Information on EC 4.2.1.115 - UDP-N-acetylglucosamine 4,6-dehydratase (configuration-inverting)

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The expected taxonomic range for this enzyme is: Bacteria

EC NUMBER
COMMENTARY hide
4.2.1.115
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RECOMMENDED NAME
GeneOntology No.
UDP-N-acetylglucosamine 4,6-dehydratase (configuration-inverting)
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REACTION
REACTION DIAGRAM
COMMENTARY hide
ORGANISM
UNIPROT
LITERATURE
UDP-N-acetyl-alpha-D-glucosamine = UDP-2-acetamido-2,6-dideoxy-beta-L-arabino-hex-4-ulose + H2O
show the reaction diagram
PATHWAY
BRENDA Link
KEGG Link
MetaCyc Link
Amino sugar and nucleotide sugar metabolism
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CMP-diacetamido-8-epilegionaminic acid biosynthesis
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CMP-pseudaminate biosynthesis
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superpathway of UDP-N-acetylglucosamine-derived O-antigen building blocks biosynthesis
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UDP-N-acetyl-beta-L-fucosamine biosynthesis
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UDP-N-acetyl-beta-L-quinovosamine biosynthesis
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SYSTEMATIC NAME
IUBMB Comments
UDP-N-acetylglucosamine hydro-lyase (inverting; UDP-2-acetamido-2,6-dideoxy-beta-L-arabino-hex-4-ulose-forming)
Contains NADP+ as a cofactor. This is the first enzyme in the biosynthetic pathway of pseudaminic acid [3], a sialic-acid-like sugar that is unique to bacteria and is used by Helicobacter pylori to modify its flagellin. This enzyme plays a critical role in H. pylori's pathogenesis, being involved in the synthesis of both functional flagella and lipopolysaccharides [1,2]. It is completely inhibited by UDP-alpha-D-galactose. The reaction results in the chirality of the C-5 atom being inverted. It is thought that Lys-133 acts sequentially as a catalytic acid, protonating the C-6 hydroxy group and as a catalytic base, abstracting the C-5 proton, resulting in the elimination of water. This enzyme belongs to the short-chain dehydrogenase/reductase family of enzymes.
CAS REGISTRY NUMBER
COMMENTARY hide
9076-60-2
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ORGANISM
COMMENTARY hide
LITERATURE
UNIPROT
SEQUENCE DB
SOURCE
strain B204, gene flaA1
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Manually annotated by BRENDA team
strain B204, gene flaA1
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Manually annotated by BRENDA team
strain 11168
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Manually annotated by BRENDA team
strain NCTC 11637; strain NCTC 11637, gene flaA1
UniProt
Manually annotated by BRENDA team
strain SS1; strain SS1, gene flaA1
UniProt
Manually annotated by BRENDA team
strain PAO1, gene wbpM
UniProt
Manually annotated by BRENDA team
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Q7A2Y4
UniProt
Manually annotated by BRENDA team
GENERAL INFORMATION
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
evolution
Q7A2Y4
CapE belongs to a distinctive subfamily of SDR enzymes of pathogenic bacteria characterized by a singular catalytic triad displaying a Met residue (instead of the canonical Tyr residue) and a dynamic element known as the latch. Although CapE and FlaA1 afford the same product, the configuration of their active sites is different. Also, the latch region is absent in FlaA1
metabolism
Q7A2Y4
conversion of UDP-D-GlcNAc into UDP-L-FucNAc, an essential precursor of capsular polysaccharide requires three enzymes CapE, CapF, and CapG in Staphylococcus aureus. CapE yields the first intermediate of the sequential reactions catalyzed by these three enzymes
physiological function
Q7A2Y4
CapE is an essential enzyme for the synthesis of capsular polysaccharide of pathogenic strains of Staphylococcus aureus
SUBSTRATE
PRODUCT                       
REACTION DIAGRAM
ORGANISM
UNIPROT
COMMENTARY
(Substrate) hide
LITERATURE
(Substrate)
COMMENTARY
(Product) hide
LITERATURE
(Product)
Reversibility
r=reversible
ir=irreversible
?=not specified
2 UDP-N-acetylglucosamine
UDP-2-acetamido-2,6-dideoxy-beta-L-arabino-4-hexulose + UDP-2-acetamido-2,6-dideoxy-alpha-D-xylo-4-hexulose + 2 H2O
show the reaction diagram
UDP-6-deoxy-6-fluoro-GlcNAc
UDP-GlcNAc + HF
show the reaction diagram
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elimination of fluoride from the substrate by the wild-type PseB, no activity by mutant enzymes K127A, D126N, and Y135F
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?
