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Information on EC 1.14.13.50 - pentachlorophenol monooxygenase and Organism(s) Sphingobium chlorophenolicum and UniProt Accession P42535
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1.14.13.50 pentachlorophenol monooxygenaseIUBMB Comments
A flavoprotein (FAD). The enzyme displaces a diverse range of substituents from the 4-position of polyhalogenated phenols but requires that a halogen substituent be present at the 2-position . If C-4 carries a halogen substituent, reaction 1 is catalysed; if C-4 is unsubstituted, reaction 2 is catalysed.
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Word Map
- 1.14.13.50
-
pcp-degrading
- flavobacterium
-
sphingobium
- chlorophenolicum
- tetrachlorohydroquinone
-
bioremediation
- tetrachlorobenzoquinone
-
dehalogenation
- environmental protection
- arthrobacter
- degradation
The taxonomic range for the selected organisms is: Sphingobium chlorophenolicum
The expected taxonomic range for this enzyme is: Bacteria, Eukaryota
The expected taxonomic range for this enzyme is: Bacteria, Eukaryota
Reaction Schemes
Synonyms
pcp hydroxylase, pcp-4-monooxygenase, pentachlorophenol-4-monooxygenase, pentachlorophenol 4-monooxygenase, pcp4mo, pentachlorophenol hydroxylase, pcp 4-monooxygenase, phe4mo, pcp-monooxygenase, 
more
topSYNONYM 
ORGANISM
UNIPROT
COMMENTARY 
LITERATURE
dechlorinase, pentachlorophenol
-
-
-
-
oxygenase, pentachlorophenol 4-mono-
-
-
-
-
PCP hydroxylase
PcpB
pentachlorophenol 4-mono-oxygenase
-
-
-
-
pentachlorophenol 4-monooxygenase
pentachlorophenol dechlorinase
-
-
-
-
pentachlorophenol dehalogenase
-
-
-
-
pentachlorophenol hydroxylase
PCP hydroxylase
-
-
-
-
pentachlorophenol 4-monooxygenase
-
-
-
-
pentachlorophenol hydroxylase
-
-
-
-
REACTION
REACTION DIAGRAM
COMMENTARY
ORGANISM
UNIPROT
LITERATURE
pentachlorophenol + NADPH + H+ + O2 = 2,3,5,6-tetrachloro-1,4-benzoquinone + NADP+ + chloride + H2O
pentachlorophenol + NADPH + H+ + O2 = 2,3,5,6-tetrachloro-1,4-benzoquinone + NADP+ + chloride + H2O
benzene rings of Phe85, Phe87, Tyr397, as well as Tyr216 and the positive charge of Arg235 are important for activity and stabilization of the active site, reaction mechanism
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pentachlorophenol + NADPH + H+ + O2 = 2,3,5,6-tetrachloro-1,4-benzoquinone + NADP+ + chloride + H2O
direct reduction by NADPH of the flavin at the active site without requirement of intermediate formation
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pentachlorophenol + NADPH + H+ + O2 = 2,3,5,6-tetrachloro-1,4-benzoquinone + NADP+ + chloride + H2O
tetrachlorobenzoquinone reductase PcpD reaction mechanism
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REACTION TYPE
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
redox reaction
-
-
-
-
oxidation
-
-
-
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reduction
-
-
-
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dehalogenation
-
-
-
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PATHWAY SOURCE
PATHWAYS
KEGG
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SYSTEMATIC NAME
IUBMB Comments
pentachlorophenol,NADPH:oxygen oxidoreductase (hydroxylating, dechlorinating)
A flavoprotein (FAD). The enzyme displaces a diverse range of substituents from the 4-position of polyhalogenated phenols but requires that a halogen substituent be present at the 2-position [2]. If C-4 carries a halogen substituent, reaction 1 is catalysed; if C-4 is unsubstituted, reaction 2 is catalysed.
CAS REGISTRY NUMBER
COMMENTARY
124148-88-5
-
136111-57-4
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SUBSTRATE
PRODUCT
REACTION DIAGRAM
ORGANISM
UNIPROT
COMMENTARY
(Substrate)
(Substrate)
LITERATURE
(Substrate)
(Substrate)
COMMENTARY
(Product)
(Product)
LITERATURE
(Product)
(Product)
Reversibility
r=reversible
ir=irreversible
?=not specified
r=reversible
ir=irreversible
?=not specified
2,3,5,6-tetrachlorophenol + 2 NADPH + H+ + O2
2,3,5,6-tetrachloro-p-hydroquinone + 2 NADP+ + chloride + H2O
-
-
-
-
?
