Information on EC 3.4.13.22 - D-Ala-D-Ala dipeptidase

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The enzyme appears in viruses and cellular organisms

EC NUMBER
COMMENTARY hide
3.4.13.22
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RECOMMENDED NAME
GeneOntology No.
D-Ala-D-Ala dipeptidase
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REACTION
REACTION DIAGRAM
COMMENTARY hide
ORGANISM
UNIPROT
LITERATURE
D-Ala-D-Ala + H2O = 2 D-Ala
show the reaction diagram
REACTION TYPE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
hydrolysis of peptide bond
PATHWAY
BRENDA Link
KEGG Link
MetaCyc Link
vancomycin resistance I
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-
vancomycin resistance II
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-
CAS REGISTRY NUMBER
COMMENTARY hide
213189-85-6
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ORGANISM
COMMENTARY hide
LITERATURE
UNIPROT
SEQUENCE DB
SOURCE
strain BM4174, bifunctional enzyme with activities of EC 3.4.17.8 and 3.4.13.22
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Manually annotated by BRENDA team
strain BM4174, bifunctional enzyme with activities of EC 3.4.17.8 and 3.4.13.22
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-
Manually annotated by BRENDA team
strain M130
Uniprot
Manually annotated by BRENDA team
strain M130
Uniprot
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
(2S)-N-D-alanyl-2-(phenylthio)-glycine + H2O
D-alanine + 2-(phenylthio)-glycine
show the reaction diagram
-
-
-
-
?
D-Ala-D-Ala + H2O
2 D-Ala
show the reaction diagram
-
D-Ala-D-Ala hydrolysis with VanX is an exothermic and spontaneous reaction and has an approximative reaction rate with the imipenem hydrolysis with metallo-beta-lactamase ImiS in vitro. The values of activation free energy DELTAG are 87.140 , 88.413 , 89.611 , and 90.823 kJ per mol at 293.15, 298.15, 303.15, and 308.15 K, respectively, activation enthalpy DELTAH is 15.332 kJ per mol, activation entropy DELTAS is -245.02 J per mol and K, apparent activation energy E is 17.830 kJ per mol, and the reaction order is 1.5
-
-
?
D-Ala-D-Ala + H2O
D-Ala
show the reaction diagram
-
-
-
-
?
D-Ala-D-Ala + H2O
D-Ala + D-Ala
show the reaction diagram
D-Ala-D-Phe + H2O
D-Ala + D-Phe
show the reaction diagram
-
-
?
D-Ala-D-Ser + H2O
D-Ala + D-Ser
show the reaction diagram
D-Ala-Gly + H2O
D-Ala + Gly
show the reaction diagram
-
-
?
D-Leu-4-nitroanilide + H2O
D-Leu + 4-nitroaniline
show the reaction diagram
specific but catalytically inefficient substrate
-
-
?
D-leucine-p-nitroanilide + H2O
D-leucine + p-nitroaniline
show the reaction diagram
substrate is discovered by construction of a library containing 35 L- and D-amino acid p-nitroanilides
-
-
?
D-Ser-D-Ala + H2O
D-Ser + D-Ala
show the reaction diagram
-
-
?
D/L-Ala-4-nitroanilide + H2O
?
show the reaction diagram
slight hydrolysis for both isomers
-
-
?
DL-Ala-DL-Asn + H2O
DL-Ala + DL-Asn
show the reaction diagram
-
-
?
DL-Ala-DL-Ser + H2O
DL-Ala + DL-Ser
show the reaction diagram
-
-
?
DL-Ala-DL-Val + H2O
DL-Ala + DL-Val
show the reaction diagram
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-
?
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-Ala-D-Ala + H2O
D-Ala + D-Ala
show the reaction diagram
additional information
?
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Q06241
substrate specificty, no activity with D-Ala-D-lactate
-
?
