BRENDA - Enzyme Database
show all sequences of 1.2.1.39

Structure and biochemistry of phenylacetaldehyde dehydrogenase from the Pseudomonas putida S12 styrene catabolic pathway

Crabo, A.G.; Singh, B.; Nguyen, T.; Emami, S.; Gassner, G.T.; Sazinsky, M.H.; Arch. Biochem. Biophys. 616, 47-58 (2017)

Data extracted from this reference:

Cloned(Commentary)
Commentary
Organism
gene styD, DNA and amino acid sequence determination and analysis, recombinant expression of N-terminally His-tagged enzyme, NPADH, in Escherichia coli strain BL21(DE3)
Pseudomonas putida
Crystallization (Commentary)
Crystallization
Organism
purified recombinant His-tagged enzyme, sitting drop vapor diffusion method, mixing of 5-7 mg/ml protein in 10 mM Tris, pH 7.5, and 1 mM 2-mercaptoethanol, with 100-200 mM trisodium citrate, pH 8.5, and 12-16% PEG 3350, at 4°C, X-ray diffraction structure determination and analysis at 2.83 A resolution, molecular replacement using ALDH1, PDB ID 1BXS, as a starting model, model building
Pseudomonas putida
Inhibitors
Inhibitors
Commentary
Organism
Structure
Mg2+
inhibits and activates
Pseudomonas putida
Mn2+
inhibits and activates
Pseudomonas putida
NAD+
substrate inhibition, competitive binding of NADH
Pseudomonas putida
NADH
product inhibition, competitive binding of NAD+. For many aldehyde dehydrogenases, NADH binds competitively with NAD+ and forms a nonproductive dead-end complex during catalysis. In the absence of styrene monooxygenase reductase, which regenerates NAD+ from NADH in the first step of styrene catabolism, NPADH is inhibited by a ternary complex involving NADH, product, and phenylacetaldehyde, substrate
Pseudomonas putida
PMSF
inactivates NPADH, presumably by modifying the active site cysteine
Pseudomonas putida
Pyridine nucleotides
titrations of NPADH with NADþ and NADH are evaluated to estimate the binding affinities of the oxidized and reduced pyridine nucleotides under equilibrium conditions. Mg2+ is included in these studies
Pseudomonas putida
KM Value [mM]
KM Value [mM]
KM Value Maximum [mM]
Substrate
Commentary
Organism
Structure
additional information
-
additional information
kinetic analysis
Pseudomonas putida
0.0487
-
NAD+
pH 8.5, 25°C
Pseudomonas putida
Metals/Ions
Metals/Ions
Commentary
Organism
Structure
Mg2+
the enzyme includes both an activating and inhibitory metal binding site in the catalytic mechanism of NPADH. The activating divalent metal binding site may be best described as the direct interaction of the metal ion with the pyrophosphate linkage joining the nicotinamide mononucleotide and adenosine mononucleotide components of the pyridine nucleotide structure. A second mononuclear metal binding site, occupied by Mg2+ is detected in this structure. The Mg2+ in this site assumes a roughly octahedral geometry and is coordinated by the backbone carbonyl oxygens of Val40, Asp109, Glu196, and Val345, as well as a monodentate interaction with a carboxylate oxygen of Asp109. The sixth ligand is a crystallographically resolved water molecule
Pseudomonas putida
Mn2+
inhibits and activates
Pseudomonas putida
Molecular Weight [Da]
Molecular Weight [Da]
Molecular Weight Maximum [Da]
Commentary
Organism
227000
-
recombinant His-tagged enzyme, gel filtration
Pseudomonas putida
Natural Substrates/ Products (Substrates)
Natural Substrates
Organism
Commentary (Nat. Sub.)
Natural Products
Commentary (Nat. Pro.)
Organism (Nat. Pro.)
Reversibility
phenylacetaldehyde + NAD+ + H2O
Pseudomonas putida
-
phenylacetate + NADH + 2 H+
-
-
?
phenylacetaldehyde + NAD+ + H2O
Pseudomonas putida S12
-
phenylacetate + NADH + 2 H+
-
-
?
