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Information on EC 6.2.1.54 - D-alanine-[D-alanyl-carrier protein] ligase
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6.2.1.54 D-alanine-[D-alanyl-carrier protein] ligaseIUBMB Comments
The enzyme is involved in the modification of wall teichoic acids, as well as type I and IV lipoteichoic acids, with D-alanine residues. It activates D-alanine using ATP to form a high-energy (D-alanyl)adenylate intermediate and subsequently transfers the alanyl moiety to the phosphopantheinyl prosthetic group of a D-alanyl-carrier protein (DltC).
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Word Map
- 6.2.1.54
-
d-alanylation
-
lipoteichoic
- cereus
-
teichoic
-
thiolation
- coa
- acetyl-coa
-
amp-forming
The enzyme appears in viruses and cellular organisms
Reaction Schemes
+
+
holo-[D-alanyl-carrier protein]
=
+
+
D-alanyl-[D-alanyl-carrier protein]
Synonyms
d-alanyl carrier protein ligase, d-alanine:d-alanyl carrier protein ligase, 
more
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SYNONYM 
ORGANISM
UNIPROT
COMMENTARY 
LITERATURE
D-alanine:D-alanyl carrier protein ligase
D-alanyl carrier protein ligase
Dcl
DltA
Dcl
-
-
-
-
DltA
-
-
-
-
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REACTION
REACTION DIAGRAM
COMMENTARY
ORGANISM
UNIPROT
LITERATURE
(D-alanyl)adenylate + holo-[D-alanyl-carrier protein] = AMP + D-alanyl-[D-alanyl-carrier protein]
(1b)
-
-
-
ATP + D-alanine + holo-[D-alanyl-carrier protein] = AMP + diphosphate + D-alanyl-[D-alanyl-carrier protein]
overall reaction
-
-
-
ATP + D-alanine = (D-alanyl)adenylate + diphosphate
(1a)
-
-
-
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REACTION TYPE
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
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PATHWAY SOURCE
PATHWAYS
-
, , , , , ,
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SYSTEMATIC NAME
IUBMB Comments
D-alanine:[D-alanyl-carrier protein] ligase
The enzyme is involved in the modification of wall teichoic acids, as well as type I and IV lipoteichoic acids, with D-alanine residues. It activates D-alanine using ATP to form a high-energy (D-alanyl)adenylate intermediate and subsequently transfers the alanyl moiety to the phosphopantheinyl prosthetic group of a D-alanyl-carrier protein (DltC).
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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-alanyl)adenylate + holo-[D-alanyl-carrier protein]
AMP + D-alanyl-[D-alanyl-carrier protein]
ATP + D-2-amino butyric acid + diphosphate
?
activity and specificity of DltA is investigated using the ATP-pyrophosphate exchange assay. Enzyme shows only minor activity 13% with D-amino butyric acid
-
-
?
ATP + D-Ala + acyl-carrier protein
D-Alanyl-acyl-carrier protein + AMP + diphosphate
-
acyl carrier protein of E. coli, Vibrio harveyi, Saccharopolyspora erythraea, not of Bacillus subtilis, Spinacia oleracea
-
?
ATP + D-Ala + D-alanyl carrier protein
D-Alanyl-D-alanyl carrier protein + AMP + diphosphate
-
-
-
-
?
ATP + D-alanine + holo-[D-alaninyl-carrier protein]
AMP + diphosphate + D-alanyl-[D-alanyl-carrier protein]
ATP + D-alanine + poly(ribitol phosphate)
AMP + diphosphate + O-D-alanyl-poly(ribitol phosphate)
ATP + D-Ser + diphosphate
?
activity and specificity of DltA is investigated using the ATP-pyrophosphate exchange assay. Enzyme shows only minor activity 13% with D-Ser
-
-
?
ATP + L-alanine + holo-[D-alaninyl-carrier protein]
AMP + diphosphate + L-alanyl-[D-alanyl-carrier protein]
-
-
-
?
ATP + L-alanine + poly(ribitol phosphate)
AMP + diphosphate + O-L-alanyl-poly(ribitol phosphate)
low activity
-
-
?
D-Alanine + ATP + ?
?
-
biosynthesis of D-alanyl-lipoteichoic acid
-
-
?
(D-alanyl)adenylate + holo-[D-alanyl-carrier protein]
AMP + D-alanyl-[D-alanyl-carrier protein]
-
-
-
?
(D-alanyl)adenylate + holo-[D-alanyl-carrier protein]
AMP + D-alanyl-[D-alanyl-carrier protein]
-
-
-
?
ATP + D-Ala + diphosphate
?
activity and specificity of DltA is investigated using the ATP-pyrophosphate exchange assay. Enzyme is highly specific for cognate D-Ala substrate
-
-
?
