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Information on EC 1.11.1.16 - versatile peroxidase and Organism(s) Pleurotus eryngii and UniProt Accession Q9UVP6
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1.11.1.16 versatile peroxidaseIUBMB Comments
A hemoprotein. This ligninolytic peroxidase combines the substrate-specificity characteristics of the two other ligninolytic peroxidases, EC 1.11.1.13, manganese peroxidase and EC 1.11.1.14, lignin peroxidase. Unlike these two enzymes, it is also able to oxidize phenols, hydroquinones and both low- and high-redox-potential dyes, due to a hybrid molecular architecture that involves multiple binding sites for substrates [2,4].
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Word Map
- 1.11.1.16
- lignin
- pleurotus
- peroxidases
-
ligninolytic
- eryngii
- laccase
-
white-rot
- bjerkandera
- ostreatus
-
veratryl
- adusta
- chrysosporium
-
phanerochaete
-
lignin-degrading
-
non-phenolic
- 2,6-dimethoxyphenol
-
delignification
-
aryl-alcohols
- degradation
- synthesis
- industry
- analysis
- paper production
The taxonomic range for the selected organisms is: Pleurotus eryngii
The expected taxonomic range for this enzyme is: Bacteria, Eukaryota
The expected taxonomic range for this enzyme is: Bacteria, Eukaryota
Reaction Schemes
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=
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+
+
=
+
+
+
2
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2
+
=
2
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2
Synonyms
myoglobin, versatile peroxidase, mb peroxidase, versatile peroxidase mnp2, metmb peroxidase, 
more
topSYNONYM 
ORGANISM
UNIPROT
COMMENTARY 
LITERATURE
VPS1
Vpl2
REACTION
REACTION DIAGRAM
COMMENTARY
ORGANISM
UNIPROT
LITERATURE
1-(4-hydroxy-3-methoxyphenyl)-2-(2-methoxyphenoxy)propane-1,3-diol + H2O2 = 4-hydroxy-3-methoxybenzaldehyde + 2-methoxyphenol + glycolaldehyde + H2O
2 manganese(II) + 2 H+ + H2O2 = 2 manganese(III) + 2 H2O
at the ferric resting state, heme peroxidases are first activated by a single molecule of H2O2 to yield an oxidized catalytic intermediate called compound I (oxoferryl IV porphyrin p-cation radical), releasing one molecule of water as the only by-product. The enzyme then catalyzes two consecutive one-electron oxidations of two reducing substrates, regenerating the ground reduced state through a second catalytic intermediate called compound II (oxoferryl) and with the concomitant production of a second molecule of water. The main limiting step within this catalytic cycle is the low conversion rate from compound II to the ground state, which allows the former to accumulate and react with a new molecule of H2O2, either in the presence (with an excess of H2O2) or absence (at catalytic concentrations of H2O2) of the reducing substrate
1-(4-hydroxy-3-methoxyphenyl)-2-(2-methoxyphenoxy)propane-1,3-diol + H2O2 = 4-hydroxy-3-methoxybenzaldehyde + 2-methoxyphenol + glycolaldehyde + H2O
NMR study of enzyme both in resting state and cyanide-inhibited form. Analysis of interaction with Mn2+
-
1-(4-hydroxy-3-methoxyphenyl)-2-(2-methoxyphenoxy)propane-1,3-diol + H2O2 = 4-hydroxy-3-methoxybenzaldehyde + 2-methoxyphenol + glycolaldehyde + H2O
the radical intermediate in the reaction of enzyme with H2O2 is a tryptophan neutral radical at W164
PATHWAY SOURCE
PATHWAYS
-
-
SYSTEMATIC NAME
IUBMB Comments
reactive-black-5:hydrogen-peroxide oxidoreductase
A hemoprotein. This ligninolytic peroxidase combines the substrate-specificity characteristics of the two other ligninolytic peroxidases, EC 1.11.1.13, manganese peroxidase and EC 1.11.1.14, lignin peroxidase. Unlike these two enzymes, it is also able to oxidize phenols, hydroquinones and both low- and high-redox-potential dyes, due to a hybrid molecular architecture that involves multiple binding sites for substrates [2,4].
CAS REGISTRY NUMBER
COMMENTARY
114995-15-2
-
42613-30-9
-
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
2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) + 2 H+ + H2O2
oxidized 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) + 2 H2O
-
-
-
?
2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) + H2O2 + H+
?
-
-
-
?
manganese(II)-substituted polyoxometalate + H2O2
manganese(III)-substituted polyoxometalate + H2O2
-
-
-
-
?
p-dimethoxybenzene + H2O2
benzoquinone + H2O
-
catalyzed by isoforms PS3, PS1
-
-
?
phenol red + H2O2
oxidized phenol red + H2O
-
Mn2+-dependent activity
-
-
?
Reactive Black 5 + 2 H+ + H2O2
oxidized Reactive Black 5 + 2 H2O
-
-
-
?
2 2,6-dimethoxyphenol + 2 H2O2
coerulignone + 2 H2O
-
Mn2+-dependent and independent activity
-
-
?
2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) + H+ + H2O2
?
-
-
-
-
?
2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) + H+ + H2O2
?
-
-
-
?
2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) + H2O2
? + H2O
-
-
-
?
2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) + H2O2
? + H2O
-
-
-
-
?
2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) + H2O2
? + H2O
-
-
-
?
2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) + H2O2
? + H2O
-
-
-
-
?
Mn2+ + H2O2
Mn3+ + H2O
the Mn2+-binding site in versatile peroxidase is formed by the side-chains of Glu36, Glu40, and Asp175 located in front of the internal (i.e. more distant from the main haem access-channel) propionate of haem, and connected to the solvent by a narrow access-channel that presents a variable geometry during catalysis
-
-
?
Reactive Black 5 + H2O2
?
versatile peroxidase activity on Reactive Black 5 is eliminated by the R257D mutation
-
-
?
Reactive Black 5 + H2O2
oxidized Reactive Black 5 + H2O
-
-
-
?
Reactive Black 5 + H2O2
oxidized Reactive Black 5 + H2O
-
-
-
?
Reactive Black 5 + H2O2
oxidized Reactive Black 5 + H2O
-
catalyzed by isoform PS1
-
-
?
veratryl alcohol + H2O2 + H+
veratraldehyde + H2O
-
catalyzed by isoforms PS2, PS1
-
-
?
veratryl alcohol + H2O2 + H+
veratraldehyde + H2O
-
Mn2+-independent activity
-
-
?
veratryl alcohol + H2O2 + H+
veratraldehyde + H2O
a solvent-exposed tryptophan is the catalytically-active residue in veratryl alcohol oxidation, initiating an electron transfer pathway to haem
-
-
?
additional information
?
-
the enzyme shows a broad substrate spectrum
-
-
-
additional information
?
-
-
the enzyme shows a broad substrate spectrum
-
-
-
additional information
?
-
-
in the absence of Mn2+, efficient hydroquinone oxidation is dependent on exogenous H2O2. In the presence of Mn2+, exogenous H2O2 is not required for complete oxidation of hydroquinones
-
-
?
additional information
?
-
versatile peroxidase is able to oxidize typical substrates of other peroxidases, these hybrid properties are due to the coexistence in a single protein of different catalytic sites reminiscent of those present in the other basidiomycete peroxidase families
-
-
?
additional information
?