Organism
Organism
Primary Accession No. (UniProt)
Commentary
Textmining
Pseudomonas putida
V4GH04
-
-
Pseudomonas putida S12
V4GH04
-
-
Purification (Commentary)
Commentary
Organism
recombinant N-terminally His-tagged enzyme, NPADH, from Escherichia coli strain BL21(DE3) by nickel affinity chromatography and ultrafiltration to over 90% purity
Pseudomonas putida
Reaction
Reaction
Commentary
Organism
phenylacetaldehyde + NAD+ + H2O = phenylacetate + NADH + 2 H+
sequential reaction mechanism in which NAD+ serves as both the leading substrate and homotropic allosteric activator, catalytic mechanism involving E169, E267, and C301, overview. The catalytic Glu has two conformations: a passive conformer that tucks away to allow hydride transfer to the nicotinamide ring, and an active conformer that abstracts a proton from the thioester-deacylating water
Pseudomonas putida
Substrates and Products (Substrate)
Substrates
Commentary Substrates
Literature (Substrates)
Organism
Products
Commentary (Products)
Literature (Products)
Organism (Products)
Reversibility
phenylacetaldehyde + NAD+ + H2O
-
741787
Pseudomonas putida
phenylacetate + NADH + 2 H+
-
-
-
?
phenylacetaldehyde + NAD+ + H2O
-
741787
Pseudomonas putida S12
phenylacetate + NADH + 2 H+
-
-
-
?
Subunits
Subunits
Commentary
Organism
homotetramer
4 * 55000, recombinant His-tagged enzyme, SDS-PAGE
Pseudomonas putida
More
the oligomerization domains of all four subunits form the core of PADH and are responsible not only for the effective dimerization observed within the homotetramer, but also for the association of these dimers to form the tetramer. Each 496 amino acid PADH subunit consists of three domains: an N-terminal NADþ-binding domain (residues 1-130 and 159-269), a catalytic domain (residues 270-471), and an oligomerization domain (131-158 and 472-496). The enzyme has a unique set of intersubunit interactions and active site tunnel for substrate entrance. Each oligomerization domain of NPADH contains a six-residue insertion that extends this loop over the substrate entrance tunnel of a neighboring subunit, thereby obstructing the active site of the adjacent subunit. This feature might be an important factor in the homotropic activation and product inhibition mechanisms. The substrate channel of NPADH is narrower and lined with more aromatic residues, which include Phe170, Phe295, Phe466, and Trp177, suggesting a means for enhancing substrate specificity
Pseudomonas putida
Temperature Optimum [°C]
Temperature Optimum [°C]
Temperature Optimum Maximum [°C]
Commentary
Organism
25
-
assay at
Pseudomonas putida
pH Optimum
pH Optimum Minimum
pH Optimum Maximum
Commentary
Organism
8
10
-
Pseudomonas putida
Cofactor
Cofactor
Commentary
Organism
Structure
NAD+
N-terminal NAD+-binding domain, comprising residues 1-130 and 159-269, structure analysis, overview
Pseudomonas putida
Cloned(Commentary) (protein specific)
Commentary
Organism
gene styD, DNA and amino acid sequence determination and analysis, recombinant expression of N-terminally His-tagged enzyme, NPADH, in Escherichia coli strain BL21(DE3)
Pseudomonas putida
Cofactor (protein specific)
Cofactor
Commentary
Organism
Structure
NAD+
N-terminal NAD+-binding domain, comprising residues 1-130 and 159-269, structure analysis, overview
Pseudomonas putida
Crystallization (Commentary) (protein specific)
Crystallization
Organism
