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Information on EC 4.2.1.46 - dTDP-glucose 4,6-dehydratase and Organism(s) Escherichia coli and UniProt Accession P27830
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4.2.1.46 dTDP-glucose 4,6-dehydrataseIUBMB Comments
Requires bound NAD+.
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Word Map
- 4.2.1.46
- dtdp-l-rhamnose
- dtdp-4-keto-6-deoxy-d-glucose
-
3,5-epimerase
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dtdp-4-dehydrorhamnose
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o-antigen
-
deoxysugars
- dtmp
-
thymidylyltransferase
-
6-deoxyhexose
- peucetius
- analysis
- synthesis
- pharmacology
The taxonomic range for the selected organisms is: Escherichia coli
The enzyme appears in selected viruses and cellular organisms
The enzyme appears in selected viruses and cellular organisms
Synonyms
dtdp-d-glucose 4,6-dehydratase, dtdp-glucose 4,6-dehydratase, dtdp-glucose-4,6-dehydratase, tdp-d-glucose 4,6-dehydratase, desiv, rml-2, udp-d-glucose 4,6-dehydratase, 
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topSYNONYM 
ORGANISM
UNIPROT
COMMENTARY 
LITERATURE
dTDP-D-glucose 4,6-dehydratase
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-
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dTDP-D-glucose oxidoreductase
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-
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TDP-D-glucose 4,6-dehydratase
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TDP-glucose oxidoreductase
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Thymidine diphosphate D-glucose oxidoreductase
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Thymidine diphospho-D-glucose 4,6-dehydratase
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Thymidine diphosphoglucose oxidoreductase
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REACTION TYPE
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
elimination
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PATHWAY SOURCE
PATHWAYS
KEGG
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-, -, -, -, -, -, -, -, -, -, -, -, -, -, -, -, -, -, -, -
SYSTEMATIC NAME
IUBMB Comments
dTDP-glucose 4,6-hydro-lyase (dTDP-4-dehydro-6-deoxy-D-glucose-forming)
Requires bound NAD+.
CAS REGISTRY NUMBER
COMMENTARY
37259-54-4
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SUBSTRATE
PRODUCT
REACTION DIAGRAM
ORGANISM
UNIPROT
COMMENTARY
(Substrate)
(Substrate)
LITERATURE
(Substrate)
(Substrate)
COMMENTARY
(Product)
(Product)
LITERATURE
(Product)
(Product)
Reversibility
r=reversible
ir=irreversible
?=not specified
r=reversible
ir=irreversible
?=not specified
dTDP-6-fluoro-6-deoxyglucose
dTDP-4-keto-6-deoxyglucose + F-
substrate undergoes fluoride ion elimination instead of dehydration
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?
dTDP-3-azido-3-deoxy-D-glucose
dTDP-3-azido-3,6-dideoxy-alpha-D-xylo-hexopyran-4-ulose
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-
-
?
dTDP-3-deoxy-D-glucose
dTDP-3,6-dideoxy-alpha-D-erythro-hexopyran-4-ulose
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-
-
?
dTDP-glucose
dTDP-4-dehydro-6-deoxy-D-glucose + H2O
the conversion takes place in three steps: dehydrogenation to dTDP-4-ketoglucose, dehydration to dTDP-4-ketoglucose-5,6-ene and rereduction of C6 to the methyl group. Asp135 and Glu136 are the acid and base catalysts, respectively of the dehydration step
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?
dTDP-glucose
dTDP-4-dehydro-6-deoxy-D-glucose + H2O
the single-turnover kinetic mechanism for the reaction is determined by rapid mix-chemical quench mass spectrometry. NAD+ initially oxidizes glucosyl C4 of dTDP-glucose to NADH and dTDP-4-ketoglucose. Next water is eliminated between C5 and C6 of dTDP-4-ketoglucose to form dTDP-4-ketoglucose-5,6-ene. Hydride transfer from NADH to C6 of dTDP-4-ketoglucose-5,6-ene regenerates NAD+ and produces the product dTDP-4-keto-4-deoxyglucose
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?
dTDP-glucose
dTDP-4-dehydro-6-deoxy-D-glucose + H2O
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the conversion takes place in three steps: dehydrogenation to dTDP-4-ketoglucose, dehydration to dTDP-4-ketoglucose-5,6-ene and rereduction of C6 to the methyl group
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?