-
presence of two independent catalytic sites for different phenols and 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) in native enzyme, characterizedby high and low specificity constants, i.e. Km in the micromolar range and Km in the millimolar range, respcetively
-
-
?
additional information
?
-
-
presence of two independent catalytic sites for different phenols and 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) in native enzyme, characterizedby high and low specificity constants, i.e. Km in the micromolar range and Km in the millimolar range, respcetively
-
-
?
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
Reactive Black 5 + 2 H+ + H2O2
oxidized Reactive Black 5 + 2 H2O
-
-
-
?
COFACTOR
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
METALS and IONS
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
Ca2+
Mn2+
Ca2+
required, binding structure, three-dimensional modeling, overview
Mn2+
-
isoenzyme Pl, putative Mn-interaction site near E35, E39, D175. Isoenzyme Ps1, putative Mn-interaction site near E36, E40, D181
INHIBITOR
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
ACTIVATING COMPOUND
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
KM VALUE [mM]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
6.4
manganese(II)-substituted polyoxometalate
-
in 0.1 M sodium tartrate, pH 5.0, at 20°C
-
0.0007
2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid)
pH 3.5, 25°C
0.0017
2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid)
pH 3.5, 25°C, mutant R257L
0.0023
2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid)
mutant P76G, high efficiency site, pH 3.5, 25°C
0.0026
2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid)
pH 3.5, 25°C, mutant M247L
0.0028
2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid)
pH 3.5, 25°C, mutant S158E
0.003
2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid)
pH 3.5, 25°C, wild-type
0.003
2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid)
wild-type, high efficiency site, pH 3.5, 25°C
0.0032
2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid)
pH 3.5, 25°C, mutant R257A/A260F
0.0035
2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid)
pH 3.5, 25°C, mutant M247F
0.0036
2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid)
pH 3.5, 25°C, mutant R257K
0.004
2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid)
pH 3.5, 25°C, mutant S158D
0.004
2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid)
mutant K176D, high efficiency site, pH 3.5, 25°C
0.0041
2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid)
mutant F142G, high efficiency site, pH 3.5, 25°C
0.0054
2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid)
mutant K176G, high efficiency site, pH 3.5, 25°C
0.0054
2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid)
mutant K215G, high efficiency site, pH 3.5, 25°C
0.0054
2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid)
mutant P141G, high efficiency site, pH 3.5, 25°C
0.0065
2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid)
pH 3.5, 25°C, mutant K264A
0.008
2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid)
mutant K215Q, high efficiency site, pH 3.5, 25°C
0.009
2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid)
pH 3.5, 25°C, mutant A260F
0.0143
2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid)
mutant E140G, high efficiency site, pH 3.5, 25°C
0.0194
2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid)
mutant E140G/P141G, high efficiency site, pH 3.5, 25°C
0.0194
2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid)
mutant E140G/P141G, low efficiency site, pH 3.5, 25°C
0.056
2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid)
mutant E37K/V160A/T184M/Q202L, pH 3.5, temperature not specified in the publication
0.0836
2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid)
mutant K215G, low efficiency site, pH 3.5, 25°C
0.305
2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid)
mutant E140G, low efficiency site, pH 3.5, 25°C
0.383
2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid)
mutant K176G, low efficiency site, pH 3.5, 25°C
0.461
2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid)
mutant P141G, low efficiency site, pH 3.5, 25°C
0.54
2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid)
wild-type, pH 3.5, temperature not specified in the publication
0.54
2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid)
at pH 3.0 and 25°C
0.828
2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid)
mutant F142G, low efficiency site, pH 3.5, 25°C
1
2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid)
mutant W164S, low efficiency site, pH 3.5, 25°C
1.09
2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid)
wild-type, low efficiency site, pH 3.5, 25°C
1.66
2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid)
mutant K215Q, low efficiency site, pH 3.5, 25°C
2.23
2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid)
mutant K176D, low efficiency site, pH 3.5, 25°C
2.86
2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid)
mutant P76G, low efficiency site, pH 3.5, 25°C
0.036
2,6-dimethoxyphenol
mutant P76G, high efficiency site, pH 3.5, 25°C
0.038
2,6-dimethoxyphenol
mutant K176D, high efficiency site, pH 3.5, 25°C
0.058
2,6-dimethoxyphenol
mutant K215G, high efficiency site, pH 3.5, 25°C
0.065
2,6-dimethoxyphenol
mutant F142G, high efficiency site, pH 3.5, 25°C
0.066
2,6-dimethoxyphenol
mutant E140G, high efficiency site, pH 3.5, 25°C
0.078
2,6-dimethoxyphenol
mutant K215Q, high efficiency site, pH 3.5, 25°C
0.078
2,6-dimethoxyphenol
wild-type, high efficiency site, pH 3.5, 25°C
0.1
2,6-dimethoxyphenol
mutant K176G, high efficiency site, pH 3.5, 25°C
0.104
2,6-dimethoxyphenol
mutant E140G/P141G, high efficiency site, pH 3.5, 25°C
0.119
2,6-dimethoxyphenol
mutant E140G/P141G/K176G, high efficiency site, pH 3.5, 25°C
0.155
2,6-dimethoxyphenol
mutant P141G, high efficiency site, pH 3.5, 25°C
0.189
2,6-dimethoxyphenol
mutant E140G/K176G, high efficiency site, pH 3.5, 25°C
2.38
2,6-dimethoxyphenol
mutant E140G/P141G/K176G, low efficiency site, pH 3.5, 25°C
2.56
2,6-dimethoxyphenol
mutant K215Q, low efficiency site, pH 3.5, 25°C
2.97
2,6-dimethoxyphenol
mutant E140G/K176G, low efficiency site, pH 3.5, 25°C
3.33
2,6-dimethoxyphenol
mutant E140G/W164S/K176G, low efficiency site, pH 3.5, 25°C
6.5
2,6-dimethoxyphenol
mutant E37K/V160A/T184M/Q202L, pH 3.5, temperature not specified in the publication
10.5
2,6-dimethoxyphenol
wild-type, low efficiency site, pH 3.5, 25°C
16
2,6-dimethoxyphenol
mutant E140G/P141G, low efficiency site, pH 3.5, 25°C
