BRENDA - Enzyme Database show
show all sequences of 1.14.11.8

Expression and purification of active, stabilized trimethyllysine hydroxylase

Kazaks, A.; Makrecka-Kuka, M.; Kuka, J.; Voronkova, T.; Akopjana, I.; Grinberga, S.; Pugovics, O.; Tars, K.; Protein Expr. Purif. 104, 1-6 (2014)

Data extracted from this reference:

Activating Compound
Activating Compound
Commentary
Organism
Structure
ascorbate
-
Homo sapiens
Cloned(Commentary)
Commentary
Organism
gene TMLH, recombinant overexpression of active and stable TMLH as a His-tagged maltose-binding protein fusion in Escherichia coli strain BL21-AI. Coexpression of the enzyme with chaperones GroES/EL increases the expression level by one order of magnitude
Homo sapiens
KM Value [mM]
KM Value [mM]
KM Value Maximum [mM]
Substrate
Commentary
Organism
Structure
additional information
-
additional information
Michaelis-Menten kinetics
Homo sapiens
0.636
-
N6,N6,N6-Trimethyl-L-lysine
recombinant MBP-tagged enzyme, pH 7.0, 37°C
Homo sapiens
0.696
-
N6,N6,N6-Trimethyl-L-lysine
recombinant detagged enzyme, pH 7.0, 37°C
Homo sapiens
Metals/Ions
Metals/Ions
Commentary
Organism
Structure
Fe2+
dependent on, required for catalysis
Homo sapiens
Natural Substrates/ Products (Substrates)
Natural Substrates
Organism
Commentary (Nat. Sub.)
Natural Products
Commentary (Nat. Pro.)
Organism (Nat. Pro.)
Reversibility
N6,N6,N6-trimethyl-L-lysine + 2-oxoglutarate + O2
Homo sapiens
-
(3S)-3-hydroxy-N6,N6,N6-trimethyl-L-lysine + succinate + CO2
-
-
?
Organism
Organism
Primary Accession No. (UniProt)
Commentary
Textmining
Homo sapiens
Q9NVH6
-
-
Purification (Commentary)
Commentary
Organism
recombinant active and stable His- and MBP-tagged TMLH from Escherichia coli strain BL21-AI by nickel or amylose affinity chromatography. It is possible that the His6-tag at the N-terminus of the fusion protein is masked and cannot be accessed by the Ni-beads. Thus, MBP affinity purification is selected as a first purification step, followed by gel filtration, His6-MBP-tag cleavage by recombinant TEV protease
Homo sapiens
Storage Stability
Storage Stability
Organism
4°C, recombinant enzyme, after storage at 4°C for 4 days, MBP-TMLH retains 96% of its activity, while detagged TMLH shows only 77% activity compared to fresh enzyme preparation
Homo sapiens
Substrates and Products (Substrate)
Substrates
Commentary Substrates
Literature (Substrates)
Organism
Products
Commentary (Products)
Literature (Products)
Organism (Products)
Reversibility
additional information
determination of the (2S,3S)-3-hydroxy-N6,N6,N6-trimethyl-L-lysine and N6,N6,N6-trimethyl-L-lysine is performed by ultraperformance liquid chromatography–tandem mass spectrometry (UPLC/MS/MS) in positive ion electrospray mode
746366
Homo sapiens
?
-
-
-
-
N6,N6,N6-trimethyl-L-lysine + 2-oxoglutarate + O2
-
746366
Homo sapiens
(3S)-3-hydroxy-N6,N6,N6-trimethyl-L-lysine + succinate + CO2
-
-
-
?