NATURAL SUBSTRATE
NATURAL PRODUCT
REACTION DIAGRAM
ORGANISM
UNIPROT
COMMENTARY
(Substrate)
(Substrate)
LITERATURE
(Substrate)
(Substrate)
COMMENTARY
(Product)
(Product)
LITERATURE
(Product)
(Product)
REVERSIBILITY
r=reversible
ir=irreversible
?=not specified
r=reversible
ir=irreversible
?=not specified
COFACTOR
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
NADH
45% of the wild-type enzyme contains NADH during steady-state turnover for each variant by adding a large excess of dTDPglucose, 9% of mutant enzyme D135N, 30% of mutant enzyme D135A, 3% of mutant enzyme E136Q and H232N, 2% of mutant enzyme K199M and K199R, 39% of mutant enzyme Y301F, 8% of mutant enzyme C187A and less than 0.5% of mutant enzymes D135N/E136Q, E198Q, N190D, N190A and H232Q
NADH
purified enzyme contains coenzyme in the reduced form. The bound NADH is oxidized to NAD+ by adding dTDP-4-keto-6-deoxyglucose. When all the coenzyme is in the oxidized form, the dTDP-sugars remove from the enzyme. In the Y160A variant, almost 98% of the coenzyme is tightly bound NADH. Variants K164M, T134V and Y160F contain 62%, 56% and 35% of bound NADH respectively
INHIBITOR
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
p-chloromercuriphenylsulfonate
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partial reactivation with cysteine and NAD+
p-hydroxymercuribenzoate
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complete inhibition after incubation with 10 mM for 15 min at 25¦C, recovery of activity with 2-mercaptoethanol
KM VALUE [mM]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
TURNOVER NUMBER [1/s]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
Ki VALUE [mM]
INHIBITOR
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
SPECIFIC ACTIVITY [µmol/min/mg]
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
pH OPTIMUM
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
pH RANGE
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
7 - 9
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at pH 7.0 50% of activity at pH 8.0, at pH 9.0 90% of activity at pH 8.0
ORGANISM
COMMENTARY
LITERATURE
UNIPROT
SEQUENCE DB
SOURCE
PDB
SCOP
CATH
UNIPROT
ORGANISM
MOLECULAR WEIGHT
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
78000
SUBUNIT
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
CRYSTALLIZATION (Commentary)
ORGANISM
UNIPROT
LITERATURE
PROTEIN VARIANTS
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
C187A
9.4fold decrease in turnover number for dTDP-glucose compared to wild-type value, 6fold decrease in KM-value for dTDP-glucose compared to wild-type value. 8% of the mutant enzyme contains NADH during steady-state turnover by adding a large excess of dTDPglucose, compared to 45% of the wild-type enzyme
C187S
4.1fold decrease in turnover number for dTDP-glucose compared to wild-type value, 4fold decrease in KM-value for dTDP-glucose compared to wild-type value. 5% of the mutant enzyme contains NADH during steady-state turnover by adding a large excess of dTDPglucose, compared to 45% of the wild-type enzyme
D135135N/E136Q
the turnover number for dTDP-6-fluoro-6-deoxyglucose is 340fold lower than that of the wild-type enzyme, the turnover number for dTDP-glucose is 204fold lower than that of the wild-type enzyme
D135A
D135N
D135N/E136Q
204fold decrease in turnover number for dTDP-glucose compared to wild-type value, 3.2fold increase in KM-value for dTDP-glucose compared to wild-type value. Less than 0.5% of the mutant enzyme contains NADH during steady-state turnover by adding a large excess of dTDPglucose, compared to 45% of the wild-type enzyme
E136A
E136Q
E198Q
H232A
57.6fold decrease in turnover number for dTDP-glucose compared to wild-type value, 1.8fold decrease in KM-value for dTDP-glucose compared to wild-type value
H232N
