Information on EC 2.4.1.217 - mannosyl-3-phosphoglycerate synthase

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The expected taxonomic range for this enzyme is: Bacteria, Archaea

EC NUMBER
COMMENTARY hide
2.4.1.217
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RECOMMENDED NAME
GeneOntology No.
mannosyl-3-phosphoglycerate synthase
REACTION
REACTION DIAGRAM
COMMENTARY hide
ORGANISM
UNIPROT
LITERATURE
GDP-mannose + 3-phospho-D-glycerate = GDP + 2-(alpha-D-mannosyl)-3-phosphoglycerate
show the reaction diagram
REACTION TYPE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
hexosyl group transfer
PATHWAY
BRENDA Link
KEGG Link
MetaCyc Link
mannosylglycerate biosynthesis I
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-
mannosylglycerate biosynthesis
-
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Fructose and mannose metabolism
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SYSTEMATIC NAME
IUBMB Comments
GDP-mannose:3-phospho-D-glycerate 3-alpha-D-mannosyltransferase
Requires Mg2+. The enzyme is absolutely specific for GDPmannose and 3-phosphoglycerate, and transfers the mannosyl group with retention of configuration. In the hyperthermophilic archaeon Pyrococcus horikoshii, the mannosyl-3-phosphoglycerate formed is subsequently dephosphorylated by a specific phosphatase, EC 3.1.3.70 (mannosyl-3-phosphoglycerate phosphatase), producing mannosylglycerate.
CAS REGISTRY NUMBER
COMMENTARY hide
393512-63-5
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ORGANISM
COMMENTARY hide
LITERATURE
UNIPROT
SEQUENCE DB
SOURCE
gene mgsD, bifunctional enzyme
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-
Manually annotated by BRENDA team
no activity in Saccharomyces cerevisiae
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-
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Manually annotated by BRENDA team
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SwissProt
Manually annotated by BRENDA team
GENERAL INFORMATION
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
evolution
metabolism
physiological function
additional information
SUBSTRATE
PRODUCT                       
REACTION DIAGRAM
ORGANISM
UNIPROT
COMMENTARY
(Substrate) hide
LITERATURE
(Substrate)
COMMENTARY
(Product) hide
LITERATURE
(Product)
Reversibility
r=reversible
ir=irreversible
?=not specified
GDP-glucose + 3-phospho-D-glycerate
GDP + 2-(alpha-D-glucosyl)-3-phosphoglycerate
show the reaction diagram
-
-
-
-
?
GDP-mannose + 3-phospho-D-glycerate
GDP + 2-(alpha-D-mannosyl)-3-phosphoglycerate
show the reaction diagram
GDPmannose + 3-phospho-D-glycerate
GDP + 2-(1-D-mannosyl)-3-phosphoglycerate
show the reaction diagram
GDPmannose + 3-phospho-D-glycerate
GDP + 2-(alpha-D-mannosyl)-3-phosphoglycerate
show the reaction diagram
additional information
?
-
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
GDP-glucose + 3-phospho-D-glycerate
GDP + 2-(alpha-D-glucosyl)-3-phosphoglycerate
show the reaction diagram
-
-
-
-
?
GDP-mannose + 3-phospho-D-glycerate
GDP + 2-(alpha-D-mannosyl)-3-phosphoglycerate
show the reaction diagram
GDPmannose + 3-phospho-D-glycerate
GDP + 2-(1-D-mannosyl)-3-phosphoglycerate
show the reaction diagram
additional information
?
-
METALS and IONS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
KCl
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or NaCl required for optimal activity
NaCl
-
or KCl, required for optimal activity
Ni2+
activates, best at 1.5 mM
additional information
INHIBITORS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
GDP
50% inhibition at 2.5 mM
Zn2+
measurement in the presence of Zn2+ (300 microM) instead Mg2+, the activity decreases about 100fold (0.4 micromol/min/mg), Zn2+ can displace Mg2+ at the second metal binding site
additional information
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ACTIVATING COMPOUND
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
additional information
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KM VALUE [mM]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
0.13 - 2.18
3-phospho-D-glycerate
1.21
GDP-glucose
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pH 7.0-8.0, 70-75C
0.17 - 0.7
GDP-mannose
TURNOVER NUMBER [1/s]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
3.9
3-phospho-D-glycerate
SPECIFIC ACTIVITY [µmol/min/mg]
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
0.0232
with GDP-mannose as a substrate
0.4
with Zn2+ as a cofactor
5.3
when 20 mM Mg2+ is added in combination with Zn2+ (300 microM), the MpgS specific activity recovered substantially
45
with Mg2+ as a cofactor
156
purified enzyme lacking the 30 amino acids of the C-terminus
pH RANGE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
5 - 10
pH-profile of full length and truncated enzyme
5 - 9
pH-profile, 30-40% of maximal activity at pH 5.0 and pH 9.0
5.5 - 8.5
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pH-profile
TEMPERATURE OPTIMUM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
50
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recombinant enzyme
70 - 75
80 - 90
90 - 100
TEMPERATURE RANGE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
10 - 60
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temperature-profile, recombinant enzyme
40 - 95
temperature-profile of full length and truncated enzyme
50 - 100
temperature-profile, 10% of maximal activity at 25C, 55% of maximal activity at 103C
80 - 100
80C: about 55% of maximal activity, 100C: about 60% of maximal activity; 80C: about 60% of maximal activity, 100C: about 60% of maximal activity
