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EC Tree
The taxonomic range for the selected organisms is: Pseudomonas knackmussii The expected taxonomic range for this enzyme is: Bacteria, Eukaryota, Archaea
Synonyms
dienelactone hydrolase, trans-dienelactone hydrolase, carboxymethylenebutenolidase, trans-dlh, carboxymethylenebutenolidase homolog, 4-carboxymethylenebut-2-en-4-olide hydrolase, cis-dienelactone hydrolase,
more
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4-carboxymethylenebut-2-en-4-olide hydrolase
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carboxymethylene butenolidase
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carboxymethylene butenolide hydrolase
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dienelactone hydrolase
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-
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maleylacetate enol-lactonase
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-
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4-carboxymethylenebut-2-en-4-olide + H2O = 4-oxohex-2-enedioate
4-carboxymethylenebut-2-en-4-olide + H2O = 4-oxohex-2-enedioate
mechanism
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4-carboxymethylenebut-2-en-4-olide + H2O = 4-oxohex-2-enedioate
esterase activity on chromophoric ester substrate, p-nitrophenyl acetate and the synthetic active-site titrant, trans-cinnamoyl imidazole
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4-carboxymethylenebut-2-en-4-olide + H2O = 4-oxohex-2-enedioate
the catalytic triad Cys123, His202 and Asp171 is a hybrid of the standard triads found in cysteine: Cys-His-Asn and serine: Ser-His-Asp proteases
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4-carboxymethylenebut-2-en-4-olide + H2O = 4-oxohex-2-enedioate
similar catalytic triads are found in alpha/beta hydrolase fold enzymes: DLH is the simplest one
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4-carboxymethylenebut-2-en-4-olide + H2O = 4-oxohex-2-enedioate
the active-site Cys123 residue is part of a triad of residues consisting of Cys123, His202 and Asp171, and is reminiscent of the serine/cysteine proteases
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hydrolysis of carboxylic ester
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4-carboxymethylenebut-2-en-4-olide lactonohydrolase
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4-nitrophenyl acetate + H2O
4-nitrophenol + acetate
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-
-
?
4-carboxymethylenebut-2-en-4-olide + H2O
?
4-fluoro-muconolactone + H2O
?
-
-
-
-
?
cis-4-carboxymethylenebut-2-en-4-olide + H2O
4-oxohex-2-enedioate
p-nitrophenyl acetate + H2O
p-nitrophenol + acetate
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esterase activity
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-
?
protoanemonin + H2O
cis-acetylacrylate
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-
-
?
trans-4-carboxymethylenebut-2-en-4-olide + H2O
4-oxohex-2-enedioate
trans-cinnamoyl imidazole + H2O
trans-cinnamate + imidazole
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esterase activity
-
-
?
additional information
?
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4-carboxymethylenebut-2-en-4-olide + H2O
?
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the enzyme is one of the enzymes of the halocatechol branch of the beta-ketoadipate pathway, a complex set of catabolic reactions used by bacteria for utilization of aromatic compounds
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?
4-carboxymethylenebut-2-en-4-olide + H2O
?
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dienelactone hydrolase catalyzes a step in the metabolic conversion of chlorocatechols to beta-ketoadipate
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-
?
cis-4-carboxymethylenebut-2-en-4-olide + H2O
4-oxohex-2-enedioate
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-
-
?
cis-4-carboxymethylenebut-2-en-4-olide + H2O
4-oxohex-2-enedioate
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-
-
?
cis-4-carboxymethylenebut-2-en-4-olide + H2O
4-oxohex-2-enedioate
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-
-
?
cis-4-carboxymethylenebut-2-en-4-olide + H2O
4-oxohex-2-enedioate
-
-
-
?
cis-4-carboxymethylenebut-2-en-4-olide + H2O
4-oxohex-2-enedioate
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-
-
?
cis-4-carboxymethylenebut-2-en-4-olide + H2O
4-oxohex-2-enedioate
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-
-
?
cis-4-carboxymethylenebut-2-en-4-olide + H2O
4-oxohex-2-enedioate
-
-
-
?