UDP-GlcNAc
UDP-2-acetamido-2,6-dideoxy-beta-L-arabino-hexos-4-ulose + H2O
show the reaction diagram
UDP-N-acetyl-alpha-D-glucosamine
UDP-2-acetamido-2,6-dideoxy-beta-L-arabino-hex-4-ulose + H2O
show the reaction diagram
UDP-N-acetylglucosamine
UDP-2-acetamido-2,6-dideoxy-beta-L-arabino-hex-4-ulose + H2O
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
2 UDP-N-acetylglucosamine
UDP-2-acetamido-2,6-dideoxy-beta-L-arabino-4-hexulose + UDP-2-acetamido-2,6-dideoxy-alpha-D-xylo-4-hexulose + 2 H2O
show the reaction diagram
UDP-GlcNAc
UDP-2-acetamido-2,6-dideoxy-beta-L-arabino-hexos-4-ulose + H2O
show the reaction diagram
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the enzyme is involved in the pseudaminic acid biosynthesis, it is responsible for the biosynthesis of 6-deoxyhexose
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?
UDP-N-acetyl-alpha-D-glucosamine
UDP-2-acetamido-2,6-dideoxy-beta-L-arabino-hex-4-ulose + H2O
show the reaction diagram
Q7A2Y4
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?
UDP-N-acetylglucosamine
UDP-2-acetamido-2,6-dideoxy-beta-L-arabino-hex-4-ulose + H2O
show the reaction diagram
additional information
?
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COFACTOR
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
NAD(P)+
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dependent on, tightly bound
NADPH
Q7A2Y4
binding and binding site structure, overview. Function of Tyr164 is related to its bulkiness in the relay mechanism
additional information
INHIBITORS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
CMP-pseudaminic acid
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mapping of the dynamic interactions of the enzyme with the inhibitor, overview
KM VALUE [mM]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
0.151 - 2.77
UDP-N-acetylglucosamine
TURNOVER NUMBER [1/s]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
0.028 - 2.8
UDP-N-acetylglucosamine
SPECIFIC ACTIVITY [µmol/min/mg]
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
0.000037
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wild-type enzyme activity with UDP-6-deoxy-6-fluoro-GlcNAc for elimination of fluoride
0.23
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wild-type enzyme activity with UDP-GlcNAc conversion to UDP-4-keto-6-deoxy-L-IdoNAc
additional information
pH OPTIMUM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
7.4
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assay at
8
Q7A2Y4
assay at
TEMPERATURE OPTIMUM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
23 - 30
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assay at
TEMPERATURE RANGE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
pI VALUE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
8.65
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sequence calculation
LOCALIZATION
ORGANISM
UNIPROT
COMMENTARY hide
GeneOntology No.
LITERATURE
SOURCE
PDB
SCOP
CATH
ORGANISM
UNIPROT
Helicobacter pylori (strain ATCC 700392 / 26695)
Helicobacter pylori (strain ATCC 700392 / 26695)
Helicobacter pylori (strain ATCC 700392 / 26695)
Helicobacter pylori (strain ATCC 700392 / 26695)
Helicobacter pylori (strain ATCC 700392 / 26695)
Staphylococcus aureus (strain Mu50 / ATCC 700699)
Staphylococcus aureus (strain Mu50 / ATCC 700699)
Staphylococcus aureus (strain Mu50 / ATCC 700699)
MOLECULAR WEIGHT
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
75000
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about, recombinant His-tagged enzyme, gel filtration
SUBUNITS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
?
