional information
evolution
-
the two amino acid substitutions Q260K/A283E via exchange at nucleotide positions 778 and 848, respectively, in VRSA-9 Ddl lead to the ability to synthesize precursors ending in D-Ala-D-Lac (72%) and D-Ala-D-Ala (21%) in the absence of vancomycin. The VRSA-9 Ddl has an altered D-Ala:D-Ala ligase activity relative to that of VRSA-6 with a Km for D-Ala of 2 mM at subsite 1 and 240 mM at subsite 2. The binding affinity for D-Ala at subsite 2 is 14fold lower than that of VRSA-6. The residues at nucleotide positions 778 and 848 are not conserved among D-Ala:DAla ligases and do not interact directly with the substrates. VRSA-9 Ddl shows the importance of conformational changes in the dimer interface which can indirectly affect the topology of the active site
evolution
-
the two amino acid substitutions Q260K/A283E via exchange at nucleotide positions 778 and 848, respectively, in VRSA-9 Ddl lead to the ability to synthesize precursors ending in D-Ala-D-Lac (72%) and D-Ala-D-Ala (21%) in the absence of vancomycin. The VRSA-9 Ddl has an altered D-Ala:D-Ala ligase activity relative to that of VRSA-6 with a Km for D-Ala of 2 mM at subsite 1 and 240 mM at subsite 2. The binding affinity for D-Ala at subsite 2 is 14fold lower than that of VRSA-6. The residues at nucleotide positions 778 and 848 are not conserved among D-Ala:DAla ligases and do not interact directly with the substrates. VRSA-9 Ddl shows the importance of conformational changes in the dimer interface which can indirectly affect the topology of the active site
-
physiological function
-
Enterococcus faecium 10/96A from Brazil is resistant to vancomycin with a minimum inhibitory concentration MIC of 0.256 mg/ml. Cytoplasmic peptidoglycan precursors from cells of strain 10/96A grown in the presence or absence of 0.004 mg of vancomycin/ml contain in both cases 95% UDP-MurNAc-pentadepsipeptide, 3% UDPMurNAc-pentapeptide, and 2% UDP-MurNAc-tetrapeptide, supporting the role of VanD4 as a D-Ala-D-Lac ligase and indicating that glycopeptide resistance is expressed constitutively
physiological function
-
in the presence of vancomycin, production of the VanB D-Ala:D-Lac ligase is induced, which overcomes the defect in the synthesis of peptidoglycan precursors ending in D-AlaD-Ala due to a lack of functional Ddl D-Ala-D-Ala ligase. Vancomycin-dependent strain BM4660 synthesizes mainly UDP-MurNAc-pentadepsipeptide, 44%, whereas large amounts of UDP-MurNActripeptide, 39%, and small amounts of pentapeptide, 12%, and tetrapeptide, 5%, are present. The presence of tripeptide in large quantity suggests that the VanB ligase may not be sufficiently active to synthesize D-AlaD-Lac as rapidly as tripeptide is produced
physiological function
-
resistance to glycopeptides in Enterococcus faecium BM4416 is due to synthesis of late peptidoglycan precursors ending in D-AlaD-Lac. Strain BM4416 mainly produces UDP-MurNAc-pentadepsipeptide, 69%, terminating in D-AlaD-Lac, UDP-MurNAc-tetrapeptide, 24%, and UDP-MurNAc-tripeptide, 7%. No significant amounts of UDP-MurNAc-pentapeptide are found. Constitutive resistance is encoded by a vanD operon closely related to that of Enterococcus faecium BM4339 and also located in the chromosome. Both VanD-type strains produce an inactivated D-Ala:D-Ala ligase due to an insertion in the ddl gene
physiological function
-
the vanD gene encodes a D-Ala:D-Lac ligase related to VanA and VanB which is not transferable by conjugation. It renders the cells constitutively resistant to vancomycin with a minimum inhibitory concentration MIC of 0.064 mg/ml and to low levels of teicoplanin, MIC 0.004 mg/ml. Cytoplasmic peptidoglycan precursors that accumulate are mainly UDP-MurNAc-pentadepsipeptide, UDP-MurNAc-tetrapeptide, and UDP-MurNAc-tripeptide. The large proportion of UDP-MurNAc-pentadepsipeptide indicates that the mechanism of vancomycin resistance in BM4339 is similar to that in VanA and VanB strains. The presence of UDP-MurNAc-tripeptide implies that the rate of synthesis of D-AlaD-Ala or D-AlaD-Lac substrates is limiting
physiological function
-