cis-4-carboxymethylenebut-2-en-4-olide + H2O
4-oxohex-2-enedioate
-
-
-
?
cis-4-carboxymethylenebut-2-en-4-olide + H2O
4-oxohex-2-enedioate
-
-
-
?
cis-4-carboxymethylenebut-2-en-4-olide + H2O
4-oxohex-2-enedioate
-
-
-
?
cis-4-carboxymethylenebut-2-en-4-olide + H2O
4-oxohex-2-enedioate
-
-
-
?
cis-4-carboxymethylenebut-2-en-4-olide + H2O
4-oxohex-2-enedioate
-
-
-
?
cis-4-carboxymethylenebut-2-en-4-olide + H2O
4-oxohex-2-enedioate
-
-
-
ir
trans-4-carboxymethylenebut-2-en-4-olide + H2O
4-oxohex-2-enedioate
-
-
-
?
trans-4-carboxymethylenebut-2-en-4-olide + H2O
4-oxohex-2-enedioate
-
-
-
?
trans-4-carboxymethylenebut-2-en-4-olide + H2O
4-oxohex-2-enedioate
-
-
-
?
trans-4-carboxymethylenebut-2-en-4-olide + H2O
4-oxohex-2-enedioate
-
-
-
?
trans-4-carboxymethylenebut-2-en-4-olide + H2O
4-oxohex-2-enedioate
-
-
-
?
trans-4-carboxymethylenebut-2-en-4-olide + H2O
4-oxohex-2-enedioate
-
-
-
?
trans-4-carboxymethylenebut-2-en-4-olide + H2O
4-oxohex-2-enedioate
-
-
-
?
trans-4-carboxymethylenebut-2-en-4-olide + H2O
4-oxohex-2-enedioate
-
-
-
?
trans-4-carboxymethylenebut-2-en-4-olide + H2O
4-oxohex-2-enedioate
-
-
-
?
trans-4-carboxymethylenebut-2-en-4-olide + H2O
4-oxohex-2-enedioate
-
-
-
?
trans-4-carboxymethylenebut-2-en-4-olide + H2O
4-oxohex-2-enedioate
-
-
-
?
trans-4-carboxymethylenebut-2-en-4-olide + H2O
4-oxohex-2-enedioate
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-
-
?
trans-4-carboxymethylenebut-2-en-4-olide + H2O
4-oxohex-2-enedioate
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-
-
ir
additional information
?
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muconolactone is not hydrolyzed to a significant extent
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-
?
additional information
?
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DLH unable to hydrolyze lactams which closely resemble its substrate, dienelactam is not a substrate, DLH has lost the ability to hydrolyze amides
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-
?
additional information
?
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substrate specificities differentiate among three types of this activity. Type I: extracts of Alcaligenes eutrophus 335, Alcaligenes eutrophus H16, Alcaligenes eutrophus JMP222 and Alcaligenes strain A7 convert trans-4-carboxymethylenebut-2-en-4-olide, trans-dienelactone, much faster than cis-isomer, type II: enzyme of Pseudomonas cepacia converts cis-dienelactone much faster, type III: enzyme of Alcaligenes eutrophus JMP134 and Pseudomonas strain B13 hydrolyze both dienelactones
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-
?
additional information
?
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substrate specificities differentiate among three types of this activity. Type I: extracts of Alcaligenes eutrophus 335, Alcaligenes eutrophus H16, Alcaligenes eutrophus JMP222 and Alcaligenes strain A7 convert trans-4-carboxymethylenebut-2-en-4-olide, trans-dienelactone, much faster than cis-isomer, type II: enzyme of Pseudomonas cepacia converts cis-dienelactone much faster, type III: enzyme of Alcaligenes eutrophus JMP134 and Pseudomonas strain B13 hydrolyze both dienelactones
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?
additional information
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substrate binding studies
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?
additional information
?
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it seems likely, that dienelactone hydrolase has the additional function of detoxification of minor amounts of protoanemonin that may be formed during chloroaromatic degradation
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?
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4-carboxymethylenebut-2-en-4-olide + H2O
?
additional information
?