6.8fold decrease in turnover number for dTDP-glucose compared to wild-type value, 1.2fold decrease in KM-value for dTDP-glucose compared to wild-type value. 3% of the mutant enzyme contains NADH during steady-state turnover by adding a large excess of dTDPglucose, compared to 45% of the wild-type enzyme
H232Q
114fold decrease in turnover number for dTDP-glucose compared to wild-type value, 1.3fold increase in KM-value for dTDP-glucose compared to wild-type value. Less than 0.5% of the mutant enzyme contains NADH during steady-state turnover by adding a large excess of dTDPglucose, compared to 45% of the wild-type enzyme
K164A
15fold increase in Km-value for dTDP-glucose, 820fold decrease in ratio of turnover number to Km-value for dTDP-glucose, 34fold decrease in turnover-number for dTDP-glucose
K164M
8.7fold increase in Km-value for dTDP-glucose, 837fold decrease in ratio of turnover number to Km-value for dTDP-glucose, 96fold decrease in turnover-number for dTDP-glucose
K199M
K199R
N190A
551fold decrease in turnover number for dTDP-glucose compared to wild-type value, 0.92fold increase in KM-value for dTDP-glucose compared to wild-type value. Less than 0.5% of the mutant enzyme contains NADH during steady-state turnover by adding a large excess of dTDPglucose, compared to 45% of the wild-type enzyme
N190D
441fold decrease in turnover number for dTDP-glucose compared to wild-type value, fold 4.2increase in KM-value for dTDP-glucose compared to wild-type value. Less than 0.5% of the mutant enzyme contains NADH during steady-state turnover by adding a large excess of dTDPglucose, compared to 45% of the wild-type enzyme
N190H
217fold decrease in turnover number for dTDP-glucose compared to wild-type value, 1.2fold increase in KM-value for dTDP-glucose compared to wild-type value
T134A
1.2fold increase in Km-value for dTDP-glucose, 283fold decrease in ratio of turnover number to Km-value for dTDP-glucose, 233fold decrease in turnover-number for dTDP-glucose
T134S
3.7fold increase in Km-value for dTDP-glucose, 7.5fold decrease in ratio of turnover number to Km-value for dTDP-glucose, 2fold decrease in turnover-number for dTDP-glucose
T134V
3.3fold increase in Km-value for dTDP-glucose, 788fold decrease in ratio of turnover number to Km-value for dTDP-glucose, 237fold decrease in turnover-number for dTDP-glucose
Y160A
2.8fold increase in Km-value for dTDP-glucose, 683fold decrease in ratio of turnover number to Km-value for dTDP-glucose, 247fold decrease in turnover-number for dTDP-glucose
Y160F
1.2fold increase in Km-value for dTDP-glucose, 234fold decrease in ratio of turnover number to Km-value for dTDP-glucose, 190fold decrease in turnover-number for dTDP-glucose
Y301F
D135A
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switch from a concerted to stepwise dehydration mechanism is due to the loss of control over the glucosyl C5C6 bond rotation in the active site
D135N
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switch from a concerted to stepwise dehydration mechanism is due to the loss of control over the glucosyl C5C6 bond rotation in the active site
D135A
222fold decrease in turnover number for dTDP-glucose compared to wild-type value, 1.2fold increase in KM-value for dTDP-glucose compared to wild-type value. 30% of the mutant enzyme contains NADH during steady-state turnover by adding a large excess of dTDPglucose, compared to 45% of the wild-type enzyme
D135A
the steady-state rate of conversion of dTDP-6-fluoro-6-deoxyglucose to dTDP-4-keto-6-deoxyglucose is identical to that of wild-type. The turnover number for dTDP-6-fluoro-6-deoxyglucose is 1.5fold lower than that of the wild-type enzyme, the turnover number for dTDP-glucose is 223fold lower than that of the wild-type enzyme
D135N
124fold decrease in turnover number for dTDP-glucose compared to wild-type value, 1.3fold increase in KM-value for dTDP-glucose compared to wild-type value. 9% of the mutant enzyme contains NADH during steady-state turnover by adding a large excess of dTDPglucose, compared to 45% of the wild-type enzyme
