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
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
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dTDP-N-acetylthomosamine biosynthesis
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dTDP-N-acetylviosamine biosynthesis
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dTDPLrhamnose biosynthesis
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Streptomycin biosynthesis
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Polyketide sugar unit biosynthesis
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Acarbose and validamycin biosynthesis
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Biosynthesis of vancomycin group antibiotics
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Biosynthesis of antibiotics
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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
gene L780
E3VXL5
UniProt
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
Q6TFC2
SwissProt
Manually annotated by BRENDA team
rmlB gene; E207-71
Q6TFC2
SwissProt
Manually annotated by BRENDA team
GENERAL INFORMATION
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
malfunction
C5J046;
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
C5J046;
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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-
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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
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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
Q6TFC2;
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
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dTDPglucose
8 - 8.5
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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
PDB
SCOP
CATH
UNIPROT
ORGANISM
A0A0F7RHQ6
Bacillus anthracis;
Q3ESA4
Bacillus thuringiensis serovar israelensis ATCC 35646;
P27830
Escherichia coli (strain K12);
O58151
Pyrococcus horikoshii (strain ATCC 700860 / DSM 12428 / JCM 9974 / NBRC 100139 / OT-3);
Q9EU31
Salmonella choleraesuis;
Salmonella typhimurium (strain LT2 / SGSC1412 / ATCC 700720);
P95780
Streptococcus mutans serotype c (strain ATCC 700610 / UA159);
Streptococcus suis;
Streptomyces venezuelae;
MOLECULAR WEIGHT
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
33000
E3VXL5;
x * 33000, SDS-PAGE
36500
C5J046;
sequence analysis
38000
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2 * 38000, SDS-PAGE
40000
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2 * 40000, gel filtration
47000
E3VXL5;
gel filtration
54000
C5J046;
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
E3VXL5;
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
Purification/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
by cobalt-immobilized affinity chromatography
C5J046;
gel filtration, SDS-PAGE
Q6TFC2;
recombinant enzyme
Cloned/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
expressed in Escherichia coli BL21(DE3) and DH5alpha
Q6TFC2;
expression in Escherichia coli
expression in Escherichia coli BL21
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gene L780, transcription profiling of UGER, phylogenetic analysis
E3VXL5;
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
C5J046;
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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