ATP + D-alanine + holo-[D-alaninyl-carrier protein]
AMP + diphosphate + D-alanyl-[D-alanyl-carrier protein]
-
-
-
?
ATP + D-alanine + holo-[D-alaninyl-carrier protein]
AMP + diphosphate + D-alanyl-[D-alanyl-carrier protein]
-
-
-
?
ATP + D-alanine + poly(ribitol phosphate)
AMP + diphosphate + O-D-alanyl-poly(ribitol phosphate)
-
-
-
?
ATP + D-alanine + poly(ribitol phosphate)
AMP + diphosphate + O-D-alanyl-poly(ribitol phosphate)
-
-
-
-
?
ATP + D-alanine + poly(ribitol phosphate)
AMP + diphosphate + O-D-alanyl-poly(ribitol phosphate)
-
-
-
?
ATP + D-alanine + poly(ribitol phosphate)
AMP + diphosphate + O-D-alanyl-poly(ribitol phosphate)
-
the biosynthesis of D-alanyl-lipoteichoic acid requires four proteins that are encoded by the dlt operon. The synthesis starts with the selection of the D-Ala by the 57000 Da D-Ala-D-Ala carrier protein ligase (DltA). Following activation by DltA, D-Ala is transferred to the 10000 Da D-alanyl carrier protein DltC which can donate D-Ala to lipoteichoic acids with the help of DltB and DltD to mediate the surface charge of the bacterium
-
-
?
additional information
?
-
the ATP and D-alanine substrates of the adenylation reaction are incorporated when the DltA enzyme cycles through its thiolation conformation
-
-
?
additional information
?
-
-
the ATP and D-alanine substrates of the adenylation reaction are incorporated when the DltA enzyme cycles through its thiolation conformation
-
-
?
additional information
?
-
the ATP and D-alanine substrates of the adenylation reaction are incorporated when the DltA enzyme cycles through its thiolation conformation
-
-
?
additional information
?
-
-
the enzyme is required for incorporation of D-alanine in teichoic acids in Gram-positive bacteria, confering resistance to cationic antimicrobial peptides nisin and gallidermin in Streptococcus pneumoniae, enzymes of D-alanine metabolism are encoded in the dltA operon, a functional dlt operon confers resistance to nisin and gallidermin
-
-
?
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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
ATP + D-alanine + poly(ribitol phosphate)
AMP + diphosphate + O-D-alanyl-poly(ribitol phosphate)
D-Alanine + ATP + ?
?
-
biosynthesis of D-alanyl-lipoteichoic acid
-
-
?
additional information
?
-
-
the enzyme is required for incorporation of D-alanine in teichoic acids in Gram-positive bacteria, confering resistance to cationic antimicrobial peptides nisin and gallidermin in Streptococcus pneumoniae, enzymes of D-alanine metabolism are encoded in the dltA operon, a functional dlt operon confers resistance to nisin and gallidermin
-
-
?
ATP + D-alanine + poly(ribitol phosphate)
AMP + diphosphate + O-D-alanyl-poly(ribitol phosphate)
-
-
-
?
ATP + D-alanine + poly(ribitol phosphate)
AMP + diphosphate + O-D-alanyl-poly(ribitol phosphate)
-
-
-
-
?
ATP + D-alanine + poly(ribitol phosphate)
AMP + diphosphate + O-D-alanyl-poly(ribitol phosphate)
-
-
-
?
ATP + D-alanine + poly(ribitol phosphate)
AMP + diphosphate + O-D-alanyl-poly(ribitol phosphate)
-
the biosynthesis of D-alanyl-lipoteichoic acid requires four proteins that are encoded by the dlt operon. The synthesis starts with the selection of the D-Ala by the 57000 Da D-Ala-D-Ala carrier protein ligase (DltA). Following activation by DltA, D-Ala is transferred to the 10000 Da D-alanyl carrier protein DltC which can donate D-Ala to lipoteichoic acids with the help of DltB and DltD to mediate the surface charge of the bacterium
-
-
?
