Information on EC 3.2.1.179 - gellan tetrasaccharide unsaturated glucuronosyl hydrolase

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The expected taxonomic range for this enzyme is: Bacillus sp.

EC NUMBER
COMMENTARY hide
3.2.1.179
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RECOMMENDED NAME
GeneOntology No.
gellan tetrasaccharide unsaturated glucuronosyl hydrolase
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REACTION
REACTION DIAGRAM
COMMENTARY hide
ORGANISM
UNIPROT
LITERATURE
beta-D-4-deoxy-DELTA4-GlcAp-(1->4)-beta-D-Glcp-(1->4)-alpha-L-Rhap-(1->3)-beta-D-Glcp + H2O = 5-dehydro-4-deoxy-D-glucuronate + beta-D-Glcp-(1->4)-alpha-L-Rhap-(1->3)-beta-D-Glcp
show the reaction diagram
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PATHWAY
BRENDA Link
KEGG Link
MetaCyc Link
chondroitin sulfate degradation I (bacterial)
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dermatan sulfate degradation I (bacterial)
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gellan degradation
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hyaluronan degradation
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SYSTEMATIC NAME
IUBMB Comments
beta-D-4-deoxy-DELTA4-GlcAp-(1->4)-beta-D-Glcp-(1->4)-alpha-L-Rhap-(1->3)-beta-D-Glcp beta-D-4-deoxy-DELTA4-GlcAp hydrolase
The enzyme releases 4-deoxy-4(5)-unsaturated D-glucuronic acid from oligosaccharides produced by polysaccharide lyases, e.g. the tetrasaccharide beta-D-4-deoxy-Delta4-GlcAp-(1->4)-beta-D-Glcp-(1->4)-alpha-L-Rhap-(1->3)-D-Glcp produced by EC 4.2.2.25, gellan lyase. The enzyme can also hydrolyse unsaturated chondroitin and hyaluronate disaccharides (beta-D-4-deoxy-Delta4-GlcAp-(1->3)-D-GalNAc, beta-D-4-deoxy-Delta4-GlcAp-(1->3)-D-GalNAc6S, beta-D-4-deoxy-Delta4-GlcAp2S-(1->3)-D-GalNAc, beta-D-4-deoxy-Delta4-GlcAp-(1->3)-D-GlcNAc), preferring the unsulfated disaccharides to the sulfated disaccharides.
ORGANISM
COMMENTARY hide
LITERATURE
UNIPROT
SEQUENCE DB
SOURCE
GENERAL INFORMATION
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
additional information
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nnnnnn
SUBSTRATE
PRODUCT                       
REACTION DIAGRAM
ORGANISM
UNIPROT
COMMENTARY
(Substrate) hide
LITERATURE
(Substrate)
COMMENTARY
(Product) hide
LITERATURE
(Product)
Reversibility
r=reversible
ir=irreversible
?=not specified
beta-D-4-deoxy-DELTA-4,5-GlcAp-(1->4)-beta-D-Glcp-(1->4)-alpha-L-Rhap-(1->3)-beta-D-Glcp + H2O
4-deoxy-L-threo-5-hexosulose-uronate + beta-D-Glcp-(1->4)-alpha-L-Rhap-(1->3)-beta-D-Glcp
show the reaction diagram
beta-D-4-deoxy-DELTA4,5-GlcAp-(1->3)-beta-D-GalNAc + H2O
4-deoxy-L-threo-5-hexosulose-uronate + beta-D-GalNAc
show the reaction diagram
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114% of the activity with beta-D-DELTA4,5-GlcAp-(1->4)-beta-D-Glcp-(1->4)-alpha-L-Rhap-(1->3)-beta-D-Glcp
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?
beta-D-4-deoxy-DELTA4,5-GlcAp-(1->3)-beta-D-GalNAc + H2O
4-deoxy-L-threo-5-hexosulose-uronate + beta-D-N-acetylgalactosamine
show the reaction diagram
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?
beta-D-4-deoxy-DELTA4,5-GlcAp-(1->3)-beta-D-GalNAc6S + H2O
4-deoxy-L-threo-5-hexosulose-uronate + beta-D-GalNAc6S
show the reaction diagram
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25.5% of the activity with beta-D-DELTA4,5-GlcAp-(1->4)-beta-D-Glcp-(1->4)-alpha-L-Rhap-(1->3)-beta-D-Glcp
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?