2,6-dichlorophenol + NADPH + H+ + O2
2,6-dichloro-p-hydroquinone + NADP+ + H2O
-
-
-
-
?
3,4,5-trichlorophenol + 2 NADPH + H+ + O2
3,5-dichloro-p-hydroquinone + 2 NADP+ + chloride + H2O
-
-
-
-
?
3,5-dichlorophenol + NADPH + H+ + O2
3,5-dichloro-p-hydroquinone + NADP+ + H2O
-
-
-
-
?
pentachlorophenol + 2 NADPH + H+ + O2
tetrachlorobenzoquinone + 2 NADP+ + chloride + H2O
-
-
-
-
?
pentachlorophenol + 2 NADPH + H+ + O2
tetrachlorohydroquinone + 2 NADP+ + chloride + H2O
-
product is not tetrachlorobenzoquinone as previously thought, ethyl acetate and glutathione can shift the redox-equilibrium in favor of tetrachlorobenzoquinone, room temperature (23-25°C), 50 mM phosphate buffer, pH 7.0, while with NADPH tetrachlorobenzoquinone is reduced to tetrachlorohydroquinone (with 0.5-1 mol NADPH/mol tetrachlorobenzoquinone 50-65% are reduced, with 2.5 mol or more almost complete reduction occurs), deionized distilled water, pH 6.8 (due to interference of phosphate or Tris-HCl buffer with the detection of tetrachlorohydroquinone)
-
?
pentachlorophenol + NADPH + H+ + O2
2,3,5,6-tetrachloro-1,4-benzoquinone + NADP+ + chloride + H2O
pentachlorophenol + NADPH + H+ + O2
2,3,5,6-tetrachlorohydroquinone + NADP+ + chloride + H2O
pentachlorophenol + NADPH + H+ + O2
2,3,5,6-tetrachloro-1,4-benzoquinone + NADP+ + chloride + H2O
degradation of pentachlorophenol
-
-
?
pentachlorophenol + NADPH + H+ + O2
2,3,5,6-tetrachloro-1,4-benzoquinone + NADP+ + chloride + H2O
the enzyme is an unusually slow flavin-dependent monooxygenase (kcat = 0.02/s) that converts pentachlorophenol to a highly reactive product, tetrachlorobenzoquinone. By stopped-flow spectroscopy, it is shown that the steps up to and including formation of tetrachlorobenzoquinone are rapid (5-30/s). Before products can be released from the active site, the strongly oxidizing tetrachlorobenzoquinone abstracts an electron from a donor at the active site, possibly a cysteine residue, resulting in an off-pathway diradical state that only slowly reverts to an intermediate capable of completing the catalytic cycle
-
-
?
pentachlorophenol + NADPH + H+ + O2
2,3,5,6-tetrachlorohydroquinone + NADP+ + chloride + H2O
-
-
-
-
?
pentachlorophenol + NADPH + H+ + O2
2,3,5,6-tetrachlorohydroquinone + NADP+ + chloride + H2O
-
initial enzyme in the PCP catabolic pathway of numerous sphingomonads, PCP-4-monooxygenase (PcpB), catalyzes the parahydroxylation of PCP to tetrachlorohydroquinone, pentachlorophenol degradation pathway, overview
-
-
?
pentachlorophenol + NADPH + H+ + O2
tetrachlorobenzoquinone + NADP+ + chloride + H2O
-
-
-
-
ir
pentachlorophenol + NADPH + H+ + O2
tetrachlorobenzoquinone + NADP+ + chloride + H2O
-
first step of pentachlorophenol degradation pathway
-
-
ir
pentachlorophenol + NADPH + H+ + O2
tetrachlorohydroquinone + NADP+ + chloride + H2O
-
-
-
-
?
pentachlorophenol + NADPH + H+ + O2
tetrachlorohydroquinone + NADP+ + chloride + H2O
-
pentachlorophenol degradation
-
-
?
tetrachlorobenzoquinone + NADPH
tetrachlorohydroquinone + NADP+
-
-
-
-
?
tetrachlorobenzoquinone + NADPH
tetrachlorohydroquinone + NADP+
-
second step in pentachlorophenol degradation, physiological role in association with pentachloro monooxygenase, overview
-
-
?
additional information
?