32
2,6-dimethoxyphenol
wild-type, pH 3.5, temperature not specified in the publication
36.2
2,6-dimethoxyphenol
mutant K176G, low efficiency site, pH 3.5, 25°C
37.4
2,6-dimethoxyphenol
mutant E140G, low efficiency site, pH 3.5, 25°C
37.6
2,6-dimethoxyphenol
mutant W164S, low efficiency site, pH 3.5, 25°C
76
2,6-dimethoxyphenol
mutant P141G, low efficiency site, pH 3.5, 25°C
0.0059
4-hydroquinone
mutant F142G, high efficiency site, pH 3.5, 25°C
0.0103
4-hydroquinone
mutant P76G, high efficiency site, pH 3.5, 25°C
0.0122
4-hydroquinone
mutant K215Q, high efficiency site, pH 3.5, 25°C
0.013
4-hydroquinone
mutant K176D, high efficiency site, pH 3.5, 25°C
0.0174
4-hydroquinone
mutant K215G, high efficiency site, pH 3.5, 25°C
0.0189
4-hydroquinone
mutant K176G, high efficiency site, pH 3.5, 25°C
0.0205
4-hydroquinone
mutant E140G, high efficiency site, pH 3.5, 25°C
0.0251
4-hydroquinone
mutant E140G/K176G, high efficiency site, pH 3.5, 25°C
0.0362
4-hydroquinone
mutant E140G/P141G/K176G, high efficiency site, pH 3.5, 25°C
0.0397
4-hydroquinone
mutant E140G/P141G, high efficiency site, pH 3.5, 25°C
0.04
4-hydroquinone
mutant P141G, high efficiency site, pH 3.5, 25°C
0.41
4-hydroquinone
mutant E140G/P141G/K176G, low efficiency site, pH 3.5, 25°C
0.618
4-hydroquinone
mutant E140G/K176G, low efficiency site, pH 3.5, 25°C
0.836
4-hydroquinone
mutant E140G, low efficiency site, pH 3.5, 25°C
0.884
4-hydroquinone
mutant E140G/W164S/K176G, low efficiency site, pH 3.5, 25°C
1.112
4-hydroquinone
mutant K215G, low efficiency site, pH 3.5, 25°C
1.114
4-hydroquinone
mutant P141G, low efficiency site, pH 3.5, 25°C
1.18
4-hydroquinone
mutant E140G/P141G, low efficiency site, pH 3.5, 25°C
0.051
H2O2
wild-type, pH 3.5, temperature not specified in the publication
0.2
H2O2
mutant E37K/V160A/T184M/Q202L, pH 3.5, temperature not specified in the publication
0.045
Mn2+
wild-type, pH 3.5, temperature not specified in the publication
0.12
Mn2+
mutant E37K/V160A/T184M/Q202L, pH 3.5, temperature not specified in the publication
0.0066
Reactive Black 5
mutant E37K/V160A/T184M/Q202L, pH 3.5, temperature not specified in the publication
0.007
Reactive Black 5
wild-type, pH 3.5, temperature not specified in the publication
additional information
additional information
kinetic parameters for wild-type enzyme and five selected mutant variants with dye substrates, comparisons, overview
-
additional information
additional information
-
kinetic parameters for wild-type enzyme and five selected mutant variants with dye substrates, comparisons, overview
-
TURNOVER NUMBER [1/s]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
47
manganese(II)-substituted polyoxometalate
-
in 0.1 M sodium tartrate, pH 5.0, at 20°C
-
0.7
2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid)
pH 3.5, 25°C, mutant M247F
4.9
2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid)
pH 3.5, 25°C, mutant M247L
5.4
2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid)
pH 3.5, 25°C
5.9
2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid)
pH 3.5, 25°C, mutant A260F
6.6
2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid)
mutant P76G, high efficiency site, pH 3.5, 25°C
7.7
2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid)
pH 3.5, 25°C, mutant S158D
7.9
2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid)
mutant K215Q, high efficiency site, pH 3.5, 25°C
8.1
2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid)
pH 3.5, 25°C, wild-type
8.1
2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid)
wild-type, high efficiency site, pH 3.5, 25°C
8.8
2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid)
pH 3.5, 25°C, mutant K264A
9.1
2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid)
pH 3.5, 25°C, mutant S158E
10.9
2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid)
mutant F142G, high efficiency site, pH 3.5, 25°C
11.8
2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid)
pH 3.5, 25°C, mutant R257K
12.6
2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid)
mutant E140G, high efficiency site, pH 3.5, 25°C
13.2
2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid)
mutant K176G, high efficiency site, pH 3.5, 25°C
13.8
2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid)
mutant K176D, high efficiency site, pH 3.5, 25°C
13.8
2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid)
mutant K215G, high efficiency site, pH 3.5, 25°C
16.1
2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid)
mutant P141G, high efficiency site, pH 3.5, 25°C
18.6
2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid)
pH 3.5, 25°C, mutant R257A/A260F
19.2
2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid)
mutant E140G/P141G, high efficiency site, pH 3.5, 25°C
22.2
2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid)
pH 3.5, 25°C, mutant R257L
93
2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid)
mutant P141G, low efficiency site, pH 3.5, 25°C
146
2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid)
mutant P76G, low efficiency site, pH 3.5, 25°C
154
2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid)
mutant F142G, low efficiency site, pH 3.5, 25°C
162
2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid)
mutant E140G/P141G, low efficiency site, pH 3.5, 25°C
165
2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid)
mutant K215Q, low efficiency site, pH 3.5, 25°C
186
2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid)
wild-type, low efficiency site, pH 3.5, 25°C
204
2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid)
mutant K215G, low efficiency site, pH 3.5, 25°C
205
2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid)
mutant W164S, low efficiency site, pH 3.5, 25°C
216
2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid)
mutant E140G, low efficiency site, pH 3.5, 25°C
220
2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid)
wild-type, pH 3.5, temperature not specified in the publication
220
2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid)
at pH 3.0 and 25°C
223
2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid)
mutant K176D, low efficiency site, pH 3.5, 25°C
227
2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid)
mutant E140G/W164S/K176G, low efficiency site, pH 3.5, 25°C
235
2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid)
mutant E140G/P141G/K176G, low efficiency site, pH 3.5, 25°C
259
2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid)
mutant K176G, low efficiency site, pH 3.5, 25°C
326
2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid)
