Literature summary for 1.14.11.8 extracted from

Kazaks, A.; Makrecka-Kuka, M.; Kuka, J.; Voronkova, T.; Akopjana, I.; Grinberga, S.; Pugovics, O.; Tars, K.
Expression and purification of active, stabilized trimethyllysine hydroxylase (2014), Protein Expr. Purif., 104, 1-6 .

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Activating Compound

Activating Compound	Comment	Organism	Structure
ascorbate	-	Homo sapiens

Cloned(Commentary)

Cloned (Comment)	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	Comment	Organism
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	Comment	Organism	Structure
Fe2+	dependent on, required for catalysis	Homo sapiens

Natural Substrates/ Products (Substrates)

Natural Substrates	Organism	Comment (Nat. Sub.)	Natural Products	Comment (Nat. Pro.)	Rev.	Reac.
N6,N6,N6-trimethyl-L-lysine + 2-oxoglutarate + O2	Homo sapiens	-	(3S)-3-hydroxy-N6,N6,N6-trimethyl-L-lysine + succinate + CO2	-	?

Organism

Organism	UniProt	Comment	Textmining
Homo sapiens	Q9NVH6	-	-

Purification (Commentary)

Purification (Comment)	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

Purification (Comment)

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	Comment Substrates	Organism	Products	Comment (Products)	Rev.	Reac.
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 chromatographytandem mass spectrometry (UPLC/MS/MS) in positive ion electrospray mode	Homo sapiens	?	-	?
N6,N6,N6-trimethyl-L-lysine + 2-oxoglutarate + O2	-	Homo sapiens	(3S)-3-hydroxy-N6,N6,N6-trimethyl-L-lysine + succinate + CO2	-	?

Subunits

Subunits	Comment	Organism
dimer	2 * 44870, recombinant detagged enzyme, SDS-PAGE	Homo sapiens

Synonyms

Synonyms	Comment	Organism
TMLH	-	Homo sapiens
trimethyllysine hydroxylase	-	Homo sapiens

Temperature Optimum [°C]

Temperature Optimum [°C]	Temperature Optimum Maximum [°C]	Comment	Organism
37	-	assay at	Homo sapiens

pH Optimum

pH Optimum Minimum	pH Optimum Maximum	Comment	Organism
7	-	assay at	Homo sapiens

General Information

General Information	Comment	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