Subunits
Subunits
Commentary
Organism
dimer
2 * 44870, recombinant detagged enzyme, SDS-PAGE
Homo sapiens
Temperature Optimum [°C]
Temperature Optimum [°C]
Temperature Optimum Maximum [°C]
Commentary
Organism
37
-
assay at
Homo sapiens
pH Optimum
pH Optimum Minimum
pH Optimum Maximum
Commentary
Organism
7
-
assay at
Homo sapiens
Activating Compound (protein specific)
Activating Compound
Commentary
Organism
Structure
ascorbate
-
Homo sapiens
Cloned(Commentary) (protein specific)
Commentary
Organism
gene TMLH, recombinant overexpression of active and stable TMLH as a His-tagged maltose-binding protein fusion in Escherichia coli strain BL21-AI. Coexpression of the enzyme with chaperones GroES/EL increases the expression level by one order of magnitude
Homo sapiens
KM Value [mM] (protein specific)
KM Value [mM]
KM Value Maximum [mM]
Substrate
Commentary
Organism
Structure
additional information
-
additional information
Michaelis-Menten kinetics
Homo sapiens
0.636
-
N6,N6,N6-Trimethyl-L-lysine
recombinant MBP-tagged enzyme, pH 7.0, 37°C
Homo sapiens
0.696
-
N6,N6,N6-Trimethyl-L-lysine
recombinant detagged enzyme, pH 7.0, 37°C
Homo sapiens
Metals/Ions (protein specific)
Metals/Ions
Commentary
Organism
Structure
Fe2+
dependent on, required for catalysis
Homo sapiens
Natural Substrates/ Products (Substrates) (protein specific)
Natural Substrates
Organism
Commentary (Nat. Sub.)
Natural Products
Commentary (Nat. Pro.)
Organism (Nat. Pro.)
Reversibility
N6,N6,N6-trimethyl-L-lysine + 2-oxoglutarate + O2
Homo sapiens
-
(3S)-3-hydroxy-N6,N6,N6-trimethyl-L-lysine + succinate + CO2
-
-
?
Purification (Commentary) (protein specific)
Commentary
Organism
recombinant active and stable His- and MBP-tagged TMLH from Escherichia coli strain BL21-AI by nickel or amylose affinity chromatography. It is possible that the His6-tag at the N-terminus of the fusion protein is masked and cannot be accessed by the Ni-beads. Thus, MBP affinity purification is selected as a first purification step, followed by gel filtration, His6-MBP-tag cleavage by recombinant TEV protease
Homo sapiens
Storage Stability (protein specific)
Storage Stability
Organism
4°C, recombinant enzyme, after storage at 4°C for 4 days, MBP-TMLH retains 96% of its activity, while detagged TMLH shows only 77% activity compared to fresh enzyme preparation
Homo sapiens
Substrates and Products (Substrate) (protein specific)
Substrates
Commentary Substrates
Literature (Substrates)
Organism
Products
Commentary (Products)
Literature (Products)
Organism (Products)
Reversibility
additional information
determination of the (2S,3S)-3-hydroxy-N6,N6,N6-trimethyl-L-lysine and N6,N6,N6-trimethyl-L-lysine is performed by ultraperformance liquid chromatography–tandem mass spectrometry (UPLC/MS/MS) in positive ion electrospray mode
746366
Homo sapiens
?
-
-
-
-
N6,N6,N6-trimethyl-L-lysine + 2-oxoglutarate + O2
-
746366
Homo sapiens
(3S)-3-hydroxy-N6,N6,N6-trimethyl-L-lysine + succinate + CO2
-
-
-
?
Subunits (protein specific)
Subunits
Commentary
Organism
dimer
2 * 44870, recombinant detagged enzyme, SDS-PAGE
Homo sapiens
Temperature Optimum [°C] (protein specific)
Temperature Optimum [°C]
Temperature Optimum Maximum [°C]
Commentary
Organism
37
-
assay at
Homo sapiens
pH Optimum (protein specific)
pH Optimum Minimum
pH Optimum Maximum
Commentary
Organism
7
-
assay at
Homo sapiens
General Information
General Information
Commentary
Organism
metabolism
trimethyllysine hydroxylase (TMLH) catalyses the first step in carnitine biosynthesis, the conversion of N6,N6,N6-trimethyl-L-lysine to 3-hydroxy-N6,N6,N6-trimethyl-L-lysine
Homo sapiens
General Information (protein specific)
General Information
Commentary
Organism
metabolism
trimethyllysine hydroxylase (TMLH) catalyses the first step in carnitine biosynthesis, the conversion of N6,N6,N6-trimethyl-L-lysine to 3-hydroxy-N6,N6,N6-trimethyl-L-lysine
Homo sapiens
Other publictions for EC 1.14.11.8
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)
744681
Lesniak
Human carnitine biosynthesis ...
