Information on EC 4.2.1.46 - dTDP-glucose 4,6-dehydratase

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The enzyme appears in viruses and cellular organisms

EC NUMBER
COMMENTARY hide
4.2.1.46
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RECOMMENDED NAME
GeneOntology No.
dTDP-glucose 4,6-dehydratase
REACTION
REACTION DIAGRAM
COMMENTARY hide
ORGANISM
UNIPROT
LITERATURE
dTDP-alpha-D-glucose = dTDP-4-dehydro-6-deoxy-alpha-D-glucose + H2O
show the reaction diagram
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REACTION TYPE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
elimination
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PATHWAY
BRENDA Link
KEGG Link
MetaCyc Link
Acarbose and validamycin biosynthesis
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Biosynthesis of antibiotics
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Biosynthesis of vancomycin group antibiotics
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dTDP-3-acetamido-3,6-dideoxy-alpha-D-glucose biosynthesis
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dTDP-3-acetamido-alpha-D-fucose biosynthesis
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dTDP-4-O-demethyl-beta-L-noviose biosynthesis
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dTDP-6-deoxy-alpha-D-allose biosynthesis
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dTDP-alpha-D-mycaminose biosynthesis
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dTDP-beta-L-4-epi-vancosamine biosynthesis
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dTDP-beta-L-digitoxose biosynthesis
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dTDP-D-beta-fucofuranose biosynthesis
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dTDP-D-desosamine biosynthesis
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dTDP-D-forosamine biosynthesis
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dTDP-D-olivose, dTDP-D-oliose and dTDP-D-mycarose biosynthesis
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dTDP-D-ravidosamine and dTDP-4-acetyl-D-ravidosamine biosynthesis
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dTDP-L-daunosamine biosynthesis
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dTDP-L-megosamine biosynthesis
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dTDP-L-mycarose biosynthesis
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dTDP-L-olivose biosynthesis
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dTDP-L-rhamnose biosynthesis I
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dTDP-L-rhamnose biosynthesis II
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dTDP-N-acetylthomosamine biosynthesis
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dTDP-N-acetylviosamine biosynthesis
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Polyketide sugar unit biosynthesis
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Streptomycin biosynthesis
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dTDPLrhamnose biosynthesis
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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 hide
37259-54-4
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ORGANISM
COMMENTARY hide
LITERATURE
UNIPROT
SEQUENCE DB
SOURCE
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Manually annotated by BRENDA team
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Manually annotated by BRENDA team
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Manually annotated by BRENDA team
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Manually annotated by BRENDA team
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Manually annotated by BRENDA team
Ensifer sp. AS08 448
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Manually annotated by BRENDA team
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Manually annotated by BRENDA team
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Manually annotated by BRENDA team
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Manually annotated by BRENDA team
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Manually annotated by BRENDA team
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Manually annotated by BRENDA team
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Manually annotated by BRENDA team
serovar typhimurium
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Manually annotated by BRENDA team
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Manually annotated by BRENDA team
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Manually annotated by BRENDA team
rmlB gene; E207-71
SwissProt
Manually annotated by BRENDA team
GENERAL INFORMATION
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
malfunction
inactivation of rmbB abolishes the production of doxorubicin while complementation of the same gene in an rmbB knockout mutant restores the doxorubicin production
physiological function
rmbB provides dTDP-4-dehydro-6-deoxy-D-glucose as a nucleotide sugar precursor for the biosynthesis of doxorubicin
SUBSTRATE
PRODUCT                       
REACTION DIAGRAM
ORGANISM
UNIPROT
COMMENTARY
(Substrate) hide
LITERATURE
(Substrate)
COMMENTARY
(Product) hide
LITERATURE
(Product)
Reversibility
r=reversible
ir=irreversible
?=not specified
dTDP-3-azido-3-deoxy-D-glucose
dTDP-3-azido-3,6-dideoxy-alpha-D-xylo-hexopyran-4-ulose
show the reaction diagram
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dTDP-3-deoxy-D-glucose
dTDP-3,6-dideoxy-alpha-D-erythro-hexopyran-4-ulose
show the reaction diagram
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dTDP-6-fluoro-6-deoxyglucose
dTDP-4-keto-6-deoxyglucose + F-
show the reaction diagram
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substrate undergoes fluoride ion elimination instead of dehydration
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?
dTDP-alpha-D-glucose
dTDP-4-dehydro-6-deoxy-alpha-D-glucose + H2O
show the reaction diagram
dTDP-glucose
?
show the reaction diagram
dTDP-glucose
dTDP-4-dehydro-6-deoxy-D-glucose + H2O
show the reaction diagram
dUDP-glucose
dUDP-4-dehydro-6-deoxy-D-glucose + H2O
show the reaction diagram
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UDP-D-glucose
UDP-4-dehydro-6-deoxy-D-glucose + 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
dTDP-alpha-D-glucose
dTDP-4-dehydro-6-deoxy-alpha-D-glucose + H2O
show the reaction diagram
dTDP-glucose
?
show the reaction diagram
dTDP-glucose
dTDP-4-dehydro-6-deoxy-D-glucose + H2O
show the reaction diagram
additional information
?