D135N
the steady-state rate of conversion of dTDP-6-fluoro-6-deoxyglucose to dTDP-4-keto-6-deoxyglucose is identical to that of wild-type. The turnover number for dTDP-6-fluoro-6-deoxyglucose is 1.2fold higher than that of the wild-type enzyme, the turnover number for dTDP-glucose is 124fold lower than that of the wild-type enzyme
E136A
288fold decrease in turnover number for dTDP-glucose compared to wild-type value, 2fold increase in KM-value for dTDP-glucose compared to wild-type value. Less than 0.5% of the mutant enzyme contains NADH during steady-state turnover by adding a large excess of dTDPglucose, compared to 45% of the wild-type enzyme
E136A
the turnover number for dTDP-6-fluoro-6-deoxyglucose is 690fold lower than that of the wild-type enzyme, the turnover number for dTDP-glucose is 288fold lower than that of the wild-type enzyme
E136Q
67fold decrease in turnover number for dTDP-glucose compared to wild-type value, 1.5fold increase in KM-value for dTDP-glucose compared to wild-type value. 3% of the mutant enzyme contains NADH during steady-state turnover by adding a large excess of dTDPglucose, compared to 45% of the wild-type enzyme
E136Q
the turnover number for dTDP-6-fluoro-6-deoxyglucose is 69fold lower than that of the wild-type enzyme, the turnover number for dTDP-glucose is 67fold lower than that of the wild-type enzyme
E198Q
258fold decrease in turnover number for dTDP-glucose compared to wild-type value, 1.8fold increase in KM-value for dTDP-glucose compared to wild-type value. Less than 0.5% of the mutant enzyme contains NADH during steady-state turnover by adding a large excess of dTDPglucose, compared to 45% of the wild-type enzyme
E198Q
the turnover number for dTDP-6-fluoro-6-deoxyglucose is 190fold lower than that of the wild-type enzyme, the turnover number for dTDP-glucose is 258fold lower than that of the wild-type enzyme
K199M
115fold decrease in turnover number for dTDP-glucose compared to wild-type value, 1.3fold increase in KM-value for dTDP-glucose compared to wild-type value. 2% of the mutant enzyme contains NADH during steady-state turnover by adding a large excess of dTDPglucose, compared to 45% of the wild-type enzyme
K199M
the turnover number for dTDP-6-fluoro-6-deoxyglucose is 260fold lower than that of the wild-type enzyme, the turnover number for dTDP-glucose is 115fold lower than that of the wild-type enzyme
K199R
288fold decrease in turnover number for dTDP-glucose compared to wild-type value, 2.2fold increase in KM-value for dTDP-glucose compared to wild-type value. 2% of the mutant enzyme contains NADH during steady-state turnover by adding a large excess of dTDPglucose, compared to 45% of the wild-type enzyme
K199R
the turnover number for dTDP-6-fluoro-6-deoxyglucose is 680fold lower than that of the wild-type enzyme, the turnover number for dTDP-glucose is 288fold lower than that of the wild-type enzyme
Y301F
117fold decrease in turnover number for dTDP-glucose compared to wild-type value, 5.2fold increase in KM-value for dTDP-glucose compared to wild-type value. 39% of the mutant enzyme contains NADH during steady-state turnover by adding a large excess of dTDPglucose, compared to 45% of the wild-type enzyme
Y301F
the turnover number for dTDP-6-fluoro-6-deoxyglucose is 31fold lower than that of the wild-type enzyme, the turnover number for dTDP-glucose is 117fold lower than that of the wild-type enzyme
STORAGE STABILITY
ORGANISM
UNIPROT
LITERATURE
stable at -12¦C, 10% loss of activity during storage at room temperature for 1 h, loss of activity upon repeated freezing and thawing
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PURIFICATION (Commentary)
ORGANISM
UNIPROT
LITERATURE
APPLICATION
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
synthesis
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hemo-enzymatic syntheses of artificial and naturally occuring deoxysugars
REF.