mutant E140G/K176G, low efficiency site, pH 3.5, 25°C
365
2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid)
mutant E37K/V160A/T184M/Q202L, pH 3.5, temperature not specified in the publication
4.6
2,6-dimethoxyphenol
mutant E140G/P141G, high efficiency site, pH 3.5, 25°C
4.8
2,6-dimethoxyphenol
mutant P141G, high efficiency site, pH 3.5, 25°C
5.6
2,6-dimethoxyphenol
wild-type, high efficiency site, pH 3.5, 25°C
6.2
2,6-dimethoxyphenol
mutant P76G, high efficiency site, pH 3.5, 25°C
7.6
2,6-dimethoxyphenol
mutant K215Q, high efficiency site, pH 3.5, 25°C
9.3
2,6-dimethoxyphenol
mutant K176G, high efficiency site, pH 3.5, 25°C
9.5
2,6-dimethoxyphenol
mutant K215G, high efficiency site, pH 3.5, 25°C
9.8
2,6-dimethoxyphenol
mutant F142G, high efficiency site, pH 3.5, 25°C
10
2,6-dimethoxyphenol
mutant E140G, high efficiency site, pH 3.5, 25°C
10.6
2,6-dimethoxyphenol
mutant K176D, high efficiency site, pH 3.5, 25°C
17.3
2,6-dimethoxyphenol
mutant E140G/K176G, high efficiency site, pH 3.5, 25°C
20.4
2,6-dimethoxyphenol
mutant E140G/P141G/K176G, high efficiency site, pH 3.5, 25°C
29.8
2,6-dimethoxyphenol
wild-type, low efficiency site, pH 3.5, 25°C
47.6
2,6-dimethoxyphenol
mutant W164S, low efficiency site, pH 3.5, 25°C
50.2
2,6-dimethoxyphenol
mutant E140G/W164S/K176G, low efficiency site, pH 3.5, 25°C
56.6
2,6-dimethoxyphenol
mutant E140G/P141G/K176G, low efficiency site, pH 3.5, 25°C
58
2,6-dimethoxyphenol
mutant E37K/V160A/T184M/Q202L, pH 3.5, temperature not specified in the publication
61
2,6-dimethoxyphenol
mutant E140G/P141G, low efficiency site, pH 3.5, 25°C
67
2,6-dimethoxyphenol
mutant E140G/K176G, low efficiency site, pH 3.5, 25°C
74.5
2,6-dimethoxyphenol
mutant K215Q, low efficiency site, pH 3.5, 25°C
98
2,6-dimethoxyphenol
wild-type, pH 3.5, temperature not specified in the publication
141
2,6-dimethoxyphenol
mutant P141G, low efficiency site, pH 3.5, 25°C
231.6
2,6-dimethoxyphenol
mutant K176G, low efficiency site, pH 3.5, 25°C
293
2,6-dimethoxyphenol
mutant E140G, low efficiency site, pH 3.5, 25°C
9.71
4-hydroquinone
mutant F142G, high efficiency site, pH 3.5, 25°C
11.4
4-hydroquinone
mutant K215Q, high efficiency site, pH 3.5, 25°C
11.6
4-hydroquinone
mutant K176G, high efficiency site, pH 3.5, 25°C
14.6
4-hydroquinone
mutant E140G/P141G, high efficiency site, pH 3.5, 25°C
15.6
4-hydroquinone
mutant P141G, high efficiency site, pH 3.5, 25°C
16.2
4-hydroquinone
mutant E140G, high efficiency site, pH 3.5, 25°C
20
4-hydroquinone
mutant E140G/K176G, high efficiency site, pH 3.5, 25°C
20.4
4-hydroquinone
mutant E140G/P141G/K176G, high efficiency site, pH 3.5, 25°C
65.5
4-hydroquinone
mutant E140G/P141G, low efficiency site, pH 3.5, 25°C
82.1
4-hydroquinone
mutant E140G/K176G, low efficiency site, pH 3.5, 25°C
82.2
4-hydroquinone
mutant E140G/P141G/K176G, low efficiency site, pH 3.5, 25°C
85.6
4-hydroquinone
mutant E140G/W164S/K176G, low efficiency site, pH 3.5, 25°C
135
H2O2
wild-type, pH 3.5, temperature not specified in the publication
490
H2O2
mutant E37K/V160A/T184M/Q202L, pH 3.5, temperature not specified in the publication
54
Mn2+
wild-type, pH 3.5, temperature not specified in the publication
75
Mn2+
mutant E37K/V160A/T184M/Q202L, pH 3.5, temperature not specified in the publication
298
Mn2+
-
0.000083 1/sec/mg, incubates 30 min at 37°C, pH 9.0, spectrophotometrically measured at 415 nm
5
Reactive Black 5
-
0.0 M NaCl, 55.6 IgG relative content, 5.5% relative DNase activity
10.6
Reactive Black 5
mutant E37K/V160A/T184M/Q202L, pH 3.5, temperature not specified in the publication
11.8
Reactive Black 5
wild-type, pH 3.5, temperature not specified in the publication
kcat/KM VALUE [1/mMs-1]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
7.36
manganese(II)-substituted polyoxometalate
-
in 0.1 M sodium tartrate, pH 5.0, at 20°C
-
1
2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid)
mutant K215Q, high efficiency site, pH 3.5, 25°C
2.45
2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid)
mutant K176G, high efficiency site, pH 3.5, 25°C
2.53
2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid)
mutant E140G/P141G, high efficiency site, pH 3.5, 25°C
2.6
2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid)
mutant K215G, high efficiency site, pH 3.5, 25°C
3.48
2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid)
mutant K176D, high efficiency site, pH 3.5, 25°C
51
2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid)
mutant P76G, low efficiency site, pH 3.5, 25°C
99
2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid)
mutant K215Q, low efficiency site, pH 3.5, 25°C
100
2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid)
mutant K176D, low efficiency site, pH 3.5, 25°C
171
2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid)
wild-type, low efficiency site, pH 3.5, 25°C
185
2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid)
mutant F142G, low efficiency site, pH 3.5, 25°C
200
2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid)
mutant W164S, low efficiency site, pH 3.5, 25°C
201
2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid)
mutant P141G, low efficiency site, pH 3.5, 25°C
244
2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid)
mutant K215G, low efficiency site, pH 3.5, 25°C
410
2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid)
wild-type, pH 3.5, temperature not specified in the publication
410
2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid)
at pH 3.0 and 25°C
676
2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid)
mutant K176G, low efficiency site, pH 3.5, 25°C
710
2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid)
mutant E140G, low efficiency site, pH 3.5, 25°C
882
2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid)
mutant E140G, high efficiency site, pH 3.5, 25°C
1120
2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid)
mutant E140G/P141G, low efficiency site, pH 3.5, 25°C
2640
2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid)
mutant F142G, high efficiency site, pH 3.5, 25°C
2700
2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid)
wild-type, high efficiency site, pH 3.5, 25°C
2830
2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid)
mutant P76G, high efficiency site, pH 3.5, 25°C
3010
2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid)
mutant P141G, high efficiency site, pH 3.5, 25°C
4090
2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid)
mutant E140G/W164S/K176G, low efficiency site, pH 3.5, 25°C
5670
2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid)
mutant E140G/P141G/K176G, low efficiency site, pH 3.5, 25°C
