Information on EC 5.1.3.37 - mannuronan 5-epimerase

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The enzyme appears in viruses and cellular organisms

EC NUMBER
COMMENTARY hide
5.1.3.37
-
RECOMMENDED NAME
GeneOntology No.
mannuronan 5-epimerase
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REACTION
REACTION DIAGRAM
COMMENTARY hide
ORGANISM
UNIPROT
LITERATURE
[mannuronan]-beta-D-mannuronate = [alginate]-alpha-L-guluronate
show the reaction diagram
PATHWAY
BRENDA Link
KEGG Link
MetaCyc Link
alginate biosynthesis I (algal)
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alginate biosynthesis II (bacterial)
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Fructose and mannose metabolism
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alginate biosynthesis
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SYSTEMATIC NAME
IUBMB Comments
[mannuronan]-beta-D-mannuronate 5-epimerase
The enzyme epimerizes the C-5 bond in some beta-D-mannuronate residues in mannuronan, converting them to alpha-L-guluronate residues, and thus modifying the mannuronan into alginate. It is found in brown algae and alginate-producing bacterial species from the Pseudomonas and Azotobacter genera.
ORGANISM
COMMENTARY hide
LITERATURE
UNIPROT
SEQUENCE DB
SOURCE
isoform AlgG
UniProt
Manually annotated by BRENDA team
GENERAL INFORMATION
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
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
hepta(beta-(1->4)-D-mannuronate)acid
?
show the reaction diagram
poor substrate
-
-
?
hexa(beta-(1->4)-D-mannuronate)
?
show the reaction diagram
poor substrate
-
-
?
octa(beta-(1->4)-D-mannuronate)
?
show the reaction diagram
-
-
-
?
[alginate]-beta-D-mannuronate
[alginate]-alpha-L-guluronate
show the reaction diagram
[mannuronan]-beta-D-mannuronate
[alginate]-alpha-L-guluronate
show the reaction diagram
additional information
?
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METALS and IONS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
Mg2+
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at Ca2+ concentrations lower than optimum, the addition of Sr2+ or Mg2+ has a stimulatory effect, but Mg2+ is not able to substitute for Ca2+ when no calcium is added
additional information
-
no Ca2+ ions required for activity, and the Ca2+-alginate complex is not a substrate for the enzyme
INHIBITORS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
EDTA
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1 mM, complete loss of activity
NaCl
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additions up to 0.1 M do not reduce the activity much, at 0.5 M NaCl less than 10% of the activity is retained
Zn2+
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1.5 mM, complete inhibition
KM VALUE [mM]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
0.018
[alginate]-beta-D-mannuronate
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pH 6.8, 37C
0.62 - 0.9
[mannuronan]-beta-D-mannuronate
additional information
additional information
-
the Km decreases from 80 microM for substrate containing fewer than 15 residues to 4 microM for substrate containing over 100 residues, pH 7.3
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TURNOVER NUMBER [1/s]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
14
[alginate]-beta-D-mannuronate
Azotobacter vinelandii
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pH 6.8, 37C
additional information
additional information
Pseudomonas aeruginosa
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the maximum velocity of the reaction increases from 0.0018 per s to 0.0218 per s as the substrate size increases from 10 to 20 residues, and no additional increase in kcat is observed for substrates up to 100 residues in length, pH 7.3, temperature not specified in the publication
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SPECIFIC ACTIVITY [µmol/min/mg]
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
0.37
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catalytic domain AlgE1-1, pH 6.9, 37C
4.4
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catalytic domain AlgE1-2, pH 6.9, 37C
4.9
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pH 6.9, 37C
pH OPTIMUM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
pH RANGE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
8
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complete loss of activity above
TEMPERATURE OPTIMUM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
pI VALUE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
4.5
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isoelectric focusing
LOCALIZATION
ORGANISM
UNIPROT
COMMENTARY hide
GeneOntology No.