AUTHORS
TITLE
JOURNAL
VOL.
PAGES
YEAR
ORGANISM (UNIPROT)
PUBMED ID
SOURCE
Glaser, L.; Zarkowsky, H.
Dehydration in nucleotide-linked deoxysugar synthesis
The Enzymes, 3rd Ed. (Boyer, P. D. , ed. )
5
465-480
1971
Escherichia coli
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Zarkowsky, H.; Lipkin, E.; Glaser, L.
The mechanism of 6-deoxyhexose synthesis. V. The relation of pyridine nucleotide to the structure of the deoxythymidine diphosphate-glucose oxidoreductase
J. Biol. Chem.
245
6599-6606
1970
Escherichia coli
Zarkowsky, H.; Lipkin, E.; Glaser, L.
The mechanism of 6-deoxyhexose synthesis. IV. The role of pyridine nucleotide in substrate release
Biochem. Biophys. Res. Commun.
38
787-793
1970
Escherichia coli
Wang, S.F.; Gabriel, O.
Biological mechanisms involved in the formation of deoxysugars. VI. Role and function of enzyme-bound nicotinamide adenine dinucleotide in thymidine diphosphate D-glucose oxidoreductase
J. Biol. Chem.
245
8-14
1970
Escherichia coli
Zarkowsky, H.; Glaser, L.
The mechanism of 6-deoxyhexose synthesis. III. Purification of deoxythymidine diphosphate-glucose oxidoreductase
J. Biol. Chem.
244
4750-4756
1969
Escherichia coli
Wang, S.F.; Gabriel, O.
Biological mechanisms involved in the formation of deoxysugars. V. Isolation and characterization of thymidine diphosphate D-glucose oxidoreductase from Escherichia coli B
J. Biol. Chem.
244
3430-3437
1969
Escherichia coli
Melo, A.; Elliott, W.H.; Glaser, L.
The mechanism of 6-deoxyhexose synthesis. I. Intramolecular hydrogen transfer catalyzed by deoxythymidine diphosphate-D-glucose oxidoreductase
J. Biol. Chem.
243
1467-1474
1968
Escherichia coli
Gilbert, J.M.; Matsuhashi, M.; Strominger, J.L.
Thymidine diphosphate 4-acetamido-4,6-dideoxyhexoses. II. Purification and properties of thymidine diphosphate D-glucose oxidoreductase
J. Biol. Chem.
240
1305-1308
1965
Escherichia coli
Naundorf, A.; Klaffke, W.
Substrate specificity of native dTDP-D-glucose-4,6-dehydratase. Chemo-enzymatic syntheses of artificial and naturally occuring deoxy sugars
Carbohydr. Res.
285
141-150
1996
Escherichia coli
Gross, J.W.; Hegeman, A.D.; Vestling, M.M.; Frey, P.A.
Characterization of enzymatic processes by rapid mix-quench mass spectrometry: the case of dTDP-glucose 4,6-dehydratase
Biochemistry
39
13633-13640
2000
Escherichia coli (P27830)
Gross, J.W.; Hegeman, A.D.; Gerratana, B.; Frey, P.A.
Dehydration is catalyzed by glutamate-136 and aspartic acid-135 active site residues in Escherichia coli dTDP-glucose 4,6-dehydratase
Biochemistry
40
12497-12504
2001
Escherichia coli (P27830), Escherichia coli
Hegeman, A.D.; Gross, J.W.; Frey, P.A.
Probing catalysis by Escherichia coli dTDP-glucose-4,6-dehydratase: identification and preliminary characterization of functional amino acid residues at the active site
Biochemistry
40
6598-6610
2001
Escherichia coli (P27830), Escherichia coli
Gerratana, B.; Cleland, W.W.; Frey, P.A.
Mechanistic roles of Thr134, Tyr160, and Lys 164 in the reaction catalyzed by dTDP-glucose 4,6-dehydratase
Biochemistry
40
9187-9195
2001
Escherichia coli (P27830), Escherichia coli
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