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it seems likely, that dienelactone hydrolase has the additional function of detoxification of minor amounts of protoanemonin that may be formed during chloroaromatic degradation
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-
?
4-carboxymethylenebut-2-en-4-olide + H2O
?
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the enzyme is one of the enzymes of the halocatechol branch of the beta-ketoadipate pathway, a complex set of catabolic reactions used by bacteria for utilization of aromatic compounds
-
-
?
4-carboxymethylenebut-2-en-4-olide + H2O
?
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dienelactone hydrolase catalyzes a step in the metabolic conversion of chlorocatechols to beta-ketoadipate
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-
?
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additional information
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no requirement
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protoanemonin
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competitive inhibitor of the transformation of cis-dienelactone
additional information
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no inactivation by iodoacetamide, iodoacetate, chloroacetamide
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dienelactam
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competitive inhibitor
dienelactam
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inhibitor binding studies
p-chloromercuribenzoate
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p-chloromercuribenzoate
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inactivation completely reversed by dithiothreitol
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additional information
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no requirement
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0.012 - 0.014
4-nitrophenyl acetate
0.381 - 0.4
cis-4-carboxymethylenebut-2-en-4-olide
0.015 - 9.9
trans-4-Carboxymethylenebut-2-en-4-olide
0.012
4-nitrophenyl acetate
mutant Q35H/F38L/C123S/Y145C/N154D/E199G/S208G/G211D, pH 7.5, 25°C
0.013
4-nitrophenyl acetate
mutant Q35H/F38L/C123S/Y137C/Y145C/E199G/S208G/G211D, pH 7.5, 25°C
0.013
4-nitrophenyl acetate
mutant Q35H/F38L/C123S/Y145C/E199G/S208G/G211D, pH 7.5, 25°C
0.013
4-nitrophenyl acetate
mutant Q35H/F38L/Q110L/C123S/Y145C/N154D/E199G/S208G/G211D, pH 7.5, 25°C
0.014
4-nitrophenyl acetate
mutant Q35H/F38L/Q110L/C123S/Y137C/Y145C/N154D/E199G/S208G/G211D, pH 7.5, 25°C
0.014
4-nitrophenyl acetate
mutant Q35H/F38L/Q110L/C123S/Y145C/E199G/S208G/G211D, pH 7.5, 25°C
0.381
cis-4-carboxymethylenebut-2-en-4-olide
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-
0.4
cis-4-carboxymethylenebut-2-en-4-olide
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-
0.015
trans-4-Carboxymethylenebut-2-en-4-olide
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-
0.17
trans-4-Carboxymethylenebut-2-en-4-olide
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-
0.22
trans-4-Carboxymethylenebut-2-en-4-olide
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C60S DLH
9.9
trans-4-Carboxymethylenebut-2-en-4-olide
-
C123S DLH
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0.57 - 0.61
4-nitrophenyl acetate
30
4-carboxymethylenebut-2-en-4-olide
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-
260
cis-4-carboxymethylenebut-2-en-4-olide
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-
2.67 - 15
trans-4-Carboxymethylenebut-2-en-4-olide
0.57
4-nitrophenyl acetate
mutant Q35H/F38L/C123S/Y137C/Y145C/E199G/S208G/G211D, pH 7.5, 25°C
0.58
4-nitrophenyl acetate
mutant Q35H/F38L/C123S/Y145C/N154D/E199G/S208G/G211D, pH 7.5, 25°C
0.58
4-nitrophenyl acetate
mutant Q35H/F38L/Q110L/C123S/Y145C/N154D/E199G/S208G/G211D, pH 7.5, 25°C
0.59
4-nitrophenyl acetate
mutant Q35H/F38L/C123S/Y145C/E199G/S208G/G211D, pH 7.5, 25°C
0.6
4-nitrophenyl acetate
mutant Q35H/F38L/Q110L/C123S/Y145C/E199G/S208G/G211D, pH 7.5, 25°C
0.61
4-nitrophenyl acetate
mutant Q35H/F38L/Q110L/C123S/Y137C/Y145C/N154D/E199G/S208G/G211D, pH 7.5, 25°C
2.67
trans-4-Carboxymethylenebut-2-en-4-olide
-
C123S DLH
9.17
trans-4-Carboxymethylenebut-2-en-4-olide