-
-
enzyme expression in response to pentachlorophenol hydroxylase PcpB
-
-
?
additional information
?
-
-
lateral gene transfer and the origin of the pentachlorophenol pathway, overview
-
-
?
additional information
?
-
-
enzyme exhibits substantial uncoupling. The C4a-hydroxyflavin intermediate, instead of hydroxylating the substrate, can decompose to produce H2O2 in a futile cycle that consumes NADPH. The extent of uncoupling varies from 0 to 100% with different substrates. The extent of uncoupling is increased by the presence of bulky substituents at position 3, 4, or 5 and decreased by the presence of a chlorine in the ortho position. The effectiveness of pentachlorophenol hydroxylase is additionally hindered by its promiscuous activity with tetrachlorohydroquinone, a downstream metabolite in the degradation pathway. The conversion of tetrachlorohydroquinone to tetrachlorobenzoquinone reverses flux through the pathway. Substantial uncoupling also occurs during the reaction with tetrachlorohydroquinone
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-
?
NATURAL SUBSTRATE
NATURAL PRODUCT
REACTION DIAGRAM
ORGANISM
UNIPROT
COMMENTARY
(Substrate)
(Substrate)
LITERATURE
(Substrate)
(Substrate)
COMMENTARY
(Product)
(Product)
LITERATURE
(Product)
(Product)
REVERSIBILITY
r=reversible
ir=irreversible
?=not specified
r=reversible
ir=irreversible
?=not specified
pentachlorophenol + 2 NADPH + H+ + O2
tetrachlorohydroquinone + 2 NADP+ + chloride + H2O
-
product is not tetrachlorobenzoquinone as previously thought, ethyl acetate and glutathione can shift the redox-equilibrium in favor of tetrachlorobenzoquinone, room temperature (23-25°C), 50 mM phosphate buffer, pH 7.0, while with NADPH tetrachlorobenzoquinone is reduced to tetrachlorohydroquinone (with 0.5-1 mol NADPH/mol tetrachlorobenzoquinone 50-65% are reduced, with 2.5 mol or more almost complete reduction occurs), deionized distilled water, pH 6.8 (due to interference of phosphate or Tris-HCl buffer with the detection of tetrachlorohydroquinone)
-
?
pentachlorophenol + NADPH + H+ + O2
2,3,5,6-tetrachloro-1,4-benzoquinone + NADP+ + chloride + H2O
degradation of pentachlorophenol
-
-
?
pentachlorophenol + NADPH + H+ + O2
2,3,5,6-tetrachlorohydroquinone + NADP+ + chloride + H2O
-
initial enzyme in the PCP catabolic pathway of numerous sphingomonads, PCP-4-monooxygenase (PcpB), catalyzes the parahydroxylation of PCP to tetrachlorohydroquinone, pentachlorophenol degradation pathway, overview
-
-
?
pentachlorophenol + NADPH + H+ + O2
tetrachlorohydroquinone + NADP+ + chloride + H2O
-
pentachlorophenol degradation
-
-
?
tetrachlorobenzoquinone + NADPH
tetrachlorohydroquinone + NADP+
-
second step in pentachlorophenol degradation, physiological role in association with pentachloro monooxygenase, overview
-
-
?
pentachlorophenol + NADPH + H+ + O2
tetrachlorobenzoquinone + NADP+ + chloride + H2O
-
-
-
-
ir
pentachlorophenol + NADPH + H+ + O2
tetrachlorobenzoquinone + NADP+ + chloride + H2O
-
first step of pentachlorophenol degradation pathway
-
-
ir
additional information
?
-
-
enzyme expression in response to pentachlorophenol hydroxylase PcpB
-
-
?
additional information
?
-
-
lateral gene transfer and the origin of the pentachlorophenol pathway, overview
-
-
?