VanA is a D-alanine:D-alanine ligase of altered substrate specificity. VanA catalyzes ester bond formation between D-alanine and the D-hydroxy acid products of VanH, the best substrate being D-2-hydroxybutyrate. The VanA product D-alanyl-D-2-hydroxybutyrate can then be incorporated into the UDPMurNAc-pentapeptide peptidoglycan precursor
physiological function
the enzyme is a key enzyme in the emergence of high level resistance to vancomycin in Enterococcus
physiological function
-
Enterococcus faecium 10/96A from Brazil is resistant to vancomycin with a minimum inhibitory concentration MIC of 0.256 mg/ml. Cytoplasmic peptidoglycan precursors from cells of strain 10/96A grown in the presence or absence of 0.004 mg of vancomycin/ml contain in both cases 95% UDP-MurNAc-pentadepsipeptide, 3% UDPMurNAc-pentapeptide, and 2% UDP-MurNAc-tetrapeptide, supporting the role of VanD4 as a D-Ala-D-Lac ligase and indicating that glycopeptide resistance is expressed constitutively
-
physiological function
-
resistance to glycopeptides in Enterococcus faecium BM4416 is due to synthesis of late peptidoglycan precursors ending in D-AlaD-Lac. Strain BM4416 mainly produces UDP-MurNAc-pentadepsipeptide, 69%, terminating in D-AlaD-Lac, UDP-MurNAc-tetrapeptide, 24%, and UDP-MurNAc-tripeptide, 7%. No significant amounts of UDP-MurNAc-pentapeptide are found. Constitutive resistance is encoded by a vanD operon closely related to that of Enterococcus faecium BM4339 and also located in the chromosome. Both VanD-type strains produce an inactivated D-Ala:D-Ala ligase due to an insertion in the ddl gene
-
physiological function
-
VanA is a D-alanine:D-alanine ligase of altered substrate specificity. VanA catalyzes ester bond formation between D-alanine and the D-hydroxy acid products of VanH, the best substrate being D-2-hydroxybutyrate. The VanA product D-alanyl-D-2-hydroxybutyrate can then be incorporated into the UDPMurNAc-pentapeptide peptidoglycan precursor
-
physiological function
-
the enzyme is a key enzyme in the emergence of high level resistance to vancomycin in Enterococcus
-
physiological function
-
the vanD gene encodes a D-Ala:D-Lac ligase related to VanA and VanB which is not transferable by conjugation. It renders the cells constitutively resistant to vancomycin with a minimum inhibitory concentration MIC of 0.064 mg/ml and to low levels of teicoplanin, MIC 0.004 mg/ml. Cytoplasmic peptidoglycan precursors that accumulate are mainly UDP-MurNAc-pentadepsipeptide, UDP-MurNAc-tetrapeptide, and UDP-MurNAc-tripeptide. The large proportion of UDP-MurNAc-pentadepsipeptide indicates that the mechanism of vancomycin resistance in BM4339 is similar to that in VanA and VanB strains. The presence of UDP-MurNAc-tripeptide implies that the rate of synthesis of D-AlaD-Ala or D-AlaD-Lac substrates is limiting
-
physiological function
-
in the presence of vancomycin, production of the VanB D-Ala:D-Lac ligase is induced, which overcomes the defect in the synthesis of peptidoglycan precursors ending in D-AlaD-Ala due to a lack of functional Ddl D-Ala-D-Ala ligase. Vancomycin-dependent strain BM4660 synthesizes mainly UDP-MurNAc-pentadepsipeptide, 44%, whereas large amounts of UDP-MurNActripeptide, 39%, and small amounts of pentapeptide, 12%, and tetrapeptide, 5%, are present. The presence of tripeptide in large quantity suggests that the VanB ligase may not be sufficiently active to synthesize D-AlaD-Lac as rapidly as tripeptide is produced
-
additional information
-
acquisition of vancomycin resistance affects expression of WalRK and PhoPR regulon genes in the phosphate-limited state. Genetic regulation of vancomycin resistance involving also VanA, detailed overview
additional information
-
acquisition of vancomycin resistance affects expression of WalRK and PhoPR regulon genes in the phosphate-limited state. Genetic regulation of vancomycin resistance involving also VanA, detailed overview
-
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UNIPROT
ENTRY NAME
ORGANISM
NO. OF AA
NO. OF TRANSM. HELICES
MOLECULAR WEIGHT[Da]
SOURCE
SEQUENCE
LOCALIZATION PREDICTION
The reliability class of the localization prediction ranges from 1 (very reliable) to 5 (not reliable).