METALS and IONS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
Fe2+
highly stimulating, optimal at 3 mM
additional information
INHIBITORS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
([(1-aminoethyl)(hydroxy)phosphoryl]oxy)acetic acid
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phosphonate dipeptide analogs of D-Ala-D-Ala
2-([(1-aminoethyl) (hydroxy) phosphoryl]oxy)propanoic acid
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phosphonate dipeptide analogs of D-Ala-D-Ala
D-3-[(1-aminoethyl)phosphinyl]-D-2-methylpropionic acid
D-3-[(1-aminoethyl)phosphonyl]-D-2-methylpropionic acid
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phosphonate analogue of the proposed tetrahedral intermediate of the hydrolysis reaction
D-Ala-D-(2-difluorothio)glycine-OH
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dipeptide-like mechanism-based inhibitor, cleavage of the substance results in formation of a highly reactive 4-thioquinone fluoromethide which covalently reacts with the enzyme resulting in irreversible inhibition, inhibition mechanism
D-Ala-D-Ala
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competitive substrate
D-Ala-D-lactate
very poor hydrolysis, blocking of the enzyme
D-Ala-L-Ala
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D-Ala-PSI[P(OOH)O]-D-Ala
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D-Ala-PSI[P(OOH)O]-D-Phe
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D-alanyl-2-[[4-(difluoromethyl)phenyl]thio]-glycine
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Kirr-value 22 microM, kinact-value 9.3/min
L-Ala-D-Ala
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L-Ala-L-Ala
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Ni2+
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3.3 mM, no residual activity
KM VALUE [mM]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
0.83
(2S)-N-D-alanyl-2-(phenylthio)-glycine
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-
1.2
Co2+
pH 7.0-9.0, 37°C
0.1 - 9
D-Ala-D-Ala
2.8 - 15.5
D-Ala-D-Ser
8.9
D-Leu-4-nitroanilide
pH 7.2, 37°C
8.9
D-leucine-p-nitroanilide
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1.7
D-Ser-D-Ala
pH 7.0-9.0, 37°C
additional information
additional information
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kinetics
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TURNOVER NUMBER [1/s]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
76
(2S)-N-D-alanyl-2-(phenylthio)-glycine
Enterococcus sp.
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-
69
Co2+
Enterococcus faecium
Q06241
pH 7.0-9.0, 37°C
0.64 - 460.1
D-Ala-D-Ala
0.35 - 1.8
D-Ala-D-Ser
0.0102
D-Leu-4-nitroanilide
Enterococcus faecium
Q06241
pH 7.2, 37°C
0.0102
D-leucine-p-nitroanilide
Enterococcus faecium
Q06241
-
0.35
D-Ser-D-Ala
Enterococcus faecium
Q06241
pH 7.0-9.0, 37°C
156
Fe2+
Enterococcus faecium
Q06241
pH 7.0-9.0, 37°C
788
Ni2+
Enterococcus faecium
Q06241
pH 7.0-9.0, 37°C
30
Zn2+
Enterococcus faecium
Q06241
pH 7.0-9.0, 37°C
Ki VALUE [mM]
INHIBITOR
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
0.03
D-Ala-D-(2-difluorothio)glycine-OH
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pH 7.0, 37°C
0.07
D-Ala-D-Ala
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242
D-Ala-D-lactate
pH 7.0-9.0, 37°C
58
D-Ala-L-Ala
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pH 8.0, 37°C
0.0165
D-Ala-PSI[P(OOH)O]-D-Ala
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koff: 0.0180 sec-1, results reveals that both dipeptide phosphonates are slow-binding inhibitors of VanX
0.00196
D-Ala-PSI[P(OOH)O]-D-Phe
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koff: 0.00231 sec-1, results reveals that both dipeptide phosphonates are slow-binding inhibitors of VanX. Moreover, in comparison with D-Ala(P,O)D-Ala phosphonate dipeptide, an additional aromatic interaction with the Phe79 residue in the active site of the enzyme may account for its higher affinity to VanX
225
L-Ala-D-Ala
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pH 8.0, 37°C
98
L-Ala-L-Ala
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pH 8.0, 37°C
additional information
additional information
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inhibition kinetics, Ki is 0.0015 mM immediately after addition of the enzyme, but is then lowered to by a relatively slow isomerization step to a second complex to Ki = 0.00047 mM
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IC50 VALUE [mM]
INHIBITOR
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
0.48
([(1-aminoethyl)(hydroxy)phosphoryl]oxy)acetic acid
Enterococcus faecium
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pH 8.0, 37°C
0.76
2-([(1-aminoethyl) (hydroxy) phosphoryl]oxy)propanoic acid
Enterococcus faecium
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pH 8.0, 37°C
SPECIFIC ACTIVITY [µmol/min/mg]
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
0.0015
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in strain overexpressing aad (NZ7100(pGIM023)), in the absence of 15 mM ZnCl2
0.0019
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wild type strain NZ7100 , in the presence of 15 mM ZnCl2
0.3872
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in strain overexpressing aad (NZ7100(pGIM023)), in the presence of 15 mM ZnCl2
10.6
recombinant enzyme
additional information
pH OPTIMUM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
7.6
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stopped-flow kinetic
pH RANGE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
TEMPERATURE OPTIMUM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
2
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stopped-flow kinetic
MOLECULAR WEIGHT
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
22700
x * 22700, recombinant enzyme, SDS-PAGE
23200
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x * 23200, SDS-PAGE
24200
x * 24200, recombinant ezyme, SDS-PAGE
SUBUNITS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
dimer
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exists as a dimer in solution but aggregates when at high concenctrations
additional information
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monomer secondary structure