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COFACTOR
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
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METALS and IONS
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
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INHIBITOR
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
additional information
-
a functional dlt operon confers resistance to nisin and gallidermin
-
5'-O-[N-(D-alanyl)-sulfamoyl]adenosine
-
inhibits D-Ala adenylylation by DltA in vitro
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ACTIVATING COMPOUND
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
coenzyme A
CoA can mimic D-alanyl carrier protein DltC. The resulting Michaelis constants in the presence of saturating CoA for both ATP and D-alanine are reduced more than 10fold as compared to the values obtained in the absence of CoA. The presence of CoA also made DltA about 100fold more selective on D-alanine over L-alanine
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KM VALUE [mM]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
0.02
D-alanine
wild-type, thiol assay, presence of coenzyme A, pH 7.2, temperature not specified in the publication
0.03
D-alanine
wild-type, diphosphate assay, presence of coenzyme A, pH 7.2, temperature not specified in the publication
0.5
D-alanine
mutant C269A, diphosphate assay, presence of coenzyme A, pH 7.2, temperature not specified in the publication
1
D-alanine
mutant C269A, thiol assay, presence of coenzyme A, pH 7.2, temperature not specified in the publication
0.109
L-alanine
wild-type, diphosphate assay, presence of coenzyme A, pH 7.2, temperature not specified in the publication
8.8
L-alanine
mutant C269A, diphosphate assay, presence of coenzyme A, pH 7.2, temperature not specified in the publication
20
L-alanine
mutant C269A, thiol assay, presence of coenzyme A, pH 7.2, temperature not specified in the publication
48
L-alanine
wild-type, thiol assay, presence of coenzyme A, pH 7.2, temperature not specified in the publication
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TURNOVER NUMBER [1/s]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
0.18
D-alanine
wild-type, diphosphate assay, presence of coenzyme A, pH 7.2, temperature not specified in the publication
0.18
D-alanine
wild-type, thiol assay, presence of coenzyme A, pH 7.2, temperature not specified in the publication
0.26
D-alanine
mutant C269A, diphosphate assay, presence of coenzyme A, pH 7.2, temperature not specified in the publication
0.33
D-alanine
mutant C269A, thiol assay, presence of coenzyme A, pH 7.2, temperature not specified in the publication
0.14
L-alanine
wild-type, thiol assay, presence of coenzyme A, pH 7.2, temperature not specified in the publication
0.28
L-alanine
mutant C269A, diphosphate assay, presence of coenzyme A, pH 7.2, temperature not specified in the publication
0.32
L-alanine
wild-type, diphosphate assay, presence of coenzyme A, pH 7.2, temperature not specified in the publication
0.41
L-alanine
mutant C269A, thiol assay, presence of coenzyme A, pH 7.2, temperature not specified in the publication
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kcat/KM VALUE [1/mMs-1]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
0.33
D-alanine
mutant C269A, thiol assay, presence of coenzyme A, pH 7.2, temperature not specified in the publication
0.53
D-alanine
mutant C269A, diphosphate assay, presence of coenzyme A, pH 7.2, temperature not specified in the publication
6.1
D-alanine
wild-type, diphosphate assay, presence of coenzyme A, pH 7.2, temperature not specified in the publication
9.2
D-alanine
wild-type, thiol assay, presence of coenzyme A, pH 7.2, temperature not specified in the publication
0.003
L-alanine
wild-type, diphosphate assay, presence of coenzyme A, pH 7.2, temperature not specified in the publication
0.003
L-alanine
wild-type, thiol assay, presence of coenzyme A, pH 7.2, temperature not specified in the publication
0.02
L-alanine
mutant C269A, thiol assay, presence of coenzyme A, pH 7.2, temperature not specified in the publication
0.03
L-alanine
mutant C269A, diphosphate assay, presence of coenzyme A, pH 7.2, temperature not specified in the publication
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Ki VALUE [mM]
INHIBITOR
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
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pH OPTIMUM
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
6.6 - 7
-
substrates D-Ala + ATP + acyl carrier protein of Escherichia coli
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TEMPERATURE OPTIMUM
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
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ORGANISM
COMMENTARY
LITERATURE
UNIPROT
SEQUENCE DB
SOURCE
laboratory strains R6, D39, and Rx,and the clinical isolate TIGR4, dltA operon, gene dltA
-
-
brenda
Show AA Sequence and Transmembrane Helices (2589 entries)
Please use the AA Sequence and Transmembrane Helices Search for a specific query.
Please use the AA Sequence and Transmembrane Helices Search for a specific query.
