Information on EC 2.1.1.20 - glycine N-methyltransferase

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The expected taxonomic range for this enzyme is: Euteleostomi

EC NUMBER
COMMENTARY hide
2.1.1.20
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RECOMMENDED NAME
GeneOntology No.
glycine N-methyltransferase
REACTION
REACTION DIAGRAM
COMMENTARY hide
ORGANISM
UNIPROT
LITERATURE
S-adenosyl-L-methionine + glycine = S-adenosyl-L-homocysteine + sarcosine
show the reaction diagram
REACTION TYPE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
methyl group transfer
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-
-
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PATHWAY
BRENDA Link
KEGG Link
MetaCyc Link
glycine betaine biosynthesis IV (from glycine)
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glycine betaine biosynthesis V (from glycine)
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Glycine, serine and threonine metabolism
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SYSTEMATIC NAME
IUBMB Comments
S-adenosyl-L-methionine:glycine N-methyltransferase
This enzyme is thought to play an important role in the regulation of methyl group metabolism in the liver and pancreas by regulating the ratio between S-adenosyl-L-methionine and S-adenosyl-L-homocysteine. It is inhibited by 5-methyltetrahydrofolate pentaglutamate [4]. Sarcosine, which has no physiological role, is converted back into glycine by the action of EC 1.5.8.3, sarcosine dehydrogenase.
CAS REGISTRY NUMBER
COMMENTARY hide
37228-72-1
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ORGANISM
COMMENTARY hide
LITERATURE
UNIPROT
SEQUENCE DB
SOURCE
-
-
-
Manually annotated by BRENDA team
GENERAL INFORMATION
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
malfunction
metabolism
physiological function
SUBSTRATE
PRODUCT                       
REACTION DIAGRAM
ORGANISM
UNIPROT
COMMENTARY
(Substrate) hide
LITERATURE
(Substrate)
COMMENTARY
(Product) hide
LITERATURE
(Product)
Reversibility
r=reversible
ir=irreversible
?=not specified
S-adenosyl-L-methionine + glycine
S-adenosyl-L-homocysteine + N-methylglycine
show the reaction diagram
S-adenosyl-L-methionine + glycine
S-adenosyl-L-homocysteine + sarcosine
show the reaction diagram
additional information
?
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NATURAL SUBSTRATES
NATURAL PRODUCTS
REACTION DIAGRAM
ORGANISM
UNIPROT
COMMENTARY
(Substrate) hide
LITERATURE
(Substrate)
COMMENTARY
(Product) hide
LITERATURE
(Product)
REVERSIBILITY
r=reversible
ir=irreversible
?=not specified
S-adenosyl-L-methionine + glycine
S-adenosyl-L-homocysteine + N-methylglycine
show the reaction diagram
S-adenosyl-L-methionine + glycine
S-adenosyl-L-homocysteine + sarcosine
show the reaction diagram
additional information
?
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INHIBITORS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
2'-deoxyadenosine
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3-deazaadenosine
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5'-N-ethylcarboxamidoadenosine
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5'-S-isobutylthio-5'-deoxyadenosine
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5'-[p-(fluorosulfonyl)benzoyl]adenosine
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5,5'-dithiobis(2-nitrobenzoate)
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5-methyl-tetrahydrofolate
5-methyltetrahydrofolate
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5-Methyltetrahydrofolate hexaglutamate
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5-methyltetrahydrofolate monoglutamate
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native N-acetylated form of the enzyme shows 50% inhibition at 0.05 mM while the recombinant non-acetylated form shows 50% inhibition at 1.89 mM
5-Methyltetrahydrofolate pentaglutamate
5-Methyltetrahydrofolate triglutamate
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5-methyltetrahydrofolic acid
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5-Methyltetrahydropteroylpentaglutamate
folate
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the native rat liver GNMT contains an acetylated N-terminal valine and is inhibited much more efficiently compared to the recombinant protein
folinic acid
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Gly
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at very high concentrations
iodoacetate
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methotrexate
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p-chloromercuribenzoate
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S-adenosyl-L-homocysteine
sarcosine
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weak
thioglycolic acid
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weak
Urea
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-
additional information
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folate-containing diets attenuate GNMT activity in diabetic rats but are without effect on abundance
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ACTIVATING COMPOUND
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
glucagon
phosphate
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in vitro phosphorylation increases activity, about 0.55 mol of phosphate present per mol of N-methyltransferase subunit
Vitamin A
KM VALUE [mM]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
0.0122 - 30
Gly
0.13 - 2.2
glycine
0.014 - 0.281
S-adenosyl-L-methionine
0.03 - 0.1
S-adenosylmethionine
TURNOVER NUMBER [1/s]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
0.105 - 1.6
Gly
0.7 - 51.9
glycine
0.105 - 1.6
S-adenosyl-L-methionine
Ki VALUE [mM]
INHIBITOR
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
0.38
2'-deoxyadenosine
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pH 8.6
0.9
3-deazaadenosine
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pH 8.6
0.51
5'-N-ethylcarboxamidoadenosine
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pH 8.6
0.056
5'-S-isobutylthio-5'-deoxyadenosine
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pH 8.6
0.28
aciclovir
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pH 8.6