80 - 105
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80C: about 60% of maximal activity, 105C: about 70% of maximal activity
PDB
SCOP
CATH
ORGANISM
UNIPROT
Pyrococcus horikoshii (strain ATCC 700860 / DSM 12428 / JCM 9974 / NBRC 100139 / OT-3)
Pyrococcus horikoshii (strain ATCC 700860 / DSM 12428 / JCM 9974 / NBRC 100139 / OT-3)
Pyrococcus horikoshii (strain ATCC 700860 / DSM 12428 / JCM 9974 / NBRC 100139 / OT-3)
Rhodothermus marinus (strain ATCC 43812 / DSM 4252 / R-10)
Rhodothermus marinus (strain ATCC 43812 / DSM 4252 / R-10)
Rhodothermus marinus (strain ATCC 43812 / DSM 4252 / R-10)
Rubrobacter xylanophilus (strain DSM 9941 / NBRC 16129)
Rubrobacter xylanophilus (strain DSM 9941 / NBRC 16129)
Rubrobacter xylanophilus (strain DSM 9941 / NBRC 16129)
Thermus thermophilus (strain HB27 / ATCC BAA-163 / DSM 7039)
Thermus thermophilus (strain HB27 / ATCC BAA-163 / DSM 7039)
Thermus thermophilus (strain HB27 / ATCC BAA-163 / DSM 7039)
MOLECULAR WEIGHT
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
43000
recombinant fusion protein, determined by SDS-PAGE
95000
determined by gel filtration, suggesting that it is dimeric in solution
SUBUNITS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
additional information
POSTTRANSLATIONAL MODIFICATION
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
proteolytic modification
the 30 amino acid C-terminal sequence of the enzyme has regulatory function, cleaving of this peptide by intracellular proteases increases the activity by 10fold
Crystallization/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
crystal structure of the unliganded MpgS and of its complexes with the nucleotide and sugar donors, at 2.2, 2.8 and 2.5 A resolution respectively
enzyme in apo-form and in complex with GMP and GDP-mannose
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for the native protein a data set extending to 2.20 A resolution is collected, for the mercury derivative a data set extending to 3.05 A, and for the ternary complex with GDP and mannose a data set extending to 2.80 A
hanging drop vapor diffusion method, using 0.2 M magnesium acetate, 0.1 M sodium cacodylate pH 6.5, 30-35% MPD with 600 mM ZnCl2, the addition of Zn2+ to the crystallization buffer is essential in order to obtain crystals
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the three-dimensional structure of mannosyl-3-phosphoglycerate synthase in its binary complex form, with GDP-alpha-D-mannose and Mg2+, shows a second metal binding site, about 6 A away from the mannose moiety, wild-type and H309A mutant
TEMPERATURE STABILITY
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
60
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half-life is 23-28 min dependent on the substrate
additional information
GENERAL STABILITY
ORGANISM
UNIPROT
LITERATURE
recombinant monofunctional mannosyl-3-phosphoglycerate synthase domain is unstable
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Purification/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
native isozymes in a multistep procedure, recombinant isozymes from Escherichia coli by 3 chromatographic steps of cation exchange chromatography and gel filtration
native mannosyl-3-phosphoglycerate synthase is purified from Rubrobacter xylanophilus cells using a Q-Sepharose FF and a Mono-Q column, recombinant MpgS is purified by immobilized metal-affinity chromatography on a Ni-Sepharose column
purified from cell extracts
Q-Sepharose column chromatography, Resource Q column chromatography, Mono Q column chromatography, and Superdex 75 gel filtration
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recombinant enzyme and enzyme domains from Escherichia coli
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recombinant enzyme from Escherichia coli strain BL21
Cloned/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
corresponding gene is expressed in Escherichia coli
DNA and amino acid sequence determination and analysis, genetic organization of genes involved in mannosylglycerate biosynthesis, functional overexpression in Escherichia coli strain BL21
expressed in Escherichia coli
expressed in Escherichia coli BL21 Rosetta cells
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gene mgsD, DNA and amino acid sequence determination and analysis, genetic organization, overexpression of the holoenzyme and of the isolated mannosyl-3-phosphoglycerate synthase and mannosyl-3-phosphoglycerate phosphatase domains of the bifunctional enzyme in Escherichia coli, the recombinant Escherichia coli strain does not accumulate mannosylglycerate, expression of active bifunctional enzyme in Saccharomyces cerevisiae
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gene mpgs, DNA and amino acid sequence determination and analysis of the 2 isozymes, expression in Escherichia coli strain BL21(DE)
into the pGEMT-Easy vector for sequencing and subcloned into pET30a for expression in Escherichia coli BL21DE3 cells
overexpression in Escherichia coli
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phylogenetic analysis
the mpgS gene from strain HB27 is cloned into the pGEM-T Easy vector
ENGINEERING
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
A164S/A208S/F211Y
MpgS triple mutant engineered to reinforce the hydrogen-bonding network
E251A
site-directed mutagenesis, no specific activity detected
H309A
site-directed mutagenesis with the absence of the metal site, negligible specific activity