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-
15
trans-4-Carboxymethylenebut-2-en-4-olide
-
C60S DLH
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43 - 49
4-nitrophenyl acetate
43
4-nitrophenyl acetate
mutant Q35H/F38L/C123S/Y137C/Y145C/E199G/S208G/G211D, pH 7.5, 25°C
43
4-nitrophenyl acetate
mutant Q35H/F38L/Q110L/C123S/Y145C/E199G/S208G/G211D, pH 7.5, 25°C
44
4-nitrophenyl acetate
mutant Q35H/F38L/Q110L/C123S/Y145C/N154D/E199G/S208G/G211D, pH 7.5, 25°C
45
4-nitrophenyl acetate
mutant Q35H/F38L/Q110L/C123S/Y137C/Y145C/N154D/E199G/S208G/G211D, pH 7.5, 25°C
46
4-nitrophenyl acetate
mutant Q35H/F38L/C123S/Y145C/E199G/S208G/G211D, pH 7.5, 25°C
49
4-nitrophenyl acetate
mutant Q35H/F38L/C123S/Y145C/N154D/E199G/S208G/G211D, pH 7.5, 25°C
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0.58
-
substrate protoanemonin
58.8
-
substrate cis-4-carboxymethylenebut-2-en-4-olide
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UniProt
brenda
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brenda
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25400 - 25800
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predicted from DNA sequences
25490
-
calculated from nucleotide sequence of the clcD gene product
28500
-
gel filtration and nondenaturing PAGE
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monomer
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1 * 28000-29000, SDS-PAGE
monomer
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1* 28000, SDS-PAGE with mercaptoethanol
monomer
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alpha,beta protein
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inhibitor, dienelactam, bound to DLH C123S
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refined crystal structure at 1.8 A resolution
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refined crystal structure of DLH at 1.8 A, C123S DLH at 2.2 A, C123A at 2.0 A resolution
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x-ray crystallographic structure at 2.8 A resolution
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Q35H/F38L/C123S/Y137C/Y145C/E199G/S208G/G211D
mutations do not influence the kinetic properties
Q35H/F38L/C123S/Y145C/E199G/S208G/G211D
mutations do not influence the kinetic properties
Q35H/F38L/C123S/Y145C/N154D/E199G/S208G/G211D
mutations do not influence the kinetic properties
Q35H/F38L/Q110L/C123S/Y137C/Y145C/N154D/E199G/S208G/G211D
mutations do not influence the kinetic properties
Q35H/F38L/Q110L/C123S/Y145C/E199G/S208G/G211D
mutations do not influence the kinetic properties
Q35H/F38L/Q110L/C123S/Y145C/N154D/E199G/S208G/G211D
mutations do not influence the kinetic properties
C60S
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no reduction in activity
D171N
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no detectable activity
E36A
-
no detectable activity
additional information
the surface mutations Q110L, Y137C and N154D do not influence the kinetic properties. Mutations Q110L and N154D have stabilizing effects, the Y137C mutation alone is destabilizing. The three mutations together increase the melting temperature by 3.4 degrees
C123S
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-
C123S
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burst kinetics with p-nitrophenyl acetate, 10% as active as DLH
C123S
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maximal activity 20% that of the wild type protein
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51.5
melting temperature, mutant Q35H/F38L/C123S/Y145C/E199G/S208G/G211D
54.9
melting temperature, mutant Q35H/F38L/Q110L/C123S/Y137C/Y145C/N154D/E199G/S208G/G211D
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4°C, 20 mM Tris-HCl, pH 7.4, 10 micromol dithiothreitol, stable for over 6 months
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4°C, converted to microcrystals, ammonium sulfate to 30% saturation, stable for more than 1 year
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3-chlorobenzoate-grown cells
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expression in Escherichia coli and Pseudomonas putida
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Schmidt, E.; Knackmuss, H.J.