0.21
adenosine
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pH 8.6
4.5
AMP
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pH 8.6
0.03
S-adenosyl-L-homocysteine
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pH 8.6
IC50 VALUE [mM]
INHIBITOR
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
0.001 - 0.59
5-Methyltetrahydrofolate pentaglutamate
SPECIFIC ACTIVITY [µmol/min/mg]
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
additional information
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pH OPTIMUM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
7.5
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assay pH
pH RANGE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
7.5 - 9.5
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pH 7.5: about 40% of maximal activity, pH 9.5: about 45% of maximal activity
TEMPERATURE OPTIMUM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
pI VALUE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
6.6
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calculation from nucleotide sequence
7.1
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calculation from nucleotide sequence
SOURCE TISSUE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
SOURCE
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high expression in normal tissue, reduced expression in tumorous tissue, some tumor samples show no enzyme expression
Manually annotated by BRENDA team
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GNMT is expressed in the epithelium of the colon under normal conditions, and with dextran sulfate sodium treatment, its expression is predominant in infiltrated leukocytes of lesions
Manually annotated by BRENDA team
in unfertilized, 2 days postfertilization, and 3 days postfertilization embryos GNMT is constitutively higher than in 4, 7, 10 or 14 days postfertilization embryos
Manually annotated by BRENDA team
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GNMT is expressed in the epithelium of the colon under normal conditions, and with dextran sulfate sodium treatment, its expression is predominant in infiltrated leukocytes of lesions
Manually annotated by BRENDA team
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DNA extracted from peripherial blood cells
Manually annotated by BRENDA team
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36.4% of tissues show loss of heterozygosity of the GNMT gene, GNMT expression diminished in 82.2% of tissues
Manually annotated by BRENDA team
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LNCaP cells and PC-3 cells
Manually annotated by BRENDA team
additional information
LOCALIZATION
ORGANISM
UNIPROT
COMMENTARY hide
GeneOntology No.
LITERATURE
SOURCE
additional information
MOLECULAR WEIGHT
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
32420 - 79000
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mass spectrometry, mass spectra of the proteins greatly depend on the method of sample preparation and storage
123500
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sedimentation equilibrium centrifugation
130000 - 132000
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sedimenation equilibrium centrifugation, gel filtration
135000
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gel filtration
SUBUNITS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
?
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x * 34000, SDS-PAGE
homotetramer
tetramer
trimer or tetramer
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3 or 4 * 27000-33000, nonidentical subunits, SDS-PAGE
POSTTRANSLATIONAL MODIFICATION
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
acetylated protein
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N-terminal valine of native GNMT is N-acetylated while in the recombinant enzyme it is not
glycoprotein
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4 residues of sialic acid and 2 residues of hexose per mol of protein
phosphoprotein
Crystallization/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
crystals are grown at 4C by hanging drop vapor diffusion method
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wild type and H176N mutant enzyme as cocrystals with a citrate molecule bound in the active site
crystals are grown at 22C by hanging drop vapor diffusion method, crystallized in two crystal forms, a monoclinic form and a tetragonal form, P2(1) and P4(1)2(1)2
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crystallized by the sitting drop method in complex with (6S)-5-methyltetrahydrofolat monoglutamate; GNMT complexed with 5-methyltetrahydrofolate, by the sitting drop method at room temperature, two folate binding sites in the intersubunit areas of the tetramer, each folate binding site is formed primarily by two 1-7 N-terminal regions of one pair of subunits and two 205-218 regions of the other pair of subunits. Both the pteridine and p-aminobenzoyl rings are located in the hydrophobic cavities formed by Tyr5, Leu207, and Met215 residues of all subunits; sitting-drop vapor diffusion method in complex with 5-methyltetrahydrofolate pentaglutamate, two molecules of inhibitor bound to a tetramer
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hanging drop method of vapor diffusion, crystal structure of the enzyme complexed with S-adenosyl-L-methionine and acetate (a potent competitive inhibitor of Gly) and the R175K mutated enzyme complexed with S-adenosyl-L-methionine are determined at 2.8 A and 3.0 A resolution, respectively
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including R175K mutant
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native and recombinant protein, to 2.55 A resolution. The native rat liver GNMT contains an acetylated N-terminal valine and is inhibited much more efficiently compared to the recombinant protein. In the folate-GNMT complexes with the native enzyme, two folate molecules establish three and four hydrogen bonds with the protein. In the folate-recombinant GNMT complex only one hydrogen bond is established. This difference results in more effective inhibition by folate of the native liver GNMT activity compared to the recombinant enzyme