beta-D-4-deoxy-DELTA4,5-GlcAp-(1->3)-beta-D-GalNAc6S + H2O
4-deoxy-L-threo-5-hexosulose-uronate + beta-D-N-acetylgalactosamine-6-O-sulfate
show the reaction diagram
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kcat/Km is 8% of the kcat/KM value for the unsulfated substrate beta-D-4-deoxy-DELTA4,5-GlcAp-(1->3)-beta-D-GalNAc
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?
beta-D-4-deoxy-DELTA4,5-GlcAp-(1->3)-beta-D-GlcNAc + H2O
4-deoxy-L-threo-5-hexosulose-uronate + beta-D-GlcNAc
show the reaction diagram
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116% of the activity with beta-D-DELTA4,5-GlcAp-(1->4)-beta-D-Glcp-(1->4)-alpha-L-Rhap-(1->3)-beta-D-Glcp
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?
beta-D-4-deoxy-DELTA4,5-GlcAp-(1->4)-beta-D-Glcp-(1->4)-alpha-L-Rhap-(1->3)-beta-D-Glcp + H2O
4-deoxy-L-threo-5-hexosulose-uronate + beta-D-Glcp-(1->4)-alpha-L-Rhap-(1->3)-beta-D-Glcp
show the reaction diagram
beta-D-4-deoxy-DELTA4,5-GlcAp2S-(1->3)-beta-D-GalNAc + H2O
? + beta-D-GalNAc
show the reaction diagram
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23.5% of the activity with beta-D-DELTA4,5-GlcAp-(1->4)-beta-D-Glcp-(1->4)-alpha-L-Rhap-(1->3)-beta-D-Glcp
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?
additional information
?
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the enzyme is specific for unsaturated glucuronides and inert to saturated glucuronides. The enzyme fails to liberate nitrophenol from p-nitrophenyl-beta-D-glucuronide. The enzyme prefers unsaturated chondroitin disaccharide without sulfate to those with sulfate
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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
beta-D-4-deoxy-DELTA4,5-GlcAp-(1->4)-beta-D-Glcp-(1->4)-alpha-L-Rhap-(1->3)-beta-D-Glcp + H2O
4-deoxy-L-threo-5-hexosulose-uronate + beta-D-Glcp-(1->4)-alpha-L-Rhap-(1->3)-beta-D-Glcp
show the reaction diagram
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degradation of oligosaccharides with unsaturated uronic acid at the nonreducing terminal produced by polysaccharide lyases
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INHIBITORS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
Ca2+
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1 mM, 46% inhibition
Co2+
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1 mM, 22% inhibition
Cu2+
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1 mM, 69% inhibition
Fe2+
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1 mM, 94% inhibition
glycine
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Hg2+
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1 mM, 77% inhibition
Zn2+
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1 mM, 21% ihibition
KM VALUE [mM]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
0.06 - 0.2
beta-D-4-deoxy-DELTA-4,5-GlcAp-(1->4)-beta-D-Glcp-(1->4)-alpha-L-Rhap-(1->3)-beta-D-Glcp
0.381 - 0.861
beta-D-4-deoxy-DELTA4,5-GlcAp-(1->3)-beta-D-GalNAc
2.2 - 18.6
beta-D-4-deoxy-DELTA4,5-GlcAp-(1->3)-beta-D-GalNAc6S
TURNOVER NUMBER [1/s]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
0.00057 - 7.3
beta-D-4-deoxy-DELTA-4,5-GlcAp-(1->4)-beta-D-Glcp-(1->4)-alpha-L-Rhap-(1->3)-beta-D-Glcp
14.1 - 23.8
beta-D-4-deoxy-DELTA4,5-GlcAp-(1->3)-beta-D-GalNAc
20.9 - 54.9
beta-D-4-deoxy-DELTA4,5-GlcAp-(1->3)-beta-D-GalNAc6S
kcat/KM VALUE [1/mMs-1]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
0.0029 - 81
beta-D-4-deoxy-DELTA-4,5-GlcAp-(1->4)-beta-D-Glcp-(1->4)-alpha-L-Rhap-(1->3)-beta-D-Glcp
12109
19.6 - 42
beta-D-4-deoxy-DELTA4,5-GlcAp-(1->3)-beta-D-GalNAc
4181
2.95 - 18.1
beta-D-4-deoxy-DELTA4,5-GlcAp-(1->3)-beta-D-GalNAc6S
2921
Ki VALUE [mM]
INHIBITOR
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
6.5
glycine
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pH 6.5, 30°C
SPECIFIC ACTIVITY [µmol/min/mg]
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
additional information
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enzyme activity is measured by monitoring the decrease in absorbance at 235 nm, corresponding to the loss of the C=C double bond of the substrate because the pyranose ring of the released DELTAGlcA readily opens so that it is nonenzymatically converted to alpha-keto acid through the loss of the double bond