The reliability class of the localization prediction ranges from 1 (very reliable) to 5 (not reliable). VANB_ENTFA
Enterococcus faecalis (strain ATCC 700802 / V583)
342
0
37332
Swiss-Prot
-
A0A1V6DVS3_9BACT
339
0
38778
TrEMBL
-
A0A645DAP7_9ZZZZ
264
0
29502
TrEMBL
other Location (Reliability: 3)
A0A645I857_9ZZZZ
120
0
13428
TrEMBL
other Location (Reliability: 1)
A0A644THU6_9ZZZZ
353
0
38238
TrEMBL
other Location (Reliability: 1)
A0A1V5F9W2_9BACT
641
0
72292
TrEMBL
-
A0A645F1S7_9ZZZZ
161
0
18551
TrEMBL
other Location (Reliability: 2)
A0A645BYJ4_9ZZZZ
350
0
37914
TrEMBL
other Location (Reliability: 3)
A0A6C0QY89_9BACL
160
0
17950
TrEMBL
-
A0A5C6C586_9BACT
341
0
38714
TrEMBL
-
A0A0J6YCT0_9MYCO
340
0
35888
TrEMBL
-
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PDB
SCOP
CATH
UNIPROT
ORGANISM
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MOLECULAR WEIGHT
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
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SUBUNIT
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
dimer
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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
-
VRSA-9 Ddl X-ray diffraction crystal structure determination and analysis
-
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PROTEIN VARIANTS
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
S150A
-
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
-
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
-
the mutant shows a complete loss of the ability to make D-alanyl-(R)-lactate
Q260K/A283E
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
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
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
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
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
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
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
additional information
Q260K/A283E
-
the two amino acid substitutions result from point mutations at nucleotide positions 778 and 848, respectively. The mutant enzyme VRSA-9 synthesizes precursors ending in D-Ala-D-Lac (72%) and D-Ala-D-Ala (21%) in the absence of vancomycin. VRSA-9 Ddl shows a 200fold loss of activity and the importance of conformational changes in the dimer interface which can indirectly affect the topology of the active site
Q260K/A283E
-
the two amino acid substitutions result from point mutations at nucleotide positions 778 and 848, respectively. The mutant enzyme VRSA-9 synthesizes precursors ending in D-Ala-D-Lac (72%) and D-Ala-D-Ala (21%) in the absence of vancomycin. VRSA-9 Ddl shows a 200fold loss of activity and the importance of conformational changes in the dimer interface which can indirectly affect the topology of the active site
-
additional information
-
construction of several mutant strains, generating a Bacillus subtilis strain BP341A expressing a depsipeptide-containing (D-Ala-D-Lac) lipid II, phenotype, overview. Strains BP341A-E, that require expression of the VanB-type operon for growth, have mutations in the endogenous Ddl ligase
additional information
-
construction of several mutant strains, generating a Bacillus subtilis strain BP341A expressing a depsipeptide-containing (D-Ala-D-Lac) lipid II, phenotype, overview. Strains BP341A-E, that require expression of the VanB-type operon for growth, have mutations in the endogenous Ddl ligase
-
additional information
-
structure-based modification of D-alanine-D-alanine ligase from strain ATCC 43589 for depsipeptide synthesis, overview
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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
expression of mutant enzymes in Escherichia coli strain BL21(DE3) as His6-tagged proteins
-
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APPLICATION
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
synthesis
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
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
-
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REF.