purified recombinant His-tagged enzyme, sitting drop vapor diffusion method, mixing of 5-7 mg/ml protein in 10 mM Tris, pH 7.5, and 1 mM 2-mercaptoethanol, with 100-200 mM trisodium citrate, pH 8.5, and 12-16% PEG 3350, at 4°C, X-ray diffraction structure determination and analysis at 2.83 A resolution, molecular replacement using ALDH1, PDB ID 1BXS, as a starting model, model building
Pseudomonas putida
Inhibitors (protein specific)
Inhibitors
Commentary
Organism
Structure
Mg2+
inhibits and activates
Pseudomonas putida
Mn2+
inhibits and activates
Pseudomonas putida
NAD+
substrate inhibition, competitive binding of NADH
Pseudomonas putida
NADH
product inhibition, competitive binding of NAD+. For many aldehyde dehydrogenases, NADH binds competitively with NAD+ and forms a nonproductive dead-end complex during catalysis. In the absence of styrene monooxygenase reductase, which regenerates NAD+ from NADH in the first step of styrene catabolism, NPADH is inhibited by a ternary complex involving NADH, product, and phenylacetaldehyde, substrate
Pseudomonas putida
PMSF
inactivates NPADH, presumably by modifying the active site cysteine
Pseudomonas putida
Pyridine nucleotides
titrations of NPADH with NADþ and NADH are evaluated to estimate the binding affinities of the oxidized and reduced pyridine nucleotides under equilibrium conditions. Mg2+ is included in these studies
Pseudomonas putida
KM Value [mM] (protein specific)
KM Value [mM]
KM Value Maximum [mM]
Substrate
Commentary
Organism
Structure
additional information
-
additional information
kinetic analysis
Pseudomonas putida
0.0487
-
NAD+
pH 8.5, 25°C
Pseudomonas putida
Metals/Ions (protein specific)
Metals/Ions
Commentary
Organism
Structure
Mg2+
the enzyme includes both an activating and inhibitory metal binding site in the catalytic mechanism of NPADH. The activating divalent metal binding site may be best described as the direct interaction of the metal ion with the pyrophosphate linkage joining the nicotinamide mononucleotide and adenosine mononucleotide components of the pyridine nucleotide structure. A second mononuclear metal binding site, occupied by Mg2+ is detected in this structure. The Mg2+ in this site assumes a roughly octahedral geometry and is coordinated by the backbone carbonyl oxygens of Val40, Asp109, Glu196, and Val345, as well as a monodentate interaction with a carboxylate oxygen of Asp109. The sixth ligand is a crystallographically resolved water molecule
Pseudomonas putida
Mn2+
inhibits and activates
Pseudomonas putida
Molecular Weight [Da] (protein specific)
Molecular Weight [Da]
Molecular Weight Maximum [Da]
Commentary
Organism
227000
-
recombinant His-tagged enzyme, gel filtration
Pseudomonas putida
Natural Substrates/ Products (Substrates) (protein specific)
Natural Substrates
Organism
Commentary (Nat. Sub.)
Natural Products
Commentary (Nat. Pro.)
Organism (Nat. Pro.)
Reversibility
phenylacetaldehyde + NAD+ + H2O
Pseudomonas putida
-
phenylacetate + NADH + 2 H+
-
-
?
phenylacetaldehyde + NAD+ + H2O
Pseudomonas putida S12
-
phenylacetate + NADH + 2 H+
-
-
?
Purification (Commentary) (protein specific)
Commentary
Organism
recombinant N-terminally His-tagged enzyme, NPADH, from Escherichia coli strain BL21(DE3) by nickel affinity chromatography and ultrafiltration to over 90% purity
Pseudomonas putida
Substrates and Products (Substrate) (protein specific)
Substrates
Commentary Substrates
Literature (Substrates)
Organism
Products
Commentary (Products)
Literature (Products)
Organism (Products)
Reversibility
phenylacetaldehyde + NAD+ + H2O
-
741787
Pseudomonas putida
phenylacetate + NADH + 2 H+
-
-
-
?
phenylacetaldehyde + NAD+ + H2O
-
741787
Pseudomonas putida S12
phenylacetate + NADH + 2 H+
-
-
-
?