COFACTOR
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
INHIBITOR
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
tetrachlorobenzoquinone
-
inactivates the enzyme, feedback control
ACTIVATING COMPOUND
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
additional information
-
pentachlorphenol induces enzyme expression
-
KM VALUE [mM]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
0.0005
Pentachlorophenol
-
pH 7.0, 25°C, recombinant wild-type enzyme
0.0008
Pentachlorophenol
-
pH 7.0, 25°C, recombinant mutant R235K
0.0029
Pentachlorophenol
-
pH 7.0, 25°C, recombinant mutant Y39F
0.0045
Pentachlorophenol
-
pH 7.0, 25°C, recombinant mutant Y216F
TURNOVER NUMBER [1/s]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
9300
Pentachlorophenol
-
pH 7.0, 25°C, recombinant wild-type enzyme
15200
Pentachlorophenol
-
pH 7.0, 25°C, recombinant mutant R235K
kcat/KM VALUE [1/mMs-1]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
SPECIFIC ACTIVITY [µmol/min/mg]
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
pH OPTIMUM
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
TEMPERATURE OPTIMUM
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
ORGANISM
COMMENTARY
LITERATURE
UNIPROT
SEQUENCE DB
SOURCE
LOCALIZATION
ORGANISM
UNIPROT
COMMENTARY
GeneOntology No.
LITERATURE
SOURCE
GENERAL INFORMATION
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
metabolism
metabolism
hydroxylation of pentachlorophenol in the pentachlorophenol biodegradation pathway
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). MOLECULAR WEIGHT
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
SUBUNIT
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
additional information
?
-
x * 62600-62750, about, native wild-type enzyme, and recombinant wild-type and mutant enzymes, overview
additional information
-
homology modeling and model building of three-dimensional structure
PROTEIN VARIANTS
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
F223A
-
site-directed mutagenesis, increased activity compared to the wild-type enzyme
F225A
-
site-directed mutagenesis, increased activity compared to the wild-type enzyme
F85A
F87A
-
site-directed mutagenesis, mutant enzyme is nearly not soluble in contrast to the wild-type, highly reduced activity compared to the wild-type enzyme
moe
-
construction of an allelic exchange mutant and a pcpD knockout mutant strains, the latter show reduced activity at high pentachlorophenol concentrations, not at low concentrations
R235A
-
site-directed mutagenesis, determination of molecular weight and comparison to wild-type enzyme
R235E
R235K
Y216A
Y216F
-
site-directed mutagenesis, 60% decreased activity compared to the wild-type enzyme
Y397A
Y397F
F85A
-
site-directed mutagenesis, 60% decreased activity compared to the wild-type enzyme
F85A
-
site-directed mutagenesis, determination of molecular weight and comparison to wild-type enzyme
R235E
-
site-directed mutagenesis, reduced activity compared to the wild-type enzyme
R235E
-
site-directed mutagenesis, determination of molecular weight and comparison to wild-type enzyme
R235K
-
site-directed mutagenesis, increased activity compared to the wild-type enzyme
R235K
-
site-directed mutagenesis, determination of molecular weight and comparison to wild-type enzyme
R235K
-
site-directed mutagenesis, mutant enzyme is nearly not soluble in contrast to the wild-type, reduced activity compared to the wild-type enzyme
Y216A
-
site-directed mutagenesis, highly reduced activity compared to the wild-type enzyme
Y216A
-
site-directed mutagenesis, determination of molecular weight and comparison to wild-type enzyme
Y397A
-
site-directed mutagenesis, highly reduced activity compared to the wild-type enzyme
Y397A
-
site-directed mutagenesis, determination of molecular weight and comparison to wild-type enzyme
Y397F
-
site-directed mutagenesis, 60% increased activity compared to the wild-type enzyme
Y397F
-
site-directed mutagenesis, determination of molecular weight and comparison to wild-type enzyme
PURIFICATION (Commentary)
ORGANISM
UNIPROT
LITERATURE
frozen pellet of cell culture is suspended in lysis buffer (50 mM Tris-HCl, pH 7.7, 025 mM phenylmethanesulfonyl fluoride, 1 microM pepstatin A, lysozyme), cells disrupted by sonication, centrifuged, supernatant purified by affinity chromatography, mixed with Ni-NTA agarose medium, washed, packed into a column, eluted, active fractions combined and stored in buffer (20 mM phosphate buffer, pH 7.0, 0.25 mM phenylmethanesulfonyl fluoride, 5% glycerol)
