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Information on EC 3.4.13.22 - D-Ala-D-Ala dipeptidase and Organism(s) Enterococcus faecium and UniProt Accession Q06241

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EC Tree
     3 Hydrolases
         3.4 Acting on peptide bonds (peptidases)
             3.4.13 Dipeptidases
                3.4.13.22 D-Ala-D-Ala dipeptidase
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This record set is specific for:
Enterococcus faecium
UNIPROT: Q06241 not found.
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The taxonomic range for the selected organisms is: Enterococcus faecium
The enzyme appears in selected viruses and cellular organisms
Reaction Schemes
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D-Ala-D-Ala
+
H2O
=
2
D-Ala
+
=
2
Synonyms
d-ala-d-ala dipeptidase, mab1843, d-alanyl-d-alanine dipeptidase, d-ala-d-ala amino dipeptidase, more
SYNONYM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
D-, D-dipeptidase
-
D-Ala-D-Ala-dipeptidase
-
Vancomycin B-type resistance protein vanX
-
D-Ala-D-Ala dipeptidase
-
-
D-alanyl-D-alanine dipeptidase
-
-
M15.011
-
Merops-ID
REACTION
REACTION DIAGRAM
COMMENTARY hide
ORGANISM
UNIPROT
LITERATURE
D-Ala-D-Ala + H2O = 2 D-Ala
show the reaction diagram
active site structure, substrate binding and mechanism
-
REACTION TYPE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
hydrolysis of peptide bond
hydrolysis of peptide bond
-
-
PATHWAY SOURCE
PATHWAYS
-
-, -
CAS REGISTRY NUMBER
COMMENTARY hide
213189-85-6
-
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-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
-
-
?
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 + D-Ala
show the reaction diagram
additional information
?
-
NATURAL SUBSTRATE
NATURAL 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-Ala + H2O
D-Ala + D-Ala
show the reaction diagram
D-Ala-D-Ala + H2O
D-Ala + D-Ala
show the reaction diagram
-
-
-
?
additional information
?
-
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
INHIBITOR
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
Cu2+
-
96% inhibition at 1 mM
D-3-[(1-aminoethyl)phosphinyl]-D-2-methylpropionic acid
-
phosphinate analogue of the proposed tetrahedral intermediate of the hydrolysis reaction, slow binding
D-Ala-D-lactate
very poor hydrolysis, blocking of the enzyme
D-Ala-L-Ala
-
-
L-Ala-D-Ala
-
-
L-Ala-L-Ala
-
-
Zn2+
-
72% inhibition at 1 mM
([(1-aminoethyl)(hydroxy)phosphoryl]oxy)acetic acid
-
phosphonate dipeptide analogs of D-Ala-D-Ala
2-([(1-aminoethyl) (hydroxy) phosphoryl]oxy)propanoic acid
-
phosphonate dipeptide analogs of D-Ala-D-Ala
D-3-[(1-aminoethyl)phosphinyl]-D-2-methylpropionic acid
-
phosphinate analogue of the proposed tetrahedral intermediate of the hydrolysis reaction
D-3-[(1-aminoethyl)phosphonyl]-D-2-methylpropionic acid
-
phosphonate analogue of the proposed tetrahedral intermediate of the hydrolysis reaction
D-Ala-PSI[P(OOH)O]-D-Ala
-
-
D-Ala-PSI[P(OOH)O]-D-Phe
-
-
KM VALUE [mM]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
1.2
Co2+
pH 7.0-9.0, 37°C
1 - 1.37
D-Ala-D-Ala
2.8
D-Ala-D-Ser
pH 7.0-9.0, 37°C
8.9
D-Leu-4-nitroanilide
pH 7.2, 37°C
8.9
D-leucine-p-nitroanilide
-
1.7
D-Ser-D-Ala
pH 7.0-9.0, 37°C
0.1 - 0.14
D-Ala-D-Ala
additional information
additional information
-
kinetics
-
TURNOVER NUMBER [1/s]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
69
Co2+
