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Information on EC 2.4.1.269 - mannosylglycerate synthase and Organism(s) Rhodothermus marinus and UniProt Accession Q9RFR0

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EC Tree
     2 Transferases
         2.4 Glycosyltransferases
             2.4.1 Hexosyltransferases
                2.4.1.269 mannosylglycerate synthase
IUBMB Comments
Rhodothermus marinus can also form mannosylglycerate via a two-step pathway catalysed by EC 2.4.1.217 (mannosyl-3-phosphoglycerate synthase) and EC 3.1.3.70 (mannosyl-3-phosphoglycerate phosphatase) . Depending on experimental conditions mannosylglycerate synthase is more or less specific for the GDP-mannose and D-glycerate [1,2].
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Rhodothermus marinus
UNIPROT: Q9RFR0
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The taxonomic range for the selected organisms is: Rhodothermus marinus
The expected taxonomic range for this enzyme is: Eukaryota, Bacteria
Synonyms
mannosylglycerate synthase, more
SYNONYM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
PATHWAY SOURCE
PATHWAYS
-
-
SYSTEMATIC NAME
IUBMB Comments
GDP-mannose:D-glycerate 2-alpha-D-mannosyltransferase
Rhodothermus marinus can also form mannosylglycerate via a two-step pathway catalysed by EC 2.4.1.217 (mannosyl-3-phosphoglycerate synthase) and EC 3.1.3.70 (mannosyl-3-phosphoglycerate phosphatase) [1]. Depending on experimental conditions mannosylglycerate synthase is more or less specific for the GDP-mannose and D-glycerate [1,2].
SUBSTRATE
PRODUCT                       
REACTION DIAGRAM
ORGANISM
UNIPROT
COMMENTARY
(Substrate) hide
LITERATURE
(Substrate)
COMMENTARY
(Product) hide
LITERATURE
(Product)
Reversibility
r=reversible
ir=irreversible
?=not specified
GDP-mannose + D-glycerate
2-O-(alpha-D-mannopyranosyl)-D-glycerate + GDP
show the reaction diagram
GDP-mannose + D-lactate
?
show the reaction diagram
-
-
-
?
GDP-alpha-D-glucose + D-glycerate
2-O-(alpha-D-glucopyranosyl)-D-glycerate + GDP
show the reaction diagram
GDP-mannose + D-glycerate
2-O-(alpha-D-mannopyranosyl)-D-glycerate + GDP
show the reaction diagram
-
at 37°C mannosylglycerate synthase shows donor flexibility and can use alpha-GDP-D-Man, alpha-GDP-D-Glc, beta-GDP-L-fucose, alpha-UDP-D-Man and alpha-UDP-D-Glc, but not Man-1-P, as substrates. The kcat/KM-value for GDP-glucose is 2.5fold lower than the value for GDP-mannose at 25°C. The kcat/KM-value for GDP-glucose is 8.6fold lower than the value for GDP-mannose at 65°C
-
-
?
GDP-mannose + D-lactate
?
show the reaction diagram
-
the kcat/KM-value for D-lactate is 70fold lower than the value for D-glycerate at 25°C
-
-
?
GDP-mannose + glycolic acid
?
show the reaction diagram
-
the kcat/KM-value for glycolic acid is 70fold lower than the value for glycolic acid at 25°C
-
-
?
NATURAL SUBSTRATE
NATURAL PRODUCT
REACTION DIAGRAM
ORGANISM
UNIPROT
COMMENTARY
(Substrate) hide
LITERATURE
(Substrate)
COMMENTARY
(Product) hide
LITERATURE
(Product)
REVERSIBILITY
r=reversible
ir=irreversible
?=not specified
GDP-mannose + D-glycerate
2-O-(alpha-D-mannopyranosyl)-D-glycerate + GDP
show the reaction diagram
Rhodothermus marinus possesses two enzymatic systems for the synthesis of mannosylglycerate. The first one is a single-step pathway in which mannosylglycerate synthase catalyses the synthesis of 2-O-(alpha-D-mannopyranosyl)-D-glycerate in one-step from GDP-mannose and D-glycerate. The second system is a two-step pathway in which mannosyl-3-phosphoglycerate synthase (EC 2.4.1.217) catalyses the conversion of GDP-mannose and 3-phospho-D-glycerate into 2-O-(alpha-D-mannopyranosyl)-3-phospho-D-glycerate, which is then converted to 2-O-(alpha-D-mannopyranosyl)-D-glycerate by mannosyl-3-phosphoglycerate phosphatase (EC 3.1.3.70)
-
-
?
GDP-alpha-D-glucose + D-glycerate
2-O-(alpha-D-glucopyranosyl)-D-glycerate + GDP
show the reaction diagram
-
the enzyme catalyzes the formation of the stress protectant 2-O-alpha-D-mannosyl glycerate
-
-
?