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GENERAL STABILITY
ORGANISM
UNIPROT
LITERATURE
urea unfolding of GNMT is a two-step process. The first transition is a reversible dissociation of the GNMT tetramer to compact monomers in 1.0-3.5 M urea with enzyme inactivation. The second step of GNMT unfolding is a reversible transition of monomers from compact to a fully unfolded state with R(S) of 50 A, exposed tryptophan residues, and disrupted secondary structure in 8 M urea
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ORGANIC SOLVENT
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
urea
H176N mutant completely inactive at 2 M urea compared with 60% remaining activity of the wild type enzyme
STORAGE STABILITY
ORGANISM
UNIPROT
LITERATURE
4C, 5.0 mM DTT, 2.0 mM EDTA, 0.02% NaN3, stable for at least 2 weeks
Purification/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
by ammonium sulfate precipitation and gel filtration; recombinant protein
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from liver by anion and cation exchange chromatography and from isolated hepatocytes and liver tissue by immunoprecipitation
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native enzyme from rat liver and recombinant enzyme from Escherichia coli
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recombinant enzymes
Cloned/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
cloning of the gene and construction of enzyme deficient mice
expressed in Escherichia coli
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expressed in Escherichia coli BL21(DE3); expressed in Escherichia coli BL21(GE3); into pET-17b vector and expressed in Escherichia coli BL21(GE3)
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expressed in Mus musculus liver and kidney
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expression in Escherichia coli
full-length human GNMT is used as the bait in a yeast two-hybrid screen system with a human kidney cDNA library
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overexpression of GNMT in SCG2-1-1 cells
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overview
PC-3 cells, LNCaP cells, HA22T/VGH cells and HEK-293A cells transfected with plasmid containing haplotype C
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when expressed in pTYB vector as a fusion protein with intein and the chitin binding domain, a cleavage of intein is found. The cleavage takes place at two sites near the N-terminus of intein and results in the appearance of an abnormal GNMT protein after one-column cleavage of the fusion protein, which can not be separated from normal GNMT. For this reason expression is done in the vector pET-17b. Expression of soluble protein in Escherichia coli at about 20-40 mg/L
wild type and H176N mutant enzyme
wild-type and mutant enzymes H176N, L49P and N140S, expressed in Escherichia coli
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EXPRESSION
ORGANISM
UNIPROT
LITERATURE
benzo[a]pyrene treatment of HepG2 cells (0.001 or 0.01 mM) induces GNMT expression
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GNMT expression is down-regulated or even completely blocked in liver and prostate tumor tissue
GNMT expression is stimulated by androgen in androgen receptor expressing cells, the stimulation occurs at the mRNA and protein levels. Androgen receptor binds to an androgen response element within the first exon of the GNMT gene in vitro and in vivo
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ENGINEERING
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
DELTA171-295
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to map the interactive domain of GNMT plasmids containing different domains GNMT are constructed: The C-terminal 171-295 amino acid fragment of GNMT is identified as interactive domain
L149P
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the mutant is inactivated by 90%
L49P
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mutant enzyme possesses 10% activity of the wild-type enzyme
R175K
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crystal structure: N-terminal domains of subunits have moved out of the active sites of adjacent subunits
Y194F
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the ratio of turnover-number to KM-value for S-adenosyl-L-methionine is 2.4fold lower than the wild-type value, the ratio of turnover-number to KM-value for Gly is 16.4fold lower than the wild-type value
Y21F
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the ratio of turnover-number to KM-value for S-adenosyl-L-methionine is 5fold lower than the wild-type value, the ratio of turnover-number to KM-value for Gly is 2.4fold lower than the wild-type value
Y220F
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the ratio of turnover-number to KM-value for S-adenosyl-L-methionine is nearly identical to the wild-type value, the ratio of turnover-number to KM-value for Gly is 179fold lower than the wild-type value
Y242F
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the ratio of turnover-number to KM-value for S-adenosyl-L-methionine is 1.14fold higher than the wild-type value, the ratio of turnover-number to KM-value for Gly is 2325fold lower than the wild-type value
Y33F
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the ratio of turnover-number to KM-value for S-adenosyl-L-methionine is nearly identical to the wild-type value, the ratio of turnover-number to KM-value for Gly is 123fold lower than the wild-type value
additional information
APPLICATION
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
diagnostics
medicine
additional information
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phosphorylated serine residues 71, 182, and 241, in GNMT prepared from liver tissue and hepatocytes an S9 additional residue is phosphorylated, in hepatocytes and in recombinant GNMT S139 is detected, serine 9 is also identified as a target for cAMP-dependent protein kinase in vitro, positions of these phosphorylated residues in the tertiary structure of GNMT indicate their possible effect on enzyme conformation and activity