x * 63000, recombinant N-terminally His-tagged WbpM, SDS-PAGE, x * 64000, recombinant C-terminally His-tagged WbpM, SDS-PAGE, x * 60000, recombinant His-tagged truncated WbpM, SDS-PAGE
dimer
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2 * 37400, recombinant His-tagged enzyme, SDS-PAGE
hexamer
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the enzyme possesses a hexameric doughnut-shaped quaternary structure, crystal stucture analysis
additional information
Crystallization/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
FlaA1-NADP+-UDP-GlcNAc and FlaA1-NADP+-UDPGlc ternary complexes, at room temperature by hanging-drop vapor diffusion in presence of NAD+, using as reservoir solution 10% v/v polyethylene glycol-200, 100 mM MES, pH 6.0, 5% v/w PEG 3000, and 4% acetone, and providing 10 mM excess of substrate, X-ray diffraction structure determination and analysis
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purified enzyme in complex with by-product UDP-xylo-sugar from 10 mM Tris-HCl. pH 9.0, 30 mM NaCl, and 1 mM DTT, suitable crystals are soaked in mother liquor supplemented with 25% v/v glycerol and 0.5 mlM UDP-D-GlcNAc, X-ray diffraction structure determination and analysis at 1.88 A resolution. The structure of CapE is virtually identical to that of apo-CapE in complex with a substrate analogue
Q7A2Y4
Purification/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
recombinant His-tagged FlaA1 9.9fold from Escherichia coli strain BL21(DE3) to homogeneity by nickel chelation and cation exchange chromatography
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recombinant His-tagged wild-type and mutant enzymes from Escherichia coli strain BL21(DE3)
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recombinant N-terminally His-tagged and C-terminally His-tagged WbpMs from Escherichia coli by selective solubilization from the inner membrane by lauryl sarcosine and nickel chelation chromatography
Cloned/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
gene flaA1, DNA and amino acid sequence determination and analysis
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gene flaA1, DNA and amino acid sequence determination, expression analysis; gene flaA1, DNA and amino acid sequence determination, expression analysis
gene flaA1, expression of the N-terminally His-tagged enzyme in Escherichia coli strain BL21(DE3), complementation of a Pseudomonas aeruginosa WbpM knockout by expression of His-tagged FlaA1
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gene pseB, expression of His-tagged wild-type and mutant enzymes in Escherichia coli strain BL21(DE3)
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overexpression of wild-type N-terminally His-tagged and C-terminally His-tagged WbpMs, and mutant truncated WbpM in membranes of Escherichia coli, reaction yields are lower with C-terminally His-tagged than with N-terminally His-tagged recombinant enzyme
ENGINEERING
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
D126N
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site-directed mutagenesis, the mutant shows about 100fold lower activity with UDP-GlcNAc and with UDP-6-deoxy-6-fluoro-GlcNAc for HF elimination compared to the wild-type enzyme. Upon addition of UDP-4-keto-6-deoxy-GlcNAc to D126N the tightly bound NADPH is immediately oxidized
K127A
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site-directed mutagenesis, the mutant shows about 100fold lower activity with UDP-GlcNAc and with UDP-6-deoxy-6-fluoro-GlcNAc for HF elimination compared to the wild-type enzyme. Upon addition of UDP-4-keto-6-deoxy-GlcNAc to K127A the tightly bound NADPH is immediately oxidized
Y135F
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site-directed mutagenesis, the mutant shows about 100fold lower activity with UDP-GlcNAc and with UDP-6-deoxy-6-fluoro-GlcNAc for HF elimination compared to the wild-type enzyme, slow oxidation of NADPH upon addition of UDP-4-keto-6-deoxy-GlcNAc to Y135F
C103M
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site-directed mutagenesis, the mutant is inactive, dimerization is prevented but the secondary structure is not significantly affected
C118M
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site-directed mutagenesis, not recombinantly expressable mutant
D149K/K150A
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site-directed mutagenesis, the mutant is inactive
D149K/K150D
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site-directed mutagenesis, the mutant is inactive
D70A
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site-directed mutagenesis, the mutant is inactive
D70N
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site-directed mutagenesis, not recombinantly expressable mutant
G20A
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site-directed mutagenesis, the mutant is inactive
H86A
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site-directed mutagenesis, insoluble protein, the mutant shows reduced activity compared to the wild-type enzyme
H86F
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site-directed mutagenesis, insoluble protein, the mutant is inactive
K133E
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site-directed mutagenesis, inactive mutant
K133M
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site-directed mutagenesis, inactive mutant
V266E
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site-directed mutagenesis, the mutant shows increased activity compared to the wild-type enzyme
Y141F
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site-directed mutagenesis, the mutant is inactive
Y141M
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site-directed mutagenesis of a FlaA1 catalytic triad mutant, the mutant shows slightly reduced activity compared to the wild-type enzyme
F78W
Q7A2Y4
site-directed mutagenesis of a cofactor binding residue, the mutant shows similar activity compared to the wild-type enzyme
L122F
Q7A2Y4
site-directed mutagenesis of a cofactor binding residue, the mutant shows reduced activity compared to the wild-type enzyme
Y164A
Q7A2Y4
site-directed mutagenesis of a cofactor binding residue, the mutant shows highly reduced activity compared to the wild-type enzyme
Y164F
Q7A2Y4
site-directed mutagenesis of a cofactor binding residue, the mutant shows markedly reduced activity compared to the wild-type enzyme
additional information
APPLICATION
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
drug development
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PseB is a potential target for the development of antibiotics