Crystallization/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
crystal structures of VanXY mutant D59S and VanXY wild-type in apo and transition state analog-bound forms and of the mutant in complex with the D-Ala-D-Ala substrate and D-Ala product. Structural and biochemical analysis identifies the molecular determinants of VanXY dual specificity acting on dipeptide D-Ala-D-Ala or pentapeptide UDP-MurNac-L-Ala-D-Glu-L-Lys-D-Ala-D-Ala, respectively. VanXY residues 110-115 form a mobile cap over the catalytic site, whose flexibility is involved in the switch between di- and pentapeptide hydrolysis. VanY pentapeptidases lack this element, which promotes binding of the penta- rather than that of the dipeptide
enzyme crystallizes as a linear homohexamer which is held together by electrostatic interactions and H-bonding, and each subunit binds one Zn2+. The active site if VanX is in a cavity of 150 A that can only accomodate small molecules which explains the narrow substrate specificity of the enzyme. The Zn2+ is coordinated in a destorted tetrahedral arrangement with the side chains of His116, His184, Asp123 and a solvent water
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enzyme-D-Ala complex, enzyme-D-Ala-D-Ala complex, and enzyme in complex with phosphonate and phosphinate transition-state analogue inhibitors, complexsitting drops, 10 mg/ml protein, plus equal volume of precipitant solution: 0.25 M ammonium sulfate, 25% w/v polyethylene glycol monomethyl ester 5000, 0.1 M MES, 1 mM ZnCl2, X-ray diffraction at 2.1 A resolution structure determination and analysis
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Purification/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
by an amylose affinity column and further separation by the DEAE ion exchange chromatography
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recombinant from Escherichia coli, to homogeneity
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soluble recombinant enzyme from Escherichia coli, to homogeneity
Cloned/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
expressed as a maltose-binding protein fusion protein in Escherichia coli
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gene vanX, genomic organization, functional expression of the soluble enzyme in Escherichia coli BL21(DE3)
gene vanX, overexpression of the soluble enzyme in Escherichia coli
overexpression in Escherichia coli
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overexpression is achieved by cloning aad under the control of the nisin-inducible promoter PnisA from Lactococcus lactis. The resulting plasmid (pGIM023) is introduced into Lactobacillus plantarum strain NZ7100, a WCFS1 derivative carrying the nisR and nisK regulatory genes required for nisin induction
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overexpression of wild-type and mutants as maltose-binding-protein fusion proteins
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ENGINEERING
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
D59A
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73% decrease in ratio kcat/KM for D-Ala-D-Ala substrate
D59S
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50% increase in ratio kcat/KM for D-Ala-D-Ala substrate
D59A
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73% decrease in ratio kcat/KM for D-Ala-D-Ala substrate
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D59S
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50% increase in ratio kcat/KM for D-Ala-D-Ala substrate
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C78S/C157S
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site-directed mutagenesis, reduced activity
D123A
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site-directed mutagenesis, inactive, nearly no remaining Zn2+
D145A/E146A
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site-directed mutagenesis, 6fold lowered kcat compared to wild-type, 11% reduced zinc content compared to wild-type
E181A
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site-directed mutagenesis, inactive, 21.8% reduced zinc content compared to wild-type
H116A
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site-directed mutagenesis, inactive, 92.1% reduced zinc content compared to wild-type
H116A/H184A
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site-directed mutagenesis, inactive, 97.5% reduced zinc content compared to wild-type
H13A
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site-directed mutagenesis, activity equivalent to wild-type, 8.3% reduced zinc content compared to wild-type
H149A
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site-directed mutagenesis, equivalent to wild-type
H149A/H150A
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site-directed mutagenesis, 11fold lowered kcat, 14.9% reduced zinc content compared to wild-type
H150A
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site-directed mutagenesis, equivalent to wild-type
H184A
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site-directed mutagenesis, inactive, 99.08% reduced zinc content compared to wild-type
additional information
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an aad knockout mutant (MD119) is constructed in NZ7100 using a two-step homologous recombination procedure. Aad inactivation results in the stable replacement of the aad open reading frame (encoding 180 amino acid residues out of 185) by an erythromycin resistance cassette
APPLICATION
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
analysis
although catalytically ineffecient, the VanX substrate D-leucine-p-nitroanilide is an alternative substrate for VanX because it can be monitored directly and assayed spectrophotometrically which facilitates the routine analysis of enzyme catalysis and the screening discovery of potential VanX inhibitors. In addition, it is with leucine in its D form that possible activities from other contaminated species (other than VanX) in Escherichia coli JM109 are greatly reduced. Moreover, D-leucine-p-nitroanilide needs essentially no sophisticated synthetic chemistry for preparation
medicine
synthesis
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