recombinant enzyme from Pseudomonas aeruginosa to over 95% homogeneity by nickel affinity chromatography
-
recombinant wild-type and mutant enzymes from Escherichia coli
-
recombinant wild-type and mutant enzymes from Escherichia coli strain Bl21(DE3)
-
CLONED (Commentary)
ORGANISM
UNIPROT
LITERATURE
DNA and amino acid sequence determination and analysis, expression of His-tagged wild-type and mutant enzymes in Escherichia coli
-
Escherichia coli M15 overexpressing recombinant hexahistidine-tagged pentachlorophenol 4-monooxygenase
expression of wild-type and mutant enzymes in Escherichia coli strain BL21(DE3)
-
gene pcpB, DNA and amino acid sequence analysis, phylogenetic analysis
-
gene pcpD, expression as C-terminally His12-tagged protein in Pseudomonas aeruginosa strain PAO1-Lac
-
APPLICATION
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
degradation
pentachlorophenol is a chloroaromatic pesticide used to protect lumber, and an environmental pollutant, Sphingobium chlorophenolicum is a microorganism that can degrade the agent to 3-oxoadipate using 5 catalytic enzymes, pentachlorophenol 4-monooxygenase catalyzes the first and rate-limiting step
environmental protection
bioaugmentation of groundwater with known Sphingobium chlorophenolicum L-1, amendment of nutrients, and air sparging result in an enhanced degradation of pentachlorophenol and hence bioremediation of PCP-contaminated groundwater. The amendments to the site undergoing air sparging may result in more effective and less time-consuming bioremediation of pentachlorophenol-contaminated groundwater without adding significantly high cost and labor
additional information
-
the enzyme can be used for development of a model system for the study of recent evolution of catabolic pathways among bacteria that degrade xenobiotic molecules introduced into the environment during the recent past
REF.
AUTHORS
TITLE
JOURNAL
VOL.
PAGES
YEAR
ORGANISM (UNIPROT)
PUBMED ID
SOURCE
Nakamura, T.; Motoyama, T.; Hirono, S.; Yamaguchi, I.
Identification, characterization, and site-directed mutagenesis of recombinant pentachlorophenol 4-monooxygenase
Biochim. Biophys. Acta
1700
151-159
2004
Sphingobium chlorophenolicum
Nakamura, T.; Motoyama, T.; Hirokawa, T.; Hirono, S.; Yamaguchi, I.
Computer-aided modeling of pentachlorophenol 4-monooxygenase and site-directed mutagenesis of its active site
Chem. Pharm. Bull.
51
1293-1298
2003
Sphingobium chlorophenolicum
Dai, M.; Rogers, J.B.; Warner, J.R.; Copley, S.D.
A previously unrecognized step in pentachlorophenol degradation in Sphingobium chlorophenolicum is catalyzed by tetrachlorobenzoquinone reductase (PcpD)
J. Bacteriol.
185
302-310
2003
Sphingobium chlorophenolicum
Crawford, R.L.; Jung, C.M.; Strap, J.L.
The recent evolution of pentachlorophenol (PCP)-4-monooxygenase (PcpB) and associated pathways for bacterial degradation of PCP
Biodegradation
18
525-539
2006
Mycolicibacterium chlorophenolicum, Sphingobium chlorophenolicum, Sphingomonas sp.
Su, Y.; Chen, L.; Bandy, B.; Yang, J.
The Catalytic Product of Pentachlorophenol 4-Monooxygenase is Tetra-chlorohydroquinone rather than Tetrachlorobenzoquinone
Open Microbiol. J.
2
100-106
2008
Sphingobium chlorophenolicum (P42535), Sphingobium chlorophenolicum
Hlouchova, K.; Rudolph, J.; Pietari, J.M.; Behlen, L.S.; Copley, S.D.
Pentachlorophenol hydroxylase, a poorly functioning enzyme required for degradation of pentachlorophenol by Sphingobium chlorophenolicum
Biochemistry
51
3848-3860
2012
Sphingobium chlorophenolicum
Rudolph, J.; Erbse, A.H.; Behlen, L.S.; Copley, S.D.
A radical intermediate in the conversion of pentachlorophenol to tetrachlorohydroquinone by Sphingobium chlorophenolicum
Biochemistry
53
6539-6549
2014
Sphingobium chlorophenolicum (P42535)
Joshi, V.; Prewitt, M.; Ma, D.; Borazjani, H.
Enhanced remediation of pentachlorophenol (PCP)-contaminated groundwater by bioaugmentation with known PCP-degrading bacteria
Bioremediat. J.
19
160-170
2015
Sphingobium chlorophenolicum (P42535), Sphingobium chlorophenolicum L-1 (P42535)
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