Chemical structure and biodegradability of halogenated aromatic compounds. Conversion of chlorinated muconic acids into maleoylacetic acid
Biochem. J.
192
339-347
1980
Pseudomonas knackmussii
brenda
Ngai, K.L.; Schlmann, M.; Knackmuss, H.J.; Ornston, L.N.
Dienelactone hydrolase from Pseudomonas sp. strain B13
J. Bacteriol.
169
699-703
1987
Pseudomonas knackmussii
brenda
Ollis, D.L.; Ngai, K.L.
Crystallization and preliminary x-ray crystallographic data of dienelactone hydrolase from Pseudomonas sp. B13
J. Biol. Chem.
260
9818-9819
1985
Burkholderia cepacia, Cupriavidus necator, Pseudomonas knackmussii
brenda
Bruckmann, M.; Blasco, R.; Timmis, K.N.; Pieper, D.H.
Detoxification of protoanemonin by dienelactone hydrolase
J. Bacteriol.
180
400-402
1998
Burkholderia cepacia, Pseudomonas knackmussii, Pseudomonas putida
brenda
Pathak, D.; Ollis, D.
Refined structure of dienelactone hydrolase at 1.8 A
J. Mol. Biol.
214
497-525
1990
Pseudomonas knackmussii
brenda
Pathak, D.; Ashley, G.; Ollis, D.
Thiol protease-like active site found in the enzyme dienelactone hydrolase: localization using biochemical, genetic, and structural tools
Proteins Struct. Funct. Genet.
9
267-279
1991
Pseudomonas knackmussii
brenda
Cheah, E.; Austin, C.; Ashley, G.W.; Ollis, D.
Substrate-induced activation of dienelactone hydrolase: an enzyme with a naturally occurring Cys-His-Asp triad
Protein Eng.
6
575-583
1993
Pseudomonas knackmussii
brenda
Cheah, E.; Ashley, G.W.; Gary, J.; Ollis, D.
Catalysis by dienelactone hydrolase: a variation on the protease mechanism
Proteins Struct. Funct. Genet.
16
64-78
1993
Pseudomonas knackmussii
brenda
Beveridge, A.J.; Ollis, D.L.
A theoretical study of substrate-induced activation of dienelactone hydrolase
Protein Eng.
8
135-142
1995
Pseudomonas knackmussii
brenda
Schlmann, M.; Schmidt, E.; Knackmuss, H.J.
Different types of dienelactone hydrolase in 4-fluorobenzoate-utilizing bacteria
J. Bacteriol.
172
5112-5118
1990
Alcaligenes sp., Alcaligenes sp. A7, Burkholderia cepacia, Cupriavidus necator, Pseudomonas knackmussii
brenda
Frantz, B.; Ngai, K.L.; Chatterjee, D.K.; Ornston, L.N.; Chakrabarty, A.M.
Nucleotide sequence and expression of clcD, a plasmid-borne dienelactone hydrolase gene from Pseudomonas sp. strain B13
J. Bacteriol.
169
704-709
1987
Pseudomonas knackmussii, Pseudomonas putida
brenda
Pathak, D.; Ngai, K.L.; Ollis, D.
X-ray crystallographic structure of dienelactone hydrolase at 2.8 A
J. Mol. Biol.
204
435-445
1988
Pseudomonas knackmussii
brenda
Schlmann, M.
Evolution of chlorocatechol catabolic pathways. Conclusions to be drawn from comparisons of lactone hydrolases
Biodegradation
5
301-321
1994
Burkholderia cepacia, Cupriavidus necator, Pseudomonas knackmussii, Pseudomonas putida, Pseudomonas sp. P51, Rhodococcus opacus
brenda
Porter, J.L.; Collyer, C.A.; Ollis, D.L.
Compensatory stabilizing role of surface mutations during the directed evolution of dienelactone hydrolase for enhanced activity
Protein J.
34
82-89
2015
Pseudomonas knackmussii (P0A115), Pseudomonas knackmussii DSM 6978 (P0A115)
brenda