Homo sapiens
Chem. Commun. (Camb.)
53
440-442
2017
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1
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1
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1
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1
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2
2
-
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745958
Vijayendar Reddy
Fluorinated trimethyllysine a ...
Homo sapiens
Org. Biomol. Chem.
15
1350-1354
2017
1
-
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-
-
-
-
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1
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1
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1
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3
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1
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1
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1
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1
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1
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3
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1
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1
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2
2
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745961
Reddy
Evidence that trimethyllysine ...
Homo sapiens
Org. Lett.
19
400-403
2017
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1
1
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1
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2
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1
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1
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2
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1
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1
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2
2
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744678
Al Temimi
Substrate scope for trimethyl ...
Homo sapiens
Chem. Commun. (Camb.)
52
12849-12852
2016
1
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1
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2
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7
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1
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1
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1
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1
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1
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7
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1
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1
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4
4
-
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-
746366
Kazaks
Expression and purification o ...
Homo sapiens
Protein Expr. Purif.
104
1-6
2014
1
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1
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3
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1
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1
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1
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1
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1
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1
2
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1
1
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697027
Fischer
A moderate excess of dietary l ...
Sus scrofa
Br. J. Nutr.
101
190-196
2009
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1
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4
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4
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1
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1
1
1
1
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691166
Furusawa
Vitamin C is not essential for ...
Mus musculus
Biol. Pharm. Bull.
31
1673-1679
2008
1
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1
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1
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1
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2
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1
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1
1
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686678
van Vlies
Submitochondrial localization ...
Rattus norvegicus
FEBS J.
274
5845-5851
2007
-
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1
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1
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1
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1
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671302
van Vlies
Measurement of carnitine biosy ...
Mus musculus, Rattus norvegicus
Anal. Biochem.
354
132-139
2006
-
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2
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2
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2
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8
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2
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2
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2
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8
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2
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675029
Monfregola
Functional analysis of TMLH va ...
Homo sapiens, Mus musculus
J. Cell. Physiol.
204
839-847
2005
4
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2
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2
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2
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3
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13
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2
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4
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2
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2
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2
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13
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2
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675561
Davis
Carnitine deficiency and suppl ...
Rattus norvegicus
J. Nutr.
135
761-764
2005
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2
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2
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1
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651370
Swiegers
Carnitine biosynthesis in neur ...
Neurospora crassa
FEMS Microbiol. Lett.
210
19-23
2002
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2
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1
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1
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652164
Vaz
Molecular and biochemical char ...
Homo sapiens, Mus musculus, Rattus norvegicus
J. Biol. Chem.
276
33512-33517
2001
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2
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2
1
1
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1
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7
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1
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1
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1
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1
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7020
Henderson
Mammalian enzymes of trimethyl ...
Bos taurus
Fed. Proc.
41
2843-2847
1982
1
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9
2
1
2
3
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1
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1
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2
1
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9
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1
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3
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1
1
1
1
1
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7021
Sachan
Carnitine biosynthesis. Hydrox ...
Rattus norvegicus
Biochem. J.
188
529-534
1980
-
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-
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3
1
1
2
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1
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7022
Sachan
Synthesis of carnitine from ep ...
Neurospora crassa
Biochem. Biophys. Res. Commun.
96
870-875
1980
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1
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7019
Hulse
Carnitine biosynthesis. beta-H ...
Rattus norvegicus
J. Biol. Chem.
253
1654-1659
1978
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2
1
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1
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1
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