pH 7.0-9.0, 37°C
4.7 - 60
D-Ala-D-Ala
1.8
D-Ala-D-Ser
pH 7.0-9.0, 37°C
0.0102
D-Leu-4-nitroanilide
pH 7.2, 37°C
0.0102
D-leucine-p-nitroanilide
-
0.35
D-Ser-D-Ala
pH 7.0-9.0, 37°C
156
Fe2+
pH 7.0-9.0, 37°C
788
Ni2+
pH 7.0-9.0, 37°C
30
Zn2+
pH 7.0-9.0, 37°C
24 - 156
D-Ala-D-Ala
Ki VALUE [mM]
INHIBITOR
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
242
D-Ala-D-lactate
pH 7.0-9.0, 37°C
58
D-Ala-L-Ala
-
pH 8.0, 37°C
225
L-Ala-D-Ala
-
pH 8.0, 37°C
98
L-Ala-L-Ala
-
pH 8.0, 37°C
0.0165
D-Ala-PSI[P(OOH)O]-D-Ala
-
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
-
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
additional information
additional information
-
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
-
IC50 VALUE [mM]
INHIBITOR
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
0.48
([(1-aminoethyl)(hydroxy)phosphoryl]oxy)acetic acid
Enterococcus faecium
-
pH 8.0, 37°C
0.76
2-([(1-aminoethyl) (hydroxy) phosphoryl]oxy)propanoic acid
Enterococcus faecium
-
pH 8.0, 37°C
SPECIFIC ACTIVITY [µmol/min/mg]
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
additional information
-
pH OPTIMUM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
7.6
-
stopped-flow kinetic
pH RANGE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
TEMPERATURE OPTIMUM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
2
-
stopped-flow kinetic
ORGANISM
COMMENTARY hide
LITERATURE
UNIPROT
SEQUENCE DB
SOURCE
UNIPROT
ENTRY NAME
ORGANISM
NO. OF AA
NO. OF TRANSM. HELICES
MOLECULAR WEIGHT[Da]
SOURCE
SEQUENCE
LOCALIZATION PREDICTION?
VANX_ENTFC
202
0
23380
Swiss-Prot
-
MOLECULAR WEIGHT
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
24200
x * 24200, recombinant enzyme, SDS-PAGE
23200
-
x * 23200, SDS-PAGE
SUBUNIT
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
?
x * 24200, recombinant enzyme, SDS-PAGE
?
-
x * 23200, SDS-PAGE
dimer
-
exists as a dimer in solution but aggregates when at high concenctrations
additional information
-
monomer secondary structure
CRYSTALLIZATION (Commentary)
ORGANISM
UNIPROT
LITERATURE
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
-
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
-
PURIFICATION (Commentary)
ORGANISM
UNIPROT
LITERATURE
by an amylose affinity column and further separation by the DEAE ion exchange chromatography
recombinant from Escherichia coli, to homogeneity
-
soluble recombinant enzyme from Escherichia coli, to homogeneity
by an amylose affinity column and further separation by the DEAE ion exchange chromatography
-
CLONED (Commentary)
ORGANISM
UNIPROT
LITERATURE
expressed as a maltose-binding protein fusion protein in Escherichia coli
gene vanX, overexpression of the soluble enzyme in Escherichia coli
overexpression in Escherichia coli
-
expressed as a maltose-binding protein fusion protein in Escherichia coli
-
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
enzyme may be a target for drug design to reverse clinical vancomycin resistance
medicine
REF.
AUTHORS
TITLE
JOURNAL
VOL.
PAGES
YEAR
ORGANISM (UNIPROT)
PUBMED ID
SOURCE
Wu, Z.; Walsh, C.T.
Phosphinate analogs of D-, D-dipeptides: slow-binding inhibition and proteolysis protection of VanX, a D-, D-dipeptidase required for vancomycin resistance in Enterococcus faecium
Proc. Natl. Acad. Sci. USA
92
11603-11607
1995
Enterococcus faecium, Enterococcus faecium BM4147
Manually annotated by BRENDA team
Wu, Z.; Wright, G.D.; Walsh, C.T.