AUTHORS
TITLE
JOURNAL
VOL.
PAGES
YEAR
ORGANISM (UNIPROT)
PUBMED ID
SOURCE
Meziane-Cherif, D.; Badet-Denisot, M.A.; Evers, S.; Courvalin, P.; Badet, B.
Purification and characterization of the VanB ligase associated with type B vancomycin resistance in Enterococcus faecalis V583
FEBS Lett.
354
140-142
1994
Enterococcus faecalis
brenda
Perichon, B.; Casadewall, B.; Reynolds, P.; Courvalin, P.
Glycopeptide-resistant Enterococcus faecium BM4416 is a VanD-type strain with an impaired D-alanine:D-alanine ligase
Antimicrob. Agents Chemother.
44
1346-1348
2000
Enterococcus faecium, Enterococcus faecium BM4416
brenda
Sova, M.; Cadez, G.; Turk, S.; Majce, V.; Polanc, S.; Batson, S.; Lloyd, A.J.; Roper, D.I.; Fishwick, C.W.; Gobec, S.
Design and synthesis of new hydroxyethylamines as inhibitors of D-alanyl-D-lactate ligase (VanA) and D-alanyl-D-alanine ligase (DdlB)
Bioorg. Med. Chem. Lett.
19
1376-1379
2009
Enterococcus faecium
brenda
Neuhaus, F.C.
Role of Arg301 in substrate orientation and catalysis in subsite 2 of D-alanine:D-alanine (D-lactate) ligase from Leuconostoc mesenteroides: A molecular docking study
J. Mol. Graph. Model.
28
728-734
2010
Leuconostoc mesenteroides
brenda
Huyton, T.; Roper, D.I.
Crystallization and preliminary X-ray characterization of VanA from Enterococcus faecium BM4147: towards the molecular basis of bacterial resistance to the glycopeptide antibiotic vancomycin
Acta Crystallogr. Sect. D
55
1481-1483
1999
Enterococcus faecium
brenda
Perichon, B.; Reynolds, P.; Courvalin, P.
VanD-type glycopeptide-resistant Enterococcus faecium BM4339
Antimicrob. Agents Chemother.
41
2016-2018
1997
Enterococcus faecium, Enterococcus faecium BM4339
brenda
Dalla Costa, L.M.; Reynolds, P.E.; Souza, H.A.; Souza, D.C.; Palepou, M.F.; Woodford, N.
Characterization of a divergent vanD-type resistance element from the first glycopeptide-resistant strain of Enterococcus faecium isolated in Brazil
Antimicrob. Agents Chemother.
44
3444-3446
2000
Enterococcus faecium, Enterococcus faecium 10/96A
brenda
San Millan, A.; Depardieu, F.; Godreuil, S.; Courvalin, P.
VanB-type Enterococcus faecium clinical isolate successively inducibly resistant to, dependent on, and constitutively resistant to vancomycin
Antimicrob. Agents Chemother.
53
1974-1982
2009
Enterococcus faecium, Enterococcus faecium BM 4660
brenda
Bugg, T.D.; Wright, G.D.; Dutka-Malen, S.; Arthur, M.; Courvalin, P.; Walsh, C.T.