Subunits (protein specific)
Subunits
Commentary
Organism
homotetramer
4 * 55000, recombinant His-tagged enzyme, SDS-PAGE
Pseudomonas putida
More
the oligomerization domains of all four subunits form the core of PADH and are responsible not only for the effective dimerization observed within the homotetramer, but also for the association of these dimers to form the tetramer. Each 496 amino acid PADH subunit consists of three domains: an N-terminal NADþ-binding domain (residues 1-130 and 159-269), a catalytic domain (residues 270-471), and an oligomerization domain (131-158 and 472-496). The enzyme has a unique set of intersubunit interactions and active site tunnel for substrate entrance. Each oligomerization domain of NPADH contains a six-residue insertion that extends this loop over the substrate entrance tunnel of a neighboring subunit, thereby obstructing the active site of the adjacent subunit. This feature might be an important factor in the homotropic activation and product inhibition mechanisms. The substrate channel of NPADH is narrower and lined with more aromatic residues, which include Phe170, Phe295, Phe466, and Trp177, suggesting a means for enhancing substrate specificity
Pseudomonas putida
Temperature Optimum [°C] (protein specific)
Temperature Optimum [°C]
Temperature Optimum Maximum [°C]
Commentary
Organism
25
-
assay at
Pseudomonas putida
pH Optimum (protein specific)
pH Optimum Minimum
pH Optimum Maximum
Commentary
Organism
8
10
-
Pseudomonas putida
General Information
General Information
Commentary
Organism
metabolism
the enzyme catalyzes a step in the styrene catabolic pathway of Pseudomonas putida
Pseudomonas putida
additional information
substrate channel and active site structure, overview. A majority of conserved residues in NPADH localize to the active site and NAD+-binding pocket. At the interface between the two pockets are the catalytic Cys 301 and Glu 267 residues, which serve as the general nucleophile and general base for the reaction, respectively
Pseudomonas putida
physiological function
phenylacetaldehyde dehydrogenase catalyzes the NAD+-dependent oxidation of phenylactealdehyde to phenylacetic acid in the styrene catabolic and detoxification pathway of Pseudomonas putida strain S12
Pseudomonas putida
General Information (protein specific)
General Information
Commentary
Organism
metabolism
the enzyme catalyzes a step in the styrene catabolic pathway of Pseudomonas putida
Pseudomonas putida
additional information
substrate channel and active site structure, overview. A majority of conserved residues in NPADH localize to the active site and NAD+-binding pocket. At the interface between the two pockets are the catalytic Cys 301 and Glu 267 residues, which serve as the general nucleophile and general base for the reaction, respectively
Pseudomonas putida
physiological function
phenylacetaldehyde dehydrogenase catalyzes the NAD+-dependent oxidation of phenylactealdehyde to phenylacetic acid in the styrene catabolic and detoxification pathway of Pseudomonas putida strain S12
Pseudomonas putida
Other publictions for EC 1.2.1.39
No.
1st author
Pub Med
title
organims
journal
volume
pages
year
Activating Compound
Application
Cloned(Commentary)
Crystallization (Commentary)
Engineering
General Stability
Inhibitors
KM Value [mM]
Localization
Metals/Ions
Molecular Weight [Da]
Natural Substrates/ Products (Substrates)
Organic Solvent Stability
Organism
Oxidation Stability
Posttranslational Modification
Purification (Commentary)
Reaction
Renatured (Commentary)
Source Tissue
Specific Activity [micromol/min/mg]
Storage Stability
Substrates and Products (Substrate)
Subunits
Temperature Optimum [°C]
Temperature Range [°C]
Temperature Stability [°C]
Turnover Number [1/s]
pH Optimum
pH Range
pH Stability
Cofactor
Ki Value [mM]
pI Value
IC50 Value
Activating Compound (protein specific)
Application (protein specific)
Cloned(Commentary) (protein specific)
Cofactor (protein specific)
Crystallization (Commentary) (protein specific)
Engineering (protein specific)
General Stability (protein specific)
IC50 Value (protein specific)
Inhibitors (protein specific)
Ki Value [mM] (protein specific)
KM Value [mM] (protein specific)
Localization (protein specific)
Metals/Ions (protein specific)
Molecular Weight [Da] (protein specific)
Natural Substrates/ Products (Substrates) (protein specific)
Organic Solvent Stability (protein specific)
Oxidation Stability (protein specific)
Posttranslational Modification (protein specific)
Purification (Commentary) (protein specific)
Renatured (Commentary) (protein specific)
Source Tissue (protein specific)
Specific Activity [micromol/min/mg] (protein specific)
Storage Stability (protein specific)
Substrates and Products (Substrate) (protein specific)
Subunits (protein specific)
Temperature Optimum [°C] (protein specific)
Temperature Range [°C] (protein specific)
Temperature Stability [°C] (protein specific)
Turnover Number [1/s] (protein specific)
pH Optimum (protein specific)
pH Range (protein specific)
pH Stability (protein specific)
pI Value (protein specific)
Expression
General Information
General Information (protein specific)
Expression (protein specific)
KCat/KM [mM/s]
KCat/KM [mM/s] (protein specific)
741787
Crabo
Structure and biochemistry of ...
