Information on EC 2.3.1.28 - chloramphenicol O-acetyltransferase

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The expected taxonomic range for this enzyme is: Bacteria, Eukaryota

EC NUMBER
COMMENTARY hide
2.3.1.28
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RECOMMENDED NAME
GeneOntology No.
chloramphenicol O-acetyltransferase
REACTION
REACTION DIAGRAM
COMMENTARY hide
ORGANISM
UNIPROT
LITERATURE
acetyl-CoA + chloramphenicol = CoA + chloramphenicol 3-acetate
show the reaction diagram
REACTION TYPE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
Acyl group transfer
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SYSTEMATIC NAME
IUBMB Comments
acetyl-CoA:chloramphenicol 3-O-acetyltransferase
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CAS REGISTRY NUMBER
COMMENTARY hide
9040-07-7
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ORGANISM
COMMENTARY hide
LITERATURE
UNIPROT
SEQUENCE DB
SOURCE
chloramphenicol resistant strains OC, NR, SIN, and T, susceptible control strains DSM8716, ATCC 21536, and ATCC 21537
UniProt
Manually annotated by BRENDA team
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Manually annotated by BRENDA team
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Manually annotated by BRENDA team
infected with either TE/3'2J/CAT or rep5/CAT/26S virus
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-
Manually annotated by BRENDA team
Escherichia coli J53(R387)
strain J53(R387)
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-
Manually annotated by BRENDA team
Escherichia coli W677/HJR66
strain W677/HJR66
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-
Manually annotated by BRENDA team
CB60
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Manually annotated by BRENDA team
CB60
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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
strain PA 103
Uniprot
Manually annotated by BRENDA team
strain Pahcr, mutations G61S and 105C contribute synergistically to the Pahcr1 resistance phenotype
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-
Manually annotated by BRENDA team
strain C22.1
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Manually annotated by BRENDA team
enzyme variant B
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Manually annotated by BRENDA team
enzyme is encoded by plasmid Rms418
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Manually annotated by BRENDA team
GENERAL INFORMATION
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
physiological function
SUBSTRATE
PRODUCT                       
REACTION DIAGRAM
ORGANISM
UNIPROT
COMMENTARY
(Substrate) hide
LITERATURE
(Substrate)
COMMENTARY
(Product) hide
LITERATURE
(Product)
Reversibility
r=reversible
ir=irreversible
?=not specified
acetyl-CoA + chloramphenicol
CoA + chloramphenicol 1,3-diacetate
show the reaction diagram
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-
-
-
?
acetyl-CoA + chloramphenicol
CoA + chloramphenicol 3-acetate
show the reaction diagram
acetyl-CoA + chloramphenicol 1-acetate
CoA + chloramphenicol 1,3-diacetate
show the reaction diagram
acetyl-CoA + D-threo-1-p-nitrophenyl-2-bromoacetamido-1,3-propanediol
CoA + ?
show the reaction diagram
acetyl-CoA + D-threo-1-phenyl-2-dichloroacetamido-1,3-propanediol
CoA + ?
show the reaction diagram
chloramphenicol + acetyl-CoA
chloramphenicol 3-acetate + CoA
show the reaction diagram
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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
acetyl-CoA + chloramphenicol
CoA + chloramphenicol 3-acetate
show the reaction diagram
chloramphenicol + acetyl-CoA
chloramphenicol 3-acetate + CoA
show the reaction diagram
Q6XD71
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-
?
