Information on EC 6.1.1.13 - D-alanine-poly(phosphoribitol) ligase

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The enzyme appears in viruses and cellular organisms

EC NUMBER
COMMENTARY hide
6.1.1.13
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RECOMMENDED NAME
GeneOntology No.
D-alanine-poly(phosphoribitol) ligase
REACTION
REACTION DIAGRAM
COMMENTARY hide
ORGANISM
UNIPROT
LITERATURE
ATP + D-alanine + poly(ribitol phosphate) = AMP + diphosphate + O-D-alanyl-poly(ribitol phosphate)
show the reaction diagram
A thioester bond is formed transiently between D-alanine and the sulfhydryl group of the 4'-phosphopantetheine prosthetic group of D-alanyl carrier protein during the activation of the alanine. Involved in the synthesis of teichoic acids
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REACTION TYPE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
Acid-thiol ligation
adenylation
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thiolation
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PATHWAY
BRENDA Link
KEGG Link
MetaCyc Link
D-Alanine metabolism
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SYSTEMATIC NAME
IUBMB Comments
D-alanine:poly(phosphoribitol) ligase (AMP-forming)
A thioester bond is formed transiently between D-alanine and the sulfhydryl group of the 4'-phosphopantetheine prosthetic group of D-alanyl carrier protein during the activation of the alanine. Involved in the synthesis of teichoic acids.
CAS REGISTRY NUMBER
COMMENTARY hide
9023-65-8
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ORGANISM
COMMENTARY hide
LITERATURE
UNIPROT
SEQUENCE DB
SOURCE
laboratory strains R6, D39, and Rx,and the clinical isolate TIGR4, dltA operon, gene dltA
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Manually annotated by BRENDA team
SUBSTRATE
PRODUCT                       
REACTION DIAGRAM
ORGANISM
UNIPROT
COMMENTARY
(Substrate) hide
LITERATURE
(Substrate)
COMMENTARY
(Product) hide
LITERATURE
(Product)
Reversibility
r=reversible
ir=irreversible
?=not specified
ATP + D-2-amino butyric acid + diphosphate
?
show the reaction diagram
activity and specificity of DltA is investigated using the ATP-pyrophosphate exchange assay. Enzyme shows only minor activity 13% with D-amino butyric acid
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?
ATP + D-2-amino-n-butyric acid + membrane fragments
D-2-Amino-n-butryl-membrane fragments + AMP + diphosphate
show the reaction diagram
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not L-2-amino-n-butyric acid
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ATP + D-Ala + acyl-carrier protein
D-Alanyl-acyl-carrier protein + AMP + diphosphate
show the reaction diagram
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acyl carrier protein of E. coli, Vibrio harveyi, Saccharopolyspora erythraea, not of Bacillus subtilis, Spinacia oleracea
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ATP + D-Ala + D-alanyl carrier protein
D-Alanyl-D-alanyl carrier protein + AMP + diphosphate
show the reaction diagram
ATP + D-Ala + diphosphate
?
show the reaction diagram
ATP + D-Ala + hydroxylamine
D-Alanine hydroxamate + AMP + diphosphate
show the reaction diagram
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D-Ala can be substituted by D-2-amino-n-butyric acid with 14.6% and D-Ser with 7.5% efficiency
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ATP + D-Ala + membrane fragments
D-Alanyl-membrane fragments + AMP + diphosphate
show the reaction diagram
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ATP can be substituted by ADP, GTP, CTP, UTP, not by AMP, D-Ala cannot be substituted by L-Ala
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ATP + D-alanine + D-alanyl lipoteichoic acid
AMP + diphosphate + D-alanyl lipoteichoic acid
show the reaction diagram
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?
ATP + D-alanine + D-alanyl teichoic acid
AMP + diphosphate + D-alanyl teichoic acid
show the reaction diagram
ATP + D-alanine + poly(ribitol phosphate)
AMP + diphosphate + O-D-alanyl-poly(ribitol phosphate)
show the reaction diagram
ATP + D-Ser + diphosphate
?
show the reaction diagram
activity and specificity of DltA is investigated using the ATP-pyrophosphate exchange assay. Enzyme shows only minor activity 13% with D-Ser
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?
ATP + diphosphate
ATP + diphosphate
show the reaction diagram
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ATP + L-Ala + diphosphate
?
show the reaction diagram
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?
D-Alanine + ATP + ?
?
show the reaction diagram
additional information
?