5680
2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid)
mutant E140G/K176G, low efficiency site, pH 3.5, 25°C
6480
2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid)
mutant E37K/V160A/T184M/Q202L, pH 3.5, temperature not specified in the publication
1.3
2,6-dimethoxyphenol
mutant W164S, low efficiency site, pH 3.5, 25°C
1.9
2,6-dimethoxyphenol
mutant P141G, low efficiency site, pH 3.5, 25°C
2.8
2,6-dimethoxyphenol
wild-type, low efficiency site, pH 3.5, 25°C
2.9
2,6-dimethoxyphenol
mutant K215Q, low efficiency site, pH 3.5, 25°C
3.1
2,6-dimethoxyphenol
wild-type, pH 3.5, temperature not specified in the publication
3.8
2,6-dimethoxyphenol
mutant E140G/P141G, low efficiency site, pH 3.5, 25°C
6.4
2,6-dimethoxyphenol
mutant K176G, low efficiency site, pH 3.5, 25°C
7.8
2,6-dimethoxyphenol
mutant E140G, low efficiency site, pH 3.5, 25°C
9.1
2,6-dimethoxyphenol
mutant E37K/V160A/T184M/Q202L, pH 3.5, temperature not specified in the publication
15.1
2,6-dimethoxyphenol
mutant E140G/W164S/K176G, low efficiency site, pH 3.5, 25°C
22.6
2,6-dimethoxyphenol
mutant E140G/K176G, low efficiency site, pH 3.5, 25°C
23.8
2,6-dimethoxyphenol
mutant E140G/P141G/K176G, low efficiency site, pH 3.5, 25°C
31
2,6-dimethoxyphenol
mutant P141G, high efficiency site, pH 3.5, 25°C
44.6
2,6-dimethoxyphenol
mutant E140G/P141G, high efficiency site, pH 3.5, 25°C
71
2,6-dimethoxyphenol
wild-type, high efficiency site, pH 3.5, 25°C
91.3
2,6-dimethoxyphenol
mutant E140G/K176G, high efficiency site, pH 3.5, 25°C
93
2,6-dimethoxyphenol
mutant K176G, high efficiency site, pH 3.5, 25°C
97
2,6-dimethoxyphenol
mutant K215Q, high efficiency site, pH 3.5, 25°C
152
2,6-dimethoxyphenol
mutant F142G, high efficiency site, pH 3.5, 25°C
153
2,6-dimethoxyphenol
mutant E140G, high efficiency site, pH 3.5, 25°C
165
2,6-dimethoxyphenol
mutant K215G, high efficiency site, pH 3.5, 25°C
171
2,6-dimethoxyphenol
mutant E140G/P141G/K176G, high efficiency site, pH 3.5, 25°C
171
2,6-dimethoxyphenol
mutant P76G, high efficiency site, pH 3.5, 25°C
283
2,6-dimethoxyphenol
mutant K176D, high efficiency site, pH 3.5, 25°C
55.6
4-hydroquinone
mutant E140G/P141G, low efficiency site, pH 3.5, 25°C
96.8
4-hydroquinone
mutant E140G/W164S/K176G, low efficiency site, pH 3.5, 25°C
132.8
4-hydroquinone
mutant E140G/K176G, low efficiency site, pH 3.5, 25°C
203.2
4-hydroquinone
mutant E140G/P141G/K176G, low efficiency site, pH 3.5, 25°C
368
4-hydroquinone
mutant E140G/P141G, high efficiency site, pH 3.5, 25°C
565
4-hydroquinone
mutant E140G/P141G/K176G, high efficiency site, pH 3.5, 25°C
800
4-hydroquinone
mutant E140G/K176G, high efficiency site, pH 3.5, 25°C
1600
4-hydroquinone
mutant F142G, high efficiency site, pH 3.5, 25°C
2400
H2O2
mutant E37K/V160A/T184M/Q202L, pH 3.5, temperature not specified in the publication
2650
H2O2
wild-type, pH 3.5, temperature not specified in the publication
630
Mn2+
mutant E37K/V160A/T184M/Q202L, pH 3.5, temperature not specified in the publication
1190
Mn2+
wild-type, pH 3.5, temperature not specified in the publication
1600
Reactive Black 5
mutant E37K/V160A/T184M/Q202L, pH 3.5, temperature not specified in the publication
1670
Reactive Black 5
wild-type, pH 3.5, temperature not specified in the publication
SPECIFIC ACTIVITY [µmol/min/mg]
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
10.8
-
substrate 2,6-dimthoxy-1,4-benzohydroquinone, pH 5.0, absence of Mn2+
8.8
substrate 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid), pH 3.5, 25°C
additional information
additional information
activities of wild-type enzyme and five selected mutant variants with dye substrates, comparisons, overview
additional information
-
activities of wild-type enzyme and five selected mutant variants with dye substrates, comparisons, overview
pH OPTIMUM
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
3
-
Mn2+-independent oxidation of substituted phenols and aromatic molecules
pI VALUE
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
3.65
ORGANISM
COMMENTARY
LITERATURE
UNIPROT
SEQUENCE DB
SOURCE
LOCALIZATION
ORGANISM
UNIPROT
COMMENTARY
GeneOntology No.
LITERATURE
SOURCE
GENERAL INFORMATION
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
evolution
versatile peroxidase (VP) is a lignin-degrading heme-containing oxidoreductase classified as a class II peroxidase, which is secreted by several species of basidiomycetes, mostly from the genera Pleurotus and Bjerkandera
physiological function
versatile peroxidase (VP) from Pleurotus eryngii is a heme-containing peroxidase with a broad substrate spectrum that can break down many structurally distinct pollutants, including azo dyes. Versatile peroxidase is a hybrid enzyme that combines the catalytic characteristics of MnP (i.e., the ability to oxidase Mn2+ to Mn3+, which when complexed by organic acids can oxidize aromatic compounds, EC 1.11.1.13) with the LiP-like ability (EC 1.11.1.14) to use the long-range electron transfer (LRET) pathway based on surface-exposed catalytic tryptophan for the oxidation of compounds with a higher redox potential. Versatile peroxidase can directly oxidize many high-redox-potential dyes, whereas LiP requires various redox mediators to complete the same reaction. Verstaile peroxidase can also oxidize veratryl alcohol but has a much lower affinity for it as compared to lignin peroxidase (LiP)
physiological function
versatile peroxidase (VP) secreted by white-rot fungi is involved in the degradation of lignin within land ecosystems, with a broad substrate scope and minor requirements
additional information
versatile peroxidase (VP) has an access channel that is open to the solvent and where low-redox potential substrates are oxidized. In addition, VP has a superficial catalytic tryptophan that, in its active state, oxidizes both low-redox and more significantly, high redox potential substrates through a long-range electron transfer pathway to the heme, like lignin peroxidase. In the sagittal plane of the protein structure there is a small heme access channel where Mn2+ is oxidized to Mn3+, the latter acting as a diffusible oxidizer as also occurs in MnP (EC 1.11.1.13)
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). VPS1_PLEER
370
0
39046
Swiss-Prot
Secretory Pathway (Reliability: 2)
MOLECULAR WEIGHT
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
41000
x * 43000, SDS-PAGE, x * 41000, SDS-PAGE of deglycosylated enzyme
43000
43000
x * 43000, SDS-PAGE, x * 41000, SDS-PAGE of deglycosylated enzyme
SUBUNIT
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
?