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enzyme of the 6-deoxyhexose biosynthetic pathway, that is also present in the aminoglycoside antibiotic biosynthetic pathway
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COFACTOR
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
METALS and IONS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
additional information
INHIBITORS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
Ag+
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irreversible
dTDP-6-deoxy-D-galactose
dTDP-6-deoxy-D-glucose
dTDP-galactose
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dTDP-xylose
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p-chloromercuribenzoate
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p-Chloromercuriphenylsulfonate
p-hydroxymercuribenzoate
KM VALUE [mM]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
0.3
dTDP-3-azido-3-deoxyglucose
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0.2
dTDP-3-deoxyglucose
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0.001 - 2.2
dTDP-glucose
0.0072 - 0.427
dTDPglucose
2.2
dUDPglucose
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0.019 - 0.2
NAD+
30
UDP-glucose
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pH 7.5, 37°C, wild-type enzyme
TURNOVER NUMBER [1/s]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
0.000064 - 0.053
dTDP-6-fluoro-6-deoxyglucose
0.0089 - 6.08
dTDP-glucose
0.024
UDP-glucose
Escherichia coli
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pH 7.5, 37°C, wild-type enzyme
Ki VALUE [mM]
INHIBITOR
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
0.097
dTDP-galactose
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pH 7.5, 37°C, wild-type enzyme
0.0112
dTDP-xylose
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pH 7.5, 37°C, wild-type enzyme
SPECIFIC ACTIVITY [µmol/min/mg]
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
0.04
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dTDPglucose
0.2
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dTDPglucose
0.31
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dTDPglucose
0.4
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dTDPglucose
1.5
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dTDP-3-azido-3-deoxyglucose
2
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dTDPglucose
2.2
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dTDP-3-deoxyglucose
4.2
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dTDPglucose
4.3
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dTDPglucose
12.3
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dTDPglucose
additional information
involved in initial steps of biosynthesis of the S-layer glycan component dTDP-3-acetamido-3,6-dideoxy-alpha-D-glucose (Quip3NAc), chromosomal organization shown, gene homologues in Gram-negative as well as in antibiotic-producing Gram-positive bacteria, also key enzyme in the biosynthetic pathway of dTDP-beta-L-rhamnose, biosynthesis pathway may also be valid for LPS (lipopolysaccharide) O-antigen structures and antibiotic precursors
pH OPTIMUM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
7.2
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dTDPglucose
7.6
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dTDPglucose
7.8
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dTDPglucose
8 - 8.5
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dTDPglucose
8
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dTDPglucose
pH RANGE
ORGANISM
UNIPROT
COMMENTARY hide
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
TEMPERATURE OPTIMUM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
MOLECULAR WEIGHT
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
33000
x * 33000, SDS-PAGE
36500
sequence analysis
38000
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2 * 38000, SDS-PAGE
40000
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2 * 40000, gel filtration
54000
recombinant enzyme
60000
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gel filtration
88000
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sedimentation equilibrium centrifugation
SUBUNITS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
monomer or dimer
x * 33000, SDS-PAGE
additional information
Crystallization/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
sitting-drop vapour-diffusion technique. Diffraction data obtained to a resolution of 2.8 A on a single frozen RmlB crystal which belongs to space group P2(1) with unit-cell parameters a = 111.85, b = 87.77, c = 145.66 A, beta = 131.53°
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structure determined to 2.47 A resolution with its cofactor NAD+ bound, sitting drop method
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wild-type and mutant enzyme D128N/E129Q crystallized as a complex with NAD+ and the substrate dTDP-glucose
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pH STABILITY
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
7.5 - 9
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rapid inactivation at acidic pH
5713
TEMPERATURE STABILITY
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
GENERAL STABILITY
ORGANISM
UNIPROT
LITERATURE
50% residual acivity after incubation at 37¦C, pH 7.0 for 2 hours, 50% residual acivity after incubation at 25¦C, pH 7.0 for 34 hours, 50% residual acivity after incubation at 25¦C, pH 8.0 for 116 hours
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enzyme can be stabilized by ammonium sulfate, 2.8 M, or bovine serum albumine, 1 mg/ml
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STORAGE STABILITY
ORGANISM
UNIPROT
LITERATURE
10% loss of activity during storage at 0¦C for 1 week
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50% loss of activity during storage at -20¦C or -80¦C for less than 1 week
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60% loss of activity during storage in 0.025 M potassium phosphate or 0.025 M Tris-HCl at 0¦C for 24 hours, stable for several days at 0¦C and buffer concentrations of 0.25 M or 20% glycerol or 34% sucrose
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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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stable for several months at -15¦C
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stable for several weeks at -20¦C
Cloned/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
expressed in Escherichia coli BL21(DE3) and DH5alpha
expression in Escherichia coli
expression in Escherichia coli BL21
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gene L780, transcription profiling of UGER, phylogenetic analysis
gene Z544R, transcription profiling of UGER, phylogenetic analysis
PCR product cloned into pET-32a(+) to construct pDH32 and transformed into Escherichia coli BL21 (DE3) to give pDH32BL for protein expression. PCR product cloned into EcoRI/HindIII of pIBR25 to construct a recombinant vector, pDHC25. The vector pDHC25 subsequently transformed into the rmbB-knockout mutant of Streptomyces peucetius DHNEO by PEG-mediated protoplast transformation
ENGINEERING
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
C187A
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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
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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
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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
D135N/E136Q
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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
H232A
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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
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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
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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
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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
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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
N190A
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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
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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
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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
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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
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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
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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
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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
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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
APPLICATION
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
analysis
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the highly conserved sequence of the dTDP-glucose-4,6-dehydratase gene is used to explore the Streptomyces tenebrarius H6 tabromycin/kanamycin biosynthetic gene cluster
synthesis
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