Overexpression, purification, and characterization of VanX, a D-, D-dipeptidase which is essential for vancomycin resistance in Enterococcus faecium BM4147
Biochemistry
34
2455-2463
1995
Enterococcus faecium (Q06241)
Manually annotated by BRENDA team
Bussiere, D.E.; Pratt, S.D.; Katz, L.; Severin, J.M.; Holzman, T.; Park, C.H.
The structure of VanX reveals a novel amino-dipeptidase involved in mediating transposon-based vancomycin resistance
Mol. Cell
2
75-84
1998
Enterococcus faecium
Manually annotated by BRENDA team
Golich, F.C.; Sigdel, T.; Breece, R.M.; Detar, L.; Herron, L.R.; Crowder, M.W.
L-Alanine-p-nitroanilide is not a substrate for VanX
Anal. Biochem.
331
398-400
2004
Enterococcus faecium
Manually annotated by BRENDA team
Hsieh, M.L.; Tseng, M.J.; Tseng, M.C.; Chu, Y.H.
Identification of a new chromophoric substrate in the library of amino acid p-nitroanilides for continuous assay of VanX, a D,D-dipeptidase essential for vancomycin resistance
Anal. Biochem.
354
104-110
2006
Enterococcus faecium (Q06241)
Manually annotated by BRENDA team
Breece, R.M.; Costello, A.; Bennett, B.; Sigdel, T.K.; Matthews, M.L.; Tierney, D.L.; Crowder, M.W.
A five-coordinate metal center in Co(II)-substituted VanX
J. Biol. Chem.
280
11074-11081
2005
Enterococcus faecium
Manually annotated by BRENDA team
Chang, Y.P.; Tseng, M.J.; Chu, Y.H.
Using surface plasmon resonance to directly measure slow binding of low-molecular mass inhibitors to a VanX chip
Anal. Biochem.
359
63-71
2006
Enterococcus faecium
Manually annotated by BRENDA team
Crowder, M.W.
Combating vancomycin resistance in bacteria: targeting the D-ala-D-ala dipeptidase VanX
Infect. Disord. Drug Targets
6
147-158
2006
Enterococcus faecium
Manually annotated by BRENDA team
Matthews, M.L.; Periyannan, G.; Hajdin, C.; Sidgel, T.K.; Bennett, B.; Crowder, M.W.
Probing the reaction mechanism of the D-ala-D-ala dipeptidase, VanX, by using stopped-flow kinetic and rapid-freeze quench EPR studies on the Co(II)-substituted enzyme
J. Am. Chem. Soc.
128
13050-13051
2006
Enterococcus faecium
Manually annotated by BRENDA team
Jia, C.; Yang, K.; Liu, C.; Feng, L.; Xiao, J.; Zhou, L.; Zhang, Y.
Synthesis, characterization and activity of new phosphonate dipeptides as potential inhibitors of VanX
Bioorg. Med. Chem. Lett.
22
482-484
2012
Enterococcus faecium
Manually annotated by BRENDA team
Liu, C.; Zhao, X.; Yang, K.; Xu, K.; Zhai, L.; Yang, X.; Gao, H.
Exploring antibiotic resistance based on enzyme hydrolysis by microcalorimetry: Part IV. Determination of thermokinetic parameters of D-Ala-D-Ala hydrolysis with dipeptidase VanX
J. Therm. Anal. Calorim.
111
1663-1667
2013
Enterococcus faecium
-
Manually annotated by BRENDA team
Hansen, T.; Pedersen, M.; Nielsen, L.; Ma, C.; Soes, L.; Worning, P.; Ostergaard, C.; Westh, H.; Pinholt, M.; Schonning, K.
Emergence of a vancomycin-variable Enterococcus faecium ST1421 strain containing a deletion in vanX
J. Antimicrob. Chemother.
73
2936-2940
2018
Enterococcus faecium
Manually annotated by BRENDA team