Molecular basis for vancomycin resistance in Enterococcus faecium BM4147: biosynthesis of a depsipeptide peptidoglycan precursor by vancomycin resistance proteins VanH and VanA
Biochemistry
30
10408-10415
1991
Enterococcus faecium, Enterococcus faecium BM4147
brenda
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
Biochemistry
35
10464-10471
1996
Escherichia coli
brenda
Healy, V.L.; Mullins, L.S.; Li, X.; Hall, S.E.; Raushel, F.M.; Walsh, C.T.
D-AlaD-X ligases: evaluation of D-alanyl phosphate intermediate by MIX, PIX and rapid quench studies
Chem. Biol.
7
505-514
2000
Enterococcus faecium
brenda
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
Biosci. Biotechnol. Biochem.
75
700-704
2011
Thermotoga maritima, Thermotoga maritima ATCC 43589
brenda
Meziane-Cherif, D.; Saul, F.A.; Moubareck, C.; Weber, P.; Haouz, A.; Courvalin, P.; Perichon, B.
Molecular basis of vancomycin dependence in VanA-type Staphylococcus aureus VRSA-9
J. Bacteriol.
192
5465-5471
2010
Staphylococcus aureus, Staphylococcus aureus VRSA-9
brenda
Bisicchia, P.; Bui, N.K.; Aldridge, C.; Vollmer, W.; Devine, K.M.
Acquisition of VanB-type vancomycin resistance by Bacillus subtilis: the impact on gene expression, cell wall composition and morphology
Mol. Microbiol.
81
157-178
2011
Bacillus subtilis, Bacillus subtilis 168
brenda
Bouvier, G.; Duclert-Savatier, N.; Desdouits, N.; Meziane-Cherif, D.; Blondel, A.; Courvalin, P.; Nilges, M.; Malliavin, T.E.
Functional motions modulating VanA ligand binding unraveled by self-organizing maps
J. Chem. Inf. Model.
54
289-301
2014
Enterococcus faecium (P25051), Enterococcus faecium, Enterococcus faecium BM4147 (P25051)
brenda
Neuhaus, F.C.
Role of the omega loop in specificity determination in subsite 2 of the D-alanine:D-alanine (D-lactate) ligase from Leuconostoc mesenteroides: a molecular docking study
J. Mol. Graph. Model.
30
31-37
2011
Leuconostoc mesenteroides
brenda
Van Der Aart, L.; Lemmens, N.; Van Wamel, W.; Van Wezel, G.
Substrate inhibition of VanA by D-alanine reduces vancomycin resistance in a VanX-dependent manner
Antimicrob. Agents Chemother.
60
4930-4939
2016
Streptomyces coelicolor, Streptomyces coelicolor M145
brenda
Nakipoglu, M.; Yilmaz, F.; Icgen, B.
vanA Gene harboring enterococcal and non-enterococcal isolates expressing high level vancomycin and teicoplanin resistance reservoired in surface waters
Bull. Environ. Contam. Toxicol.
98
712-719
2017
Comamonas testosteroni, Enterococcus faecalis, Enterococcus faecium, Raoultella planticola, Pseudomonas fluorescens, Pseudomonas plecoglossicida, Pseudomonas koreensis, Enterococcus faecalis Cr07, Comamonas testosteroni Ni11, Enterococcus faecium 330, Pseudomonas plecoglossicida Ag10, Pseudomonas koreensis Hg11, Pseudomonas koreensis Hg10, Pseudomonas fluorescens SDS3, Enterococcus faecalis Pb06, Enterococcus faecalis E07, Raoultella planticola Ag11, Pseudomonas koreensis Cu12
brenda
Kruse, T.; Levisson, M.; de Vos, W.M.; Smidt, H.
vanI a novel D-Ala-D-Lac vancomycin resistance gene cluster found in Desulfitobacterium hafniense
Microb. Biotechnol.
7
456-466
2014
Desulfitobacterium hafniense, Desulfitobacterium hafniense Y51
brenda
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