METALS and IONS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
HgCl2
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at 0.1 M and 0.3 mM, no influence on the activity of the pSCS6-encoded enzyme. The activity of the pSCS7-encoded enzyme decreases to 10-15%. At 1.0 mM HgCl2, the pSCS6-encoded enzyme retains 50% of ist original activity, complete inactivation of the enzyme from pSCS7-encoded enzyme
INHIBITORS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
1,5-Difluoro-2,4-dinitrobenzene
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2,2'-dithiobis(pyridine)
4,4'-dithiodipyridine
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5,5'-dithiobis(2-nitrobenzoic acid)
Crystal violet
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strong, competitive for chloramphenicol, non-competitive for acetyl-CoA
EDTA
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0.1 mM, 24% inhibition
ethyl violet
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strong, competitive for chloramphenicol, non-competitive for acetyl-CoA
Hg2+
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0.01 mM, complete inhibition
I2
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0.1 mM, 20% inhibition
methyl methanethiolsulfonate
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Zn2+
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0.1 mM, 15% inhibition
ACTIVATING COMPOUND
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
KM VALUE [mM]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
0.028 - 0.821
acetyl-CoA
0.00247 - 11
Chloramphenicol
0.0037 - 0.022
D-threo-1-p-nitrophenyl-2-bromoacetamido-1,3-propanediol
0.021 - 0.068
D-threo-1-phenyl-2-dichloroacetamido-1,3-propanediol
additional information
additional information
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TURNOVER NUMBER [1/s]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
additional information
additional information
Ki VALUE [mM]
INHIBITOR
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
additional information
additional information
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SPECIFIC ACTIVITY [µmol/min/mg]
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
additional information
pH OPTIMUM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
pH RANGE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
5 - 9
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about 50% of maximal activity at pH 5.0 and 9.0
6 - 8.8
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pH 6.0: about 55% of maximal activity, pH 8.8: about 85% of maximal activity
TEMPERATURE OPTIMUM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
additional information
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enzyme expression is increased at 42C. At 42C the enzyme is still soluble and active
TEMPERATURE RANGE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
30 - 45
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30C: about 20% of maximal activity, 45C: about 55% of maximal activity
SOURCE TISSUE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
SOURCE
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Culex pipiens infected with either TE/3'2J/CAT or rep5/CAT/26S virus
Manually annotated by BRENDA team
LOCALIZATION
ORGANISM
UNIPROT
COMMENTARY hide
GeneOntology No.
LITERATURE
SOURCE
MOLECULAR WEIGHT
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
26660
predicted from sequence analysis, 347 bp, 228 amino acid protein, chromosomal location
69000
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gel filtration
70000
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gel filtration
80000
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gel filtration
SUBUNITS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
tetramer
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1 * 19500 + 1 * 19000 + 1 * 18000 + 1 * 17500, SDS-PAGE
trimer
Crystallization/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
crystal structure of CATI in the unbound (apo) and CAM-bound forms are refined to 3.2 A and 2.9 A resolution, respectively
hanging drop vapour diffusion method
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TEMPERATURE STABILITY
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
53
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0.1 mM chloramphenicol and 0.1 mM acetyl-CoA, 50 mM Tris-HCl buffer, pH 7.8, half-life: 100 min
55
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,30 min, 82% loss of activity, wild-type enzyme
60
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nearly complete inactivation within 10 min
70
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15 min, 40% loss of activity of enzyme variant from plasmid pSCS6, 45% loss of activity of enzyme variant pSCS7
GENERAL STABILITY
ORGANISM
UNIPROT
LITERATURE
loss of activity by freezing and thawing can be prevented by adding 10% glycerol
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OXIDATION STABILITY
ORGANISM
UNIPROT
LITERATURE
photooxidation of enzyme type C in presence of methylene blue, progressive loss of activity with increase in pH, inclusion of 1 mM chloramphenicol affords complete protection against loss of activity
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486724
STORAGE STABILITY
ORGANISM
UNIPROT
LITERATURE
-20 C, activity in elution buffer is stable for at least 1 year
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-20 C, stable for several months
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-80 C, 20% glycerol, stable for more than 3 months
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Purification/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
ELP (elastin-like polypeptide) fusion tag technique to purify ultra-low levels of chloramphenicol acetyltransferase-ELP fusion protein
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enzyme variant A