x * 42000, wild-type enzyme, sequence calculation x * 51500, Aga2-fusion enzyme, sequence calculation, x * 50000-65000, recombinant hyperglycosylated Aga2-fusion enzyme, SDS-PAGE
POSTTRANSLATIONAL MODIFICATION
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
glycoprotein
glycoprotein
-
MP-1, up to 5% carbohydrate content, MP-2, up to 7% carbohydrate
CRYSTALLIZATION (Commentary)
ORGANISM
UNIPROT
LITERATURE
hanging drop vapor diffusion method, crystal structures of untreated versatile peroxidase (immediately after expression in Escherichia coli and in vitro reconstitution), native versatile peroxidase (treated with Mn2+), D175A variant, and wild-type verstile peroxidase (from Pleurotus eryngii culture)
mutant enzyme W164Y, sitting-drop vapor diffusion method, resolution 1.94 A
mutants E140G, P141G, K176G, and E140G/K176G, to 1.6, 2.0, 1.5, 1.7 and 2.35 A resolution, respectively
sitting drop vapor diffusion method, using 0.1 M sodium MES buffer at pH 6.5, 25% (w/v) PEG 4000 and 0.2 M MgCl2
PROTEIN VARIANTS
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
V160I/A260G
site-directed mutagenesis, the mutant MV4 shows increased dye degradation activity compared to the wild-type enzymen with Evans blue, Amido black 10B, and especially with Guinea green B
V160I/A260V
site-directed mutagenesis, the mutant MV5 shows increased dye degradation activity compared to the wild-type enzyme with Evans blue, and Guinea green B, but not with Amido black 10B
V160L/A260S
site-directed mutagenesis, the mutant MV1 shows increased dye degradation activity compared to the wild-type enzyme
V160Y
site-directed mutagenesis, the mutant MV2 shows increased dye degradation activity compared to the wild-type enzyme with Evans blue and Amido black 10B, but not with Guinea green B
V160Y/A260R
site-directed mutagenesis, the mutant MV3 shows increased dye degradation activity compared to the wild-type enzyme with Evans blue and Amido black 10B, but not with Guinea green B
A173R
kcat/KM for Mn2+ is 1.4fold lower than wild-type value, kcat/Km for veratryl alcohol is 1.4fold higher than wild-type value, kcat/Km for Reactive Black 5 is 1.3fold higher than wild-type value
D175A
kcat/KM for Mn2+ is 842fold lower than wild-type value, kcat/Km for veratryl alcohol is3.2 fold higher than wild-type value, kcat/Km for Reactive Black 5 is 1.8fold higher than wild-type value
E140G
substitution of bulky residue at the main heme access channel, kinetic analysis
E140G/K176G
variant attains catalytic efficiencies for oxidation of 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonate) at the heme channel similar to those of the exposed tryptophan site W164
E140G/P141G
substitution of bulky residue at the main heme access channel, kinetic analysis
E140G/P141G/K176G
variant attains catalytic efficiencies for oxidation of 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonate) at the heme channel similar to those of the exposed tryptophan site W164
E140G/P182S/Q229P
site-directed mutagenesis, the mutant BB-8 is active over an enhanced pH range compared to wild-type and displays strong hyperactivation after incubation at alkaline pH with a 3fold increase in activity, The active pH range for mutant BB-8 is expanded considerably for several substrates, including ABTS, sinapic acid and guaiacol. Consequently, BB-8 is active in the acid range (pH 3-4) and remarkably, in the pH interval from 5 to 9 in which the activity of the parental VP is negligible. The kinetic parameters measured for ABTS reveals enhanced catalytic efficiency at acid pH as result of increased affinity, which permits BB-8 to remain active at basic pHs. This effect is mostly attributed to the E140G mutation that enables the mutant to work with similar catalytic efficiency at pH 6 as the parental type at pH 3.5, due to the widening of the heme channel. Whilst the activity against Mn2+ is diminished due to the P182S mutation introduced close to this catalytic site, this mutation offers the first experimental insight into the role of the Mn2+ site for the direct (non-mediated) oxidation of ABTS at neutral/basic pH
E140G/W164S/K176G
variant attains catalytic efficiencies for oxidation of 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonate) at the heme channel similar to those of the exposed tryptophan site W164
E36A
kcat/KM for Mn2+ is 258fold lower than wild-type value, kcat/Km for veratryl alcohol is identical to wild-type value, kcat/Km for Reactive Black 5 is 1.2fold higher than wild-type value
E36A/E40A
kcat/KM for Mn2+ is 16000fold lower than wild-type value, kcat/Km for veratryl alcohol is 1.3fold higher than wild-type value, kcat/Km for Reactive Black 5 is 1.1fold higher than wild-type value
E36A/E40A/D175A
kcat for Mn2+ is 149fold lower than wild-type value, kcat/Km for veratryl alcohol is nearly identical to wild-type value, kcat/Km for Reactive Black 5 is 2fold higher than wild-type value
E36A/E40A/D175A/P327ter
kcat for Mn2+ is 149fold lower than wild-type value, kcat/Km for veratryl alcohol is 1.6fold lower than wild-type value, kcat/Km for Reactive Black 5 is 2.4fold higher than wild-type value
E36D
kcat/KM for Mn2+ is 77fold lower than wild-type value, kcat/Km for veratryl alcohol is 1.3fold higher than wild-type value, kcat/Km for Reactive Black 5 is 3.5fold higher than wild-type value
E37K/H39R/V160A/T184M/Q202L/D213A/G330R
site-directed mutagenesis of enzyme mutant E37K/V160A/T184M/Q202L introducing three additional stabilizing point mutations, the final mutant (2-1B) shows an overall enhancement of 8°C in kinetic thermostability compared to wild-type enzyme, the specific activity increases 2.5fold, and the expression rate is enhanced by 52 fold. The thermostability mutant 2-1B displays remarkable stability at alkaline pH (with a residual activity above 60% at pH 9 after 120 h of incubation), which is rather unusual in fungal peroxidases. Although 2-1B is stable at alkaline conditions, there is hardly any activity at its three catalytic sites at basic pH
E37K/V160A/T184M/Q202L
E40A
kcat/KM for Mn2+ is 1231fold lower than wild-type value, kcat/Km for veratryl alcohol is 1.2fold lower than wild-type value, kcat/Km for Reactive Black 5 is nearly identical to wild-type value
E40D
kcat/KM for Mn2+ is 54fold lower than wild-type value, kcat/Km for veratryl alcohol is 1.3fold lower than wild-type value, kcat/Km for Reactive Black 5 is 2.4fold higher than wild-type value
F142G
substitution of bulky residue at the main heme access channel, kinetic analysis
K176D
substitution of bulky residue at the main heme access channel, kinetic analysis
K176G
substitution of bulky residue at the main heme access channel, kinetic analysis
K215G
substitution of bulky residue at the main heme access channel, kinetic analysis
K215Q
substitution of bulky residue at the main heme access channel, kinetic analysis
N11D/G35K/E40K/T45A/S86R/P141A/F186L/T323I
site-directed mutagenesis
P141G
substitution of bulky residue at the main heme access channel, kinetic analysis
P76G
substitution of bulky residue at the main heme access channel, kinetic analysis
R257A/A260F
R257D
W164H
W164S
W164X
site-directed mutagenesis, no activity at the catalytic Trp164 at basic pH due to the fact that the reduction potential of the Trp164 radical decreases as the pH increases, hindering the oxidation of high-redox potential substrates at neutral/basic pH. The long-range electron transfer pathway from Trp164 to the heme is permanently cancelled out at pHs above pH 5.0, thereby diverting the oxidative route for the oxidation of low-redox potential substrates to the other two catalytic sites at the time that the oxidation of high-redox potential compounds is supressed
W164Y
site-directed mutagenesis, substitution of Trp-164 by a histidine, serine, or tyrosine residues causes a complete loss of activity on veratryl alcohol and Reactive Black 5
W164Y/R257A/A260F
site-directed mutagenesis, substitution of Trp-164 by a histidine, serine, or tyrosine residues causes a complete loss of activity on veratryl alcohol and Reactive Black 5
additional information
E37K/V160A/T184M/Q202L
mutant obtained by directed evolution, increase in activity and temperature stability
E37K/V160A/T184M/Q202L