Pseudomonas putida, Pseudomonas putida S12
Arch. Biochem. Biophys.
616
47-58
2017
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6
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1
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2
2
1
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1
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3
3
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742786
Debnar-Daumler
Simultaneous involvement of a ...
Aromatoleum aromaticum, Aromatoleum aromaticum EbN1
J. Bacteriol.
196
483-492
2014
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-
1
-
-
-
4
3
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2
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4
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1
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2
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6
1
1
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2
2
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5
2
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1
5
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4
2
3
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1
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2
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6
1
1
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-
2
2
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-
1
1
-
2
2
724020
Koma
Production of aromatic compoun ...
Escherichia coli
Appl. Environ. Microbiol.
78
6203-6216
2012
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-
-
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2
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5
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2
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2
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1
1
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725180
Satoh
Engineering of a tyrosol-produ ...
Escherichia coli
J. Agric. Food Chem.
60
979-984
2012
-
-
-
-
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1
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2
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1
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2
2
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686425
Arias
Genetic analyses and molecular ...
Pseudomonas putida
Environ. Microbiol.
10
413-432
2008
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-
1
-
-
-
-
-
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1
2
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5
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1
1
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2
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1
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1
2
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2
2
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1
1
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2
-
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-
-
-
-
-
-
-
-
-
-
690567
Hirano
Purification and characterizat ...
Brevibacterium sp., Brevibacterium sp. KU1309
Appl. Microbiol. Biotechnol.
76
357-363
2007
-
-
-
-
-
-
14
2
1
4
2
2
-
5
-
-
1
-
-
-
2
1
13
1
1
-
-
-
1
1
1
1
-
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-
1
-
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-
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14
-
2
1
4
2
2
-
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-
1
-
-
2
1
13
1
1
-
-
-
1
1
1
-
-
-
-
-
-
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670833
Rodriguez-Zavala
Characterization of E. coli te ...
Escherichia coli
Protein Sci.
15
1387-1396
2006
-
-
-
-
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8
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1
1
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1
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8
1
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9
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2
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2
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8
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1
1
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8
1
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9
-
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718
Schneider
Anaerobic metabolism of L-phen ...
Thauera aromatica
Arch. Microbiol.
168
310-320
1997
-
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-
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1
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4
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2
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390298
Long
Enzymology of oxidation of tro ...
Pseudomonas sp., Pseudomonas sp. AT3
J. Bacteriol.
179
1044-1050
1997
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-
-
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2
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6
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4
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1
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4
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390299
Ferrandez
Molecular characterization of ...
Escherichia coli
FEBS Lett.
406
23-27
1997
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1
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1
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3
1
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2
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1
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1
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1
1
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1
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3
1
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2
1
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390300
Hanlon
2-Phenylethylamine catabolism ...
Escherichia coli
Microbiology
143
513-518
1997
-
-
-
-
-
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3
3
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1
2
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3
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1
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1
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5
1
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1
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1
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3
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3
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1
2
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1
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1
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5
1
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390301
O'Connor
The effect of nutrient limitat ...
Pseudomonas putida, Pseudomonas putida CA-3
Appl. Environ. Microbiol.
62
3594-3599
1996
-
-
-
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2
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9
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4
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1
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1
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2
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4
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390293
Hartmans
Bacterial degradation of styre ...
Bacteria, Bacteria S5
Appl. Environ. Microbiol.
56
1347-1351
1990
-
-
-
-
-
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2
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5
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4
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1
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1
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2
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4
-
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-
-
-
390294
Hartmans
Metabolism of styrene oxide an ...
Xanthobacter sp. 124X, Xanthobacter sp.
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1989
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7426
Van den Tweel
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Catabolism of DL-alpha-phenylh ...
Flavobacterium sp.
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1988
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390296
Parrott
2-Phenylethylamine catabolism ...
Escherichia coli
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1987
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390292
Fujioka
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Metabolism of phenylalanine (A ...
Achromobacter eurydice
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1970
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