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enzyme variant B
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enzyme variant C and D
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plasmid Rms418 is transferred from Vibrio anguillarum to E. coli K-12
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Cloned/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
cloning of enzyme form CATB7
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cloning of mutant cat-86 in pTB361 and transformation of Escherichia coli JM109
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expressed in Escherichia coli
overexpression in Escherichia coli harboring the plasmid pYT1
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PCR-amplification, clinging into Escherichia coli MC1061, electrotransformaion of Leuconostoc mesenteroides DRC (isolated from fermenting salted Chinese cabbage/Kimchi)
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PCR-amplification, electrotransformation into Escherichia coli DH10B to test for chloramphenicol inactivation with Micrococcus lutes ATCC 9341 as indicator organism
plasmid Rms418 is transferred from Vibrio anguillarum to Escherichia coli K-12
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ENGINEERING
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
A203G
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mutant enzyme is less stable than wild-type enzyme
A203I
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mutant enzyme is more thermostable than wild-type
I191V
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mutant enzyme is less stable than wild-type enzyme
Y33F/A203V
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mutant enzyme is more thermostable than wild-type
C214A
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95% of the in vivo produced mutant polypeptide is soluble compared to 90% for the wild-type enzyme. Mutant enzyme loses 31% of activity after 30 min at 65C compared to 15% for the wild-type enzyme
C214D
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50% of the in vivo produced mutant polypeptide is soluble compared to 90% for the wild-type enzyme. Mutant enzyme loses 85% of activity after 30 min at 65C compared to 15% for the wild-type enzyme
C214E
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75% of the in vivo produced mutant polypeptide is soluble compared to 90% for the wild-type enzyme. Mutant enzyme loses 84% of activity after 30 min at 65C compared to 15% for the wild-type enzyme
C214F/G219S
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95% of the in vivo produced mutant polypeptide is soluble compared to 90% for the wild-type enzyme. Mutant enzyme loses 81% of activity after 30 min at 65C compared to 15% for the wild-type enzyme
C214G
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80% of the in vivo produced mutant polypeptide is soluble compared to 90% for the wild-type enzyme. Mutant enzyme loses 44% of activity after 30 min at 65C compared to 15% for the wild-type enzyme
C214L
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100% of the in vivo produced mutant polypeptide is soluble compared to 90% for the wild-type enzyme. Mutant enzyme loses 33% of activity after 30 min at 65C compared to 15% for the wild-type enzyme
C214P
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95% of the in vivo produced mutant polypeptide is soluble compared to 90% for the wild-type enzyme. Mutant enzyme loses 88% of activity after 30 min at 65C compared to 15% for the wild-type enzyme
C214Q
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95% of the in vivo produced mutant polypeptide is soluble compared to 90% for the wild-type enzyme. Mutant enzyme loses 73% of activity after 30 min at 65C compared to 15% for the wild-type enzyme
C214R
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55% of the in vivo produced mutant polypeptide is soluble compared to 90% for the wild-type enzyme. Mutant enzyme loses 84% of activity after 30 min at 65C compared to 15% for the wild-type enzyme
C214S
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95% of the in vivo produced mutant polypeptide is soluble compared to 90% for the wild-type enzyme. Mutant enzyme loses 32% of activity after 30 min at 65C compared to 15% for the wild-type enzyme
C214T
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90% of the in vivo produced mutant polypeptide is soluble compared to 90% for the wild-type enzyme. Mutant enzyme loses 59% of activity after 30 min at 65C compared to 15% for the wild-type enzyme
C214V
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95% of the in vivo produced mutant polypeptide is soluble compared to 90% for the wild-type enzyme. Mutant enzyme loses 45% of activity after 30 min at 65C compared to 15% for the wild-type enzyme
C214W
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50% of the in vivo produced mutant polypeptide is soluble compared to 90% for the wild-type enzyme. Mutant enzyme loses 70% of activity after 30 min at 65 C compared to 15% for the wild-type enzyme
C214Y
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90% of the in vivo produced mutant polypeptide is soluble compared to 90% for the wild-type enzyme. Mutant enzyme loses 81% of activity after 30 min at 65C compared to 15% for the wild-type enzyme
CATIII (F24A/Y25F/L29A)
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Km-value for acetyl-CoA is 0.095 mM compared to 0.093 mM for wild-type CATIII, Km-value for chloramphenicol is 0.023 mM compared to 0.012 mM for the wild-type CATIII, turnover number is 30% of the wild-type enzyme CAT III
CATIII(K14E/H195A/K217A)
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no activity
CATIII(Q92C/N146F/Y169F/I172V)
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Km-value for acetyl-CoA is 0.165 mM compared to 0.093 mM for