site-directed mutagenesis, the secretion of the mutant enzyme from recombinant Saccharomyces cerevisiae improves 129fold compared to wild-type, yielding 22 mg/l of active, soluble and stable enzyme, overexpression in Pichia pastoris, the enzyme is secreted
R257A/A260F
43% decrease in efficiency for oxidizing veratryl alcohol
R257D
versatile peroxidase activity on Reactive Black 5 is eliminated by the R257D mutation
W164H
site-directed mutagenesis, substitution of Trp-164 by a histidine, serine, or tyrosine residues causes a complete loss of activity on veratryl alcohol and Reactive Black 5
W164S
site-directed mutagenesis, substitution of Trp-164 by a histidine, serine, or tyrosine residues causes a complete loss of activity on veratryl alcohol and Reactive Black 5
W164S
loss activity. Residue is responsible for high redox potential substrate oxidation
additional information
improvement of degradation of azo dyes by versatile peroxidase through saturation mutagenesis and application in form of VP-coated yeast cell walls. Via saturation mutagenesis, two amino acids in the catalytic tryptophan environment of the enzyme are altered (V160 and A260). Library screening with three azo dyes reveals that these two positions have a significant influence on substrate specificity. Enzyme variants with up to 16fold higher catalytic efficiency for different azo dyes are isolated and sequenced. Immobilization of versatile peroxidase on the surface of yeast cells in purified cell wall fragments after lysis, the enzyme VP embedded in the cell wall retains about 70 % of its initial activity after 10 cycles of dye degradation each lasting 12 h
additional information
-
improvement of degradation of azo dyes by versatile peroxidase through saturation mutagenesis and application in form of VP-coated yeast cell walls. Via saturation mutagenesis, two amino acids in the catalytic tryptophan environment of the enzyme are altered (V160 and A260). Library screening with three azo dyes reveals that these two positions have a significant influence on substrate specificity. Enzyme variants with up to 16fold higher catalytic efficiency for different azo dyes are isolated and sequenced. Immobilization of versatile peroxidase on the surface of yeast cells in purified cell wall fragments after lysis, the enzyme VP embedded in the cell wall retains about 70 % of its initial activity after 10 cycles of dye degradation each lasting 12 h
additional information
an engineered N-terminally truncated variant of mutant E37K/V160A/T184M/Q202L displays similar biochemical properties to those of the non-truncated counterpart in terms of kinetics, stability and spectroscopic features. Additional cycles of evolution raised the melting temperature by 8 degrees and significantly increased the enzyme's stability at alkaline pHs. In addition, the Km for H2O2 is enhanced up to 15fold while the catalytic efficiency is maintained, and there is an improvement in peroxide stability
additional information
-
an engineered N-terminally truncated variant of mutant E37K/V160A/T184M/Q202L displays similar biochemical properties to those of the non-truncated counterpart in terms of kinetics, stability and spectroscopic features. Additional cycles of evolution raised the melting temperature by 8 degrees and significantly increased the enzyme's stability at alkaline pHs. In addition, the Km for H2O2 is enhanced up to 15fold while the catalytic efficiency is maintained, and there is an improvement in peroxide stability
additional information
due to its broad substrate scope and minor requirements, versatile peroxidase is an extremely attractive blueprint to be designed by the directed evolution tool-box, directed evolution for functional expression in Saccharomyces cerevisiae, directed evolution for activity at alkaline pH, overview
TEMPERATURE STABILITY
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
STORAGE STABILITY
ORGANISM
UNIPROT
LITERATURE
PURIFICATION (Commentary)
ORGANISM
UNIPROT
LITERATURE
extraction and purification of Aga2-VP fusion enzyme proteins from Saccharomyces cerevisiae strain EBY100 cell walls by dialysis, and ultrafiltration, followed by ion exchange chromatography
Resource-Q column chromatography
CLONED (Commentary)
ORGANISM
UNIPROT
LITERATURE
generation of an enzyme saturation mutagenesis library by site-directed muztagenesis, recombinant expression of enzyme mutants in Saccharomyces cerevisiae strain EBY100, DNA and amino acid sequence determination and analysis.The recombinant enzyme shows microheterogeneity due to hyperglycosylation in Saccharomyces cerevisiae
all attempts to express a functional versatile peroxidase as soluble protein in Escherichia coli fail, recombinant enzyme expression and secretion from Saccharomyces cerevisiae, overexpression of enzyme mutant E37K/V160A/T184M/Q202L in Pichia pastoris
expression in Escherichia coli
expression in Escherichia coli fused to a thioredoxin-hexahistidine tag. Activity of the enzyme increases after removing the tag
RENATURED/Commentary
ORGANISM
UNIPROT
LITERATURE
using 0.15 M urea, 5 mM Ca2+, 0.02 mM hemin, a 4:1 oxidized-glutathione/reduced-glutathione ratio and 0.1 mg/ml protein at pH 9.5
-
using 0.16 M urea, 0.02 mM hemin, 5 mM CaCl2, 0.5 mM GSSG, 0.1 mM dithiothreitol, and 0.1 mg/ml protein in 20 mM Tris-HCl at pH 9.5
APPLICATION
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
environmental protection
the enzyme immobilized on yeast cell wall fragments can be used for longterm bioremediation of environments contaminated with azo dyes
degradation
versatile peroxidase presents particular interest due to its catalytic versatility including the degradation of compounds that other peroxidases are not able to oxidize directly, versatile peroxidase versatility permits its application in Mn3+-mediated or Mn-independent reactions on both low and high redox-potential aromatic substrates and dyes, versatile peroxidase can be used to reoxidize Mn-containing polyoxometalates, which are efficient oxidizers in paper pulp delignification
industry
-
the enzyme can be used in the treatment of industrial dye effluents. Paper pulp industries presently employ these ligninolytic enzymes for their pulp bleaching applications
synthesis
synthesis
-
expression of enzyme under control of the alcohol dehydrogenase promoter of Aspergillus nidulans in this host. Culture temperature of 19°C enhances the level of peroxidase 5.8-fold and reduces the effective proteolytic activity twofold giving a peroxidase activity of 466 units per l. Application of the same conditions to expression in Aspergillus niger does not result in improved peroxidase activity, while the effective proteolytic activity is increased
synthesis
-
enzyme is produced by solid-state fermentation, using the agricultural residue banana peel as growth medium. An enzymatic activity of 10800 U/l i.e. 36 U/g of substrate is detected after 18 days, whereas only 1800 U/l are reached by conventional submerged fermentation with glucose-based medium
REF.
AUTHORS
TITLE
JOURNAL
VOL.
PAGES
YEAR
ORGANISM (UNIPROT)
PUBMED ID
SOURCE
Martinez, M.J.; Ruiz-Duenas, F.J.; Guillen, F.; Martinez, A.T.
Purification and catalytic properties of two manganese peroxidase isoenzymes from Pleurotus eryngii
Eur. J. Biochem.
237
424-432
1996
Pleurotus eryngii
Ruiz-Duenas, F.J.; Guillen, F.; Camarero, S.; Perez-Boada, M.; Martinez, M.J.; Martinez, A.T.
Regulation of peroxidase transcript levels in liquid cultures of the ligninolytic fungus Pleurotus eryngii
Appl. Environ. Microbiol.
65
4458-4463
1999
Pleurotus eryngii
Ruiz-Duenas, F.J.; Martinez, M.J.; Martinez, A.T.
Heterologous expression of Pleurotus eryngii peroxidase confirms its ability to oxidize Mn(2+) and different aromatic substrates
Appl. Environ. Microbiol.
65
4705-4707
1999
Pleurotus eryngii (O94753)
Ruiz-Duenas, F.J.; Camarero, S.; Perez-Boada, M.; Martinez, M.J.; Martinez, A.T.
A new versatile peroxidase from Pleurotus
Biochem. Soc. Trans.
29
116-122
2001
Pleurotus eryngii
Gomez-Toribio, V.; Martinez, A.T.; Martinez, M.J.; Guillen, F.
Oxidation of hydroquinones by the versatile ligninolytic peroxidase from Pleurotus eryngii. H2O2 generation and the influence of Mn2+
Eur. J. Biochem.
268
4787-4793
2001
Pleurotus eryngii
Camarero, S.; Ruiz-Duenas, F.J.; Sarkar, S.; Martinez, M.J.; Martinez, A.T.