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PDB
SCOP
CATH
UNIPROT
ORGANISM
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CRYSTALLIZATION (Commentary)
ORGANISM
UNIPROT
LITERATURE
crystal structure of a DltA is determined at 2.0 A resolution in complex with the D-alanine adenylate intermediate of the first reaction. Despite the low level of sequence similarity, the DltA structure resembles known structures of adenylation domains such as the acetyl-CoA synthetase. The enantiomer selection appears to be enhanced by the medium-sized side chain of Cys-269
hanging drop vapor diffusion method, using 0.1 M MgCl2, 0.5 M KCl, 16% (w/v) polyethylene glycol 3350 and 0.05 M HEPES-NaOH buffer at pH 7.2
the crystal structure of Bacillus cereus D-alanyl carrier protein ligase (DltA) is determined in complex with ATP to 1.9 A resolution
protein is crystallized in the presence of substrates D-Ala, ATP and MgCl2. The structures of the reported DltA reveal the determinants for D-Ala substrate specificity, and confirm that the PCP-activating domains are able to adopt multiple conformational states, in this case corresponding to the thiolation reaction
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PROTEIN VARIANTS
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
C269A
D383N
mutant, an invariant amino residue in the ATP-binding pocket is choosen for mutagenesis studies
E298Q
mutant, an invariant amino residue in the ATP-binding pocket is choosen for mutagenesis studies
R397Q
mutant, an invariant amino residue in the ATP-binding pocket is choosen for mutagenesis studies
C269A
C268A
mutant shows strong preference for D-Ala as a substrate, a high activity is also observed towards D-amino butyric acid (56%). Most significantly, the variant accepts the L-amino acids LAla (9.7%), L-amino butyric acid (6%) and L-Ser (7%). Turnover of D-Ser is reduced to 3%
C268D
mutant demonstrates a similar specificity profile to wild-type DltA, although with significantly lower activity
additional information
-
insertional inactivation of dltA in D39 and Rx yielded pairs of dltA-deficient and dltA-proficient strains, phenotypes with enhanced sensitivity to the cationic antimicrobial peptides nisin and gallidermin, overview
C269A
C269A mutation relaxes D-Ala preference. Km (mM): (D-Ala) 3.1, (L-Ala) 6.6, kcat (1/sec): (D-Ala) 0.103, (L-Ala) 0.12
C269A
the mutant shows strongly reduced activity compared to the wild type enzyme
C269A
mutant displays a relaxed preference for D-alanine over L-alanine
C269A
-
the mutant shows strongly reduced activity compared to the wild type enzyme
-
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TEMPERATURE STABILITY
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
45
-
45% loss of activity in 15 min, ATP and/or D-alanyl carrier protein enhance thermostability, acyl carrier protein facilitates inactivation
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PURIFICATION (Commentary)
ORGANISM
UNIPROT
LITERATURE
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CLONED (Commentary)
ORGANISM
UNIPROT
LITERATURE
dltA, enzymes of D-alanine metabolism are encoded in the dltA operon, DNA and amino acid sequence determination and analysis, a functional dlt operon confers resistance to nisin and gallidermin
-
expressed in Escherichia coli as a His-tagged fusion protein
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REF.
AUTHORS
TITLE
JOURNAL
VOL.
PAGES
YEAR
ORGANISM (UNIPROT)
PUBMED ID
SOURCE
Heaton, M.P.; Neuhaus, F.C.
Role of D-alanyl carrier protein in the biosynthesis of D-alanyl-lipoteichoic acid
J. Bacteriol.
176
681-690
1994
Lacticaseibacillus casei
brenda
May, J.J.; Finking, R.; Wiegeshoff, F.; Weber, T.T.; Bandur, N.; Koert, U.; Marahiel, M.A.
Inhibition of the D-alanine:D-alanyl carrier protein ligase from Bacillus subtilis increases the bacterium's susceptibility to antibiotics that target the cell wall
FEBS J.
272
2993-3003
2005
Bacillus subtilis
brenda
Kovacs, M.; Halfmann, A.; Fedtke, I.; Heintz, M.; Peschel, A.; Vollmer, W.; Hakenbeck, R.; Brueckner, R.
A functional dlt operon, encoding proteins required for incorporation of D-alanine in teichoic acids in Gram-positive bacteria, confers resistance to cationic antimicrobial peptides in Streptococcus pneumoniae
J. Bacteriol.
188
5797-5805
2006
Streptococcus pneumoniae
brenda
Du, L.; He, Y.; Luo, Y.
Crystal Structure and Enantiomer Selection by D-Alanyl Carrier Protein Ligase DltA from Bacillus cereus
Biochemistry
47
11473-11480
2008
Bacillus cereus (Q81G39), Bacillus cereus
brenda
Yonus, H.; Neumann, P.; Zimmermann, S.; May, J.J.; Marahiel, M.A.; Stubbs, M.T.
Crystal structure of DltA: implications for the reaction mechanism of non-ribosomal peptide synthetase (NRPS) adenylation domains
J. Biol. Chem.
283
32484-32491
2008
Bacillus subtilis (P39581)
brenda
Osman, K.T.; Du, L.; He, Y.; Luo, Y.
Crystal structure of Bacillus cereus D-alanyl carrier protein ligase (DltA) in complex with ATP
J. Mol. Biol.
388
345-355
2009
Bacillus cereus (Q81G39), Bacillus cereus
brenda
Luo, Y.; Du, L.
Thiolation-enhanced substrate recognition by D-alanyl carrier protein ligase DltA from Bacillus cereus
F1000Res.
3
106
2014
Bacillus cereus (Q81G39), Bacillus cereus, Bacillus cereus DSM 31 (Q81G39)
brenda
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