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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 SUBSTRATES
NATURAL PRODUCTS
REACTION DIAGRAM
ORGANISM
UNIPROT
COMMENTARY
(Substrate) hide
LITERATURE
(Substrate)
COMMENTARY
(Product) hide
LITERATURE
(Product)
REVERSIBILITY
r=reversible
ir=irreversible
?=not specified
ATP + D-alanine + D-alanyl lipoteichoic acid
AMP + diphosphate + D-alanyl lipoteichoic acid
show the reaction diagram
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?
ATP + D-alanine + D-alanyl teichoic acid
AMP + diphosphate + D-alanyl teichoic acid
show the reaction diagram
ATP + D-alanine + poly(ribitol phosphate)
AMP + diphosphate + O-D-alanyl-poly(ribitol phosphate)
show the reaction diagram
D-Alanine + ATP + ?
?
show the reaction diagram
additional information
?
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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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COFACTOR
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
METALS and IONS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
Ba2+
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Ba2+ can replace Mg2+ in activity enhancement
Ca2+
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Ca2+ can replace Mg2+ in activity enhancement
Co2+
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Co2+ can replace Mg2+ in activity enhancement
K+
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K+ can replace Mg2+ in activity enhancement
Na+
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Na+ can replace Mg2+ in activity enhancement
INHIBITORS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
5'-O-[N-(D-alanyl)-sulfamoyl]adenosine
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inhibits D-Ala adenylylation by DltA in vitro
D-2-Amino-n-butyric acid hydroxamate
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DL-2-Amino-n-butyric acid hydroxamate
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DL-Alanine amide
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DL-Alanine hydroxamate
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additional information
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ACTIVATING COMPOUND
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
DltD
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facilitator of D-Ala ligation to the carrier protein Dcp
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DTT
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stimulation
KM VALUE [mM]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
0.01 - 3.3
ATP
0.85
D-2-amino-n-butyric acid
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0.018 - 70
D-Ala
0.13
diphosphate
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ATP-diphosphate exchange reaction
6.6 - 14.4
L-Ala
TURNOVER NUMBER [1/s]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
0.011 - 0.215
ATP
0.025 - 0.103
D-Ala
0.11 - 0.12
L-Ala
Ki VALUE [mM]
INHIBITOR
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
0.000232
5'-O-[N-(D-alanyl)-sulfamoyl]adenosine
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SPECIFIC ACTIVITY [µmol/min/mg]
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
pH OPTIMUM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
5 - 8
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substrates D-Ala + ATP + D-alanyl carrier protein
6.6 - 7
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substrates D-Ala + ATP + acyl carrier protein of Escherichia coli
7 - 8
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substrates D-Ala + hydroxylamine + ATP
7.5 - 8
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ATP-diphosphate exchange reaction
TEMPERATURE OPTIMUM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
LOCALIZATION
ORGANISM
UNIPROT
COMMENTARY hide
GeneOntology No.
LITERATURE
SOURCE
PDB
SCOP
CATH
ORGANISM
UNIPROT
Bacillus subtilis (strain 168)
Bacillus subtilis (strain 168)
Bacillus subtilis (strain 168)
Streptococcus pyogenes serotype M6 (strain ATCC BAA-946 / MGAS10394)
MOLECULAR WEIGHT
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
39000
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gel filtration
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
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the crystal structure of Bacillus cereus D-alanyl carrier protein ligase (DltA) is determined in complex with ATP to 1.9 A resolution
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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
sitting drop vapor diffusion method
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TEMPERATURE STABILITY
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
45
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45% loss of activity in 15 min, ATP and/or D-alanyl carrier protein enhance thermostability, acyl carrier protein facilitates inactivation
STORAGE STABILITY
ORGANISM
UNIPROT
LITERATURE
-10°C, ammonium sulfate fraction stable for 6 months
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-20°C, chromatographed fraction loses 25% of activity in 1 week
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Purification/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
partial
using Ni-NTA chromatography
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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
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expressed in Escherichia coli as a His-tagged fusion protein
overexpression of DltA and DltC in Escherichia coli
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the pET28a vector is used
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ENGINEERING
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
C269A
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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
D383N
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mutant, an invariant amino residue in the ATP-binding pocket is choosen for mutagenesis studies
E298Q
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mutant, an invariant amino residue in the ATP-binding pocket is choosen for mutagenesis studies
R397Q
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mutant, an invariant amino residue in the ATP-binding pocket is choosen for mutagenesis studies
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
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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
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