pH OPTIMUM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
6 - 6.5
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sodium acetate buffer
6.5
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assay at
7.5
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assay at
TEMPERATURE OPTIMUM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
30
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assay at
TEMPERATURE RANGE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
30 - 55
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30°C: about 90% of maximal activity, 55°C: about 35% of maximal activity
SOURCE TISSUE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
SOURCE
PDB
SCOP
CATH
ORGANISM
UNIPROT
Bacillus sp. (strain GL1)
Bacillus sp. (strain GL1)
Bacillus sp. (strain GL1)
Bacillus sp. (strain GL1)
Bacillus sp. (strain GL1)
Bacillus sp. (strain GL1)
Bacillus sp. (strain GL1)
MOLECULAR WEIGHT
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
SUBUNITS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
monomer
Crystallization/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
crystallized at 20°C from a droplet containing 56% 2-methyl-2,4-pentanediol, 0.1 M NaCl, 0.1 M glycine/NaOH pH 8.2 and 0.1 M dithiothreitol using the hanging-drop vapour-diffusion method. The crystals are hexagonal and belonged to space group P6122 or P6522, with unit-cell parameters a = b = 102.8, c = 223.4 A. Diffraction data to 2.4 A are collected from a single crystal
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crystals are obtained by using hanging drop vapor diffusion and microseeding, wild-type enzyme, mutant enzyme D88N and mutant enzyme D88N in complex with beta-D-4-deoxy-DELTA4,5-GlcAp-(1->3)-beta-D-GalNAc
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sitting-drop vapor duffusion method, X-ray crystallographic structure of the enzyme at 1.8 A resolution
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vapor diffusion method, demonstration of substrate recognition mechanism of the unsaturated glucuronyl hydrolase by determining the X-ray crystallographic structure of its substrate-enzyme complexes (D88N/DELTAGlcA-Glc-Pha-Glc and D99N/DELTAGlcA-GlcNAc). The tetrasaccharide-enzyme complex demonstrates that at least four subsites are present in the active pocket
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TEMPERATURE STABILITY
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
Purification/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
Cloned/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
expression in Escherichia coli
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EXPRESSION
ORGANISM
UNIPROT
LITERATURE
induced in the bacterial cells grown on either gellan or xanthan. Expression levels of unsaturated glucuronyl hydrolase in the bacterium grown on gellan and xanthan are 20.7 and 16.4 mU/mg protein, respectively, while the bacterium grown in the glucose, pectin, or LB medium fails to produce the enzyme
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ENGINEERING
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
D149N
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almost inactive mutant enzyme, kcat/Km is significantly lower than that of the wild-type enzyme for gellan tetrasaccharide
D88N
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almost inactive mutant enzyme, kcat/Km is significantly lower than that of the wild-type enzyme for gellan tetrasaccharide
G342S
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kcat/KM for beta-D-4-deoxy-DELTA4,5-GlcAp-(1->3)-beta-D-GalNAc6S is similar to the wild-type value, kcat/Km for beta-D-4-deoxy-DELTA4,5-GlcAp-(1->3)-beta-D-GalNAc is similar to wild-type value
H339S
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kcat/KM for beta-D-4-deoxy-DELTA4,5-GlcAp-(1->3)-beta-D-GalNAc6S is 1.2fold higher than the kcat/Km-value for the wild-type enzyme, kcat/Km for beta-D-4-deoxy-DELTA4,5-GlcAp-(1->3)-beta-D-GalNAc is 53% of the wild-type value
I344K
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kcat/KM for beta-D-4-deoxy-DELTA4,5-GlcAp-(1->3)-beta-D-GalNAc6S is 6.1fold higher than the wild-type value, kcat/Km for beta-D-4-deoxy-DELTA4,5-GlcAp-(1->3)-beta-D-GalNAc is similar to the wild-type value
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