LITERATURE
SOURCE
MOLECULAR WEIGHT
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
55000
x * 60000, preform, x * 55000, mature protein, SDS-PAGE
60000
x * 60000, preform, x * 55000, mature protein, SDS-PAGE
79000
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x * 57700, calculated, x * 79000, SDS-PAGE
120000
150000
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gel filtration
SUBUNITS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
monomer
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1 * 120000, SDS-PAGE
additional information
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enzyme is composed of repeats of two protein modules designated A (385 amino acids) and R (153 amino acids). The modular structure of isoform AlgE1 is A1R1R2R3A2R4. AlgE1 has two catalytic sites for epimerization, each site introducing a different G distribution pattern
POSTTRANSLATIONAL MODIFICATION
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
proteolytic modification
Crystallization/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
15N-HSQC spectrum of the 17.7 kDa R2 module of AlgE6, together with the assignments of the resonances. The backbone and the side-chain assignments are essentially complete, side-chain amide groups of all Asn and Gln are assigned. Most of the protons and the carbon atoms of aromatic side-chains are assigned
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dynamic force spectroscopy and comparison of epimeases AlgE4, AlgE6, recombinant construct PKA1 composed of A- and R-modules from various AlgE's, as well as separate R- and A-modules. The strength of the protein-mannuronan interaction, at a loading rate of 0.6 nN/s, variy from 73 pN (AlgE4) to 144 pN (A-module). The potential width, that is, the distance from the activation barrier to the bound substrate molecule, is 0.23 nm for AlgE4, 0.19 nm for AlgE6 and 0.1 nm for the A-module. No attraction is observed between the R-module and the substrate. The observations indicate that the A-module contains the substrate binding site and that the R-module modulates the enzyme-substrate binding strength; dynamic force spectroscopy and comparison of epimerases AlgE4, AlgE6, recombinant construct PKA1 composed of A- and R-modules from various AlgE's, as well as separate R- and A-modules. The strength of the protein-mannuronan interaction, at a loading rate of 0.6 nN/s, vary from 73 pN (AlgE4) to 144 pN (A-module). The potential width, that is, the distance from the activation barrier to the bound substrate molecule, is 0.23 nm for AlgE4, 0.19 nm for AlgE6 and 0.1 nm for the A-module. No attraction is observed between the R-module and the substrate. The observations indicate that the A-module contains the substrate binding site and that the R-module modulates the enzyme-substrate binding strength
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Dynamic Force Spectroscopy. The position of the activation barrier is 0.23 nm for the AlgE4 and 0.10 nm for its A-module. The lack of interaction observed between the R-module and mannuronan suggest that the A-module contains the binding site for the polymer substrate. The ratio between the epimerase-mannuronan dissociation rate and the catalytic rate for epimerization of single hexose residues suggests a processive mode of action of the AlgE4 epimerase yielding the observed sequence pattern in the uronan associated with the A-module of this enzyme
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NMR structure of the R-module from AlgE4. The R-module folds into a right-handed parallel beta-roll. Its overall shape is an elongated molecule with a positively charged patch that interacts with the substrate. Titration of the R-module with thulium indicates possible calcium binding sites in the loops formed by the nonarepeat sequences in the N-terminal part of the molecule. Calcium binding is important for the stability of the R-module. Calcium ions can be incorporated in these loops without structural violations and changes
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to 2.1 A resolution. Isoform AlgE4A folds into a right-handed parallel beta-helix structure. The alpha-helix is composed of four parallel beta-sheets, comprising 12 complete turns, and has an amphipathic alpha-helix near the N terminus. The catalytic site is positioned in a positively charged cleft formed by loops extending from the surface encompassing Asp152, an amino acid important for the reaction
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to 2.1 A resolution. Isoform AlgG is a long right-handed parallel beta-helix with an elaborate lid structure. Residue His319 acts as the catalytic base and that Arg345 neutralizes the acidic group during the epimerase reaction. Water is the likely catalytic acid. Substrate docking studies suggest that a conserved electropositive groove facilitates polymannuronate binding and contains at least nine substrate binding subsite
TEMPERATURE STABILITY
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
50
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stable up to
STORAGE STABILITY
ORGANISM
UNIPROT
LITERATURE
-80 C, 10% (v/v) glycerol, retains full catalytic activity for at least two months
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Purification/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
rapid purification procedure based on fast protein liquid chromatography on MonoQ resin
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using a Sepharose 6B column containing alginate covalently linked to the hydroxyl groups
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Cloned/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
erxpression in Escherichia coli
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expression in Escherichia coli
expression of the gene encoding for the isoform AlgE6 R2 module, residues 534-693, in Escherichia coli
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ENGINEERING
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
D152G
mutation eliminates almost all of both the lyase and epimerase activities
D173E
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54% residual activity
D178E
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complete loss of activity
D178N
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complete loss of activity
F122Y
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65% residual activity
H154F
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complete loss of activity
H154R
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complete loss of activity
K117 R
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24% residual activity
K117A
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16% residual activity
K255A
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8% residual activity
K255R
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51% residual activity
P153A
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10% residual activity
P153A/D173E
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4% residual activity
Q156A
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10% residual activity
Q225A
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9% residual activity
Q225E
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4% residual activity
Q225N
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6% residual activity
R249A
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46% residual activity
Y149F
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complete loss of activity
Y149H
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complete loss of activity
D317A
about 5% of wild-type activity
D320A
complete loss of activity
D368N
about 5% of wild-type activity
D452A
about 70% of wild-type activity
H319A
complete loss of activity
H339A
about 50% of wild-type activity
K338A
about 90% of wild-type activity
R321K
about 75% of wild-type activity
R345A
about 10% of wild-type activity
R345K
about 40% of wild-type activity
R345Q
about125% of wild-type activity
R353E
complete loss of activity
R369A
about 25% of wild-type activity
R415C
complete loss of activity
S344A
about 55% of wild-type activity
Y291F
about 85% of wild-type activity
Y294A
about 25% of wild-type activity
Y294F
about 65% of wild-type activity
Y296A
about 65% of wild-type activity
Y314F
about 5% of wild-type activity
Y392A
about 65% of wild-type activity
Y392F
about 70% of wild-type activity
additional information
Renatured/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
epimerase activity of AlgE4 preincubated with 1 mM Na2EDTA can be restored by the addition of a molar excess of Ca2+
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APPLICATION
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
analysis