METALS and IONS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
Ca2+
may partially substitute for Mg2+
Mn2+
may partially substitute for Mg2+
Mg2+
-
absolute requirement for divalent metal ions. 200 mM of Mg2+, Ca2+, Mn2+, Ni2+ and Co2+ giving relative rates of 1.0, 0.6, 0.33, 0.16 and 0.15, respectively
additional information
INHIBITOR
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
KM VALUE [mM]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
0.0014 - 3.8
D-glycerate
0.0022
D-lactate
wild-type, pH 7.0, 23°C, presence of Mn2+
0.00031 - 0.3
GDP-mannose
96.5 - 121.9
D-glycerate
21.3
D-lactate
-
pH 7.8, 25°C, cosubstrate: GDP-mannose
124.9 - 138.6
GDP-glucose
81.2 - 89.4
GDP-mannose
23.7
glycolic acid
-
pH 7.8, 25°C, cosubstrate: GDP-mannose
TURNOVER NUMBER [1/s]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
0.017 - 1.92
D-glycerate
0.017
D-lactate
wild-type, pH 7.0, 23°C, presence of Mn2+
0.018 - 0.967
GDP-mannose
1.02 - 6.5
D-glycerate
0.0026
D-lactate
-
pH 7.8, 25°C, cosubstrate: GDP-mannose
0.63 - 1.1
GDP-glucose
1.02 - 6.1
GDP-mannose
0.0052
glycolic acid
-
pH 7.8, 25°C, cosubstrate: GDP-mannose
SPECIFIC ACTIVITY [µmol/min/mg]
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
pH OPTIMUM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
5.5 - 7
activity remains nearly constant between pH 5.5 and 7.0
7.8
-
assay at
TEMPERATURE OPTIMUM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
85 - 90
native and recombinant enzyme
TEMPERATURE RANGE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
75 - 95
75°C: native enzyme shows about 70% of maximal activity, recombinant enzyme shows about 40% of maximal activity, 95°C: native enzyme shows about 60% of maximal activity, recombinant enzyme shows about 70% of maximal activity
pI VALUE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
4.8
isoelectric focusing
ORGANISM
COMMENTARY hide
LITERATURE
UNIPROT
SEQUENCE DB
SOURCE
UNIPROT
ENTRY NAME
ORGANISM
NO. OF AA
NO. OF TRANSM. HELICES
MOLECULAR WEIGHT[Da]
SOURCE
SEQUENCE
LOCALIZATION PREDICTION?
MGS_RHOMR
397
0
46126
Swiss-Prot
-
MOLECULAR WEIGHT
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
122000
gel filtration
45000
x * 45000, SDS-PAGE
46125
x * 46125, calculated from sequence
SUBUNIT
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
CRYSTALLIZATION (Commentary)
ORGANISM
UNIPROT
LITERATURE
in complex with GDP-Man/Mg2+, GDP/Mg2+, GDP/Mn2+/malonate and lactate, to 2.42-2.9 A resolution. The guanine base of GDP is housed in a hydrophobic pocket between the side chains of Lys9 and Tyr37 on one face and the aliphatic portion of the side chain of Lys76 on the opposing face, with Gln66 accepting hydrogen bonds from the endocyclic N1 and exocyclic N2
hanging-drop method
-
PROTEIN VARIANTS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
D102A
completely inactive in the presence of all metals tested
D135A
very high Km value for D-glycerate and a slight increase in kcat
E166A
15-fold increase in Km for glycerate
H217A
significant change in metal preference. Mutant is essentially inactive in the presence of Ca2+, whereas the kcat value is reduced 23-fold in the presence of Mg2+
K76A
3-4-fold increase in kcat coupled with an elevation in Km for D-glycerate and GDP-Man of 15- and 3-fold, respectively
K9A
significant increase in kcat, little effect on the Km value for GDP-Man
M229A
minor influence on catalytic performance
Q66A
substantial increases in kcat and Km for both the acceptor and donor substrates
R218A
significant increase in kcat and Km values for GDP-Man, only a modest influence on the Km value for the acceptor substrate
R73A
minor influence on catalytic performance
R73G
minor influence on catalytic performance
T139A
Km value for lactate above 100 mM, modest decrease in kcat but a 1500-fold increase in the Km values for D-glycerate
W189A
3- and 7-fold increase in kcat and Km for D-glycerate, compared with the wild-type enzyme, no influence on utilization of GDP-Man
Y220A
500-fold increase in Km for D-glycerate
Y220F
1500-fold increase in Km for D-glycerate
Y37A
significant increase in kcat, little effect on the Km value for GDP-Man
D100A
-
inactive mutant enzyme
D102A
-
inactive mutant enzyme
H217A
-
inactive mutant enzyme
R131A
-
inactive mutant enzyme
TEMPERATURE STABILITY
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
65
half-life: 170 min
90
half-life: 30 min for the native enzyme, 17 min for the recombinant enzyme
additional information
PURIFICATION (Commentary)
ORGANISM
UNIPROT
LITERATURE
CLONED (Commentary)
ORGANISM
UNIPROT
LITERATURE
expression in Escherichia coli
REF.
AUTHORS
TITLE
JOURNAL
VOL.
PAGES
YEAR
ORGANISM (UNIPROT)
PUBMED ID
SOURCE
Martins, L.O.; Empadinhas, N.; Marugg, J.D.; Miguel, C.; Ferreira, C.; Da Costa, M.S.; Santos, H.
Biosynthesis of mannosylglycerate in the thermophilic bacterium Rhodothermus marinus. Biochemical and genetic characterization of a mannosylglycerate synthase
J. Biol. Chem.
274
35407-35414
1999
Rhodothermus marinus (Q9RFR0), Rhodothermus marinus
Manually annotated by BRENDA team
Flint, J.; Taylor, E.; Yang, M.; Bolam, D.N.; Tailford, L.E.; Martinez-Fleites, C.; Dodson, E.J.; Davis, B.G.; Gilbert, H.J.; Davies, G.J.
Structural dissection and high-throughput screening of mannosylglycerate synthase
Nat. Struct. Mol. Biol.
12
608-614
2005
Rhodothermus marinus
Manually annotated by BRENDA team
Nielsen, M.M.; Suits, M.D.; Yang, M.; Barry, C.S.; Martinez-Fleites, C.; Tailford, L.E.; Flint, J.E.; Dumon, C.; Davis, B.G.; Gilbert, H.J.; Davies, G.J.
Substrate and metal ion promiscuity in mannosylglycerate synthase
J. Biol. Chem.
286
15155-15164
2011
Rhodothermus marinus (Q9RFR0), Rhodothermus marinus
Manually annotated by BRENDA team