The cloning of a new peroxidase found in lignocellulose cultures of Pleurotus eryngii and sequence comparison with other fungal peroxidases
FEMS Microbiol. Lett.
191
37-43
2000
Pleurotus eryngii (Q9UVP6), Pleurotus eryngii
Camarero, S.; Sarkar, S.; Ruiz-Duenas, F.J.; Martinez, M.J.; Martinez, A.T.
Description of a versatile peroxidase involved in the natural degradation of lignin that has both manganese peroxidase and lignin peroxidase substrate interaction sites
J. Biol. Chem.
274
10324-10330
1999
Pleurotus eryngii
Pogni, R.; Baratto, M.C.; Teutloff, C.; Giansanti, S.; Ruiz-Duenas, F.J.; Choinowski, T.; Piontek, K.; Martinez, A.T.; Lendzian, F.; Basosi, R.
A tryptophan neutral radical in the oxidized state of versatile peroxidase from Pleurotus eryngii: a combined multifrequency EPR and density functional theory study
J. Biol. Chem.
281
9517-9526
2006
Pleurotus eryngii (O94753), Pleurotus eryngii
Banci, L.; Camarero, S.; Martinez, A.T.; Martinez, M.J.; Perez-Boada, M.; Pierattelli, R.; Ruiz-Duenas, F.J.
NMR study of manganese(II) binding by a new versatile peroxidase from the white-rot fungus Pleurotus eryngii
J. Biol. Inorg. Chem.
8
751-760
2003
Pleurotus eryngii
Perez-Boada, M.; Ruiz-Duenas, F.J.; Pogni, R.; Basosi, R.; Choinowski, T.; Martinez, M.J.; Piontek, K.; Martinez, A.T.
Versatile peroxidase oxidation of high redox potential aromatic compounds: site-directed mutagenesis, spectroscopic and crystallographic investigation of three long-range electron transfer pathways
J. Mol. Biol.
354
385-402
2005
Pleurotus eryngii
Ruiz-Duenas, F.J.; Morales, M.; Perez-Boada, M.; Choinowski, T.; Martinez, M.J.; Piontek, K.; Martinez, A.T.
Manganese oxidation site in Pleurotus eryngii versatile peroxidase: a site-directed mutagenesis, kinetic, and crystallographic study
Biochemistry
46
66-77
2007
Pleurotus eryngii (O94753), Pleurotus eryngii
Ruiz-Duenas, F.J.; Morales, M.; Mate, M.J.; Romero, A.; Martinez, M.J.; Smith, A.T.; Martinez, A.T.
Site-directed mutagenesis of the catalytic tryptophan environment in Pleurotus eryngii versatile peroxidase
Biochemistry
47
1685-1695
2008
Pleurotus eryngii (O94753), Pleurotus eryngii
Eibes, G.M.; Lu-Chau, T.A.; Ruiz-Duenas, F.J.; Feijoo, G.; Martinez, M.J.; Martinez, A.T.; Lema, J.M.
Effect of culture temperature on the heterologous expression of Pleurotus eryngii versatile peroxidase in Aspergillus hosts
Bioprocess Biosyst. Eng.
32
129-134
2009
Pleurotus eryngii
Ruiz-Duenas, F.J.; Pogni, R.; Morales, M.; Giansanti, S.; Mate, M.J.; Romero, A.; Martinez, M.J.; Basosi, R.; Martinez, A.T.
Protein radicals in fungal versatile peroxidase: catalytic tryptophan radical in both compound I and compound II and studies on W164Y, W164H, and W164S variants
J. Biol. Chem.
284
7986-7994
2009
Pleurotus eryngii (O94753), Pleurotus eryngii
Ruiz-Duenas, F.J.; Morales, M.; Garcia, E.; Miki, Y.; Martinez, M.J.; Martinez, A.T.
Substrate oxidation sites in versatile peroxidase and other basidiomycete peroxidases
J. Exp. Bot.
60
441-452
2009
Pleurotus eryngii (O94753)
Marques, G.; Gamelas, J.A.; Ruiz-Duenas, F.J.; del Rio, J.C.; Evtuguin, D.V.; Martinez, A.T.; Gutierrez, A.
Delignification of eucalypt kraft pulp with manganese-substituted polyoxometalate assisted by fungal versatile peroxidase
Biores. Technol.
101
5935-5940
2010
Pleurotus eryngii
Garcia-Ruiz, E.; Gonzalez-Perez, D.; Ruiz-Duenas, F.J.; Martinez, A.T.; Alcalde, M.
Directed evolution of a temperature-, peroxide- and alkaline pH-tolerant versatile peroxidase
Biochem. J.
441
487-498
2012
Pleurotus eryngii (O94753), Pleurotus eryngii
Bao, X.; Liu, A.; Lu, X.; Li, J.J.
Direct over-expression, characterization and H2O2 stability study of active Pleurotus eryngii versatile peroxidase in Escherichia coli
Biotechnol. Lett.
34
1537-1543
2012
Pleurotus eryngii (O94753), Pleurotus eryngii
Morales, M.; Mate, M.J.; Romero, A.; Martinez, M.J.; Martinez, A.T.; Ruiz-Duenas, F.J.
Two oxidation sites for low redox potential substrates: a directed mutagenesis, kinetic, and crystallographic study on Pleurotus eryngii versatile peroxidase
J. Biol. Chem.
287
41053-41067
2012
Pleurotus eryngii (O94753), Pleurotus eryngii
Gonzalez-Perez, D.; Garcia-Ruiz, E.; Ruiz-Duenas, F.; Martinez, A.; Alcalde, M.
Structural determinants of oxidative stabilization in an evolved versatile peroxidase
ACS Catal.
4
3891-3901
2014
Pleurotus eryngii (O94753)
-
Ravichandran, A.; Sridhar, M.
Insights into the mechanism of lignocellulose degradation by versatile peroxidases
Curr. Sci.
113
35-42
2017
Bjerkandera adusta, Pleurotus eryngii, Pleurotus ostreatus, Bjerkandera fumosa
-
Saez-Jimenez, V.; Fernandez-Fueyo, E.; Medrano, F.J.; Romero, A.; Martinez, A.T.; Ruiz-Duenas, F.J.
Improving the pH-stability of versatile peroxidase by comparative structural analysis with a naturally-stable manganese peroxidase
PLoS ONE
10
e0140984
2015
Pleurotus eryngii (O94753), Pleurotus eryngii
Palma, C.; Lloret, L.; Sepulveda, L.; Contreras, E.
Production of versatile peroxidase from Pleurotus eryngii by solid-state fermentation using agricultural residues and evaluation of its catalytic properties
Prep. Biochem. Biotechnol.
46
200-207
2016
Pleurotus eryngii
Gonzalez-Perez, D.; Alcalde, M.
The making of versatile peroxidase by directed evolution
Biocatal. Biotransform.
36
1-11
2018
Pleurotus eryngii (O94753)
-
Durdic, K.I.; Ostafe, R.; Durdevic Delmas, A.; Popovic, N.; Schillberg, S.; Fischer, R.; Prodanovic, R.
Saturation mutagenesis to improve the degradation of azo dyes by versatile peroxidase and application in form of VP-coated yeast cell walls
Enzyme Microb. Technol.
136
109509
2020
Pleurotus eryngii (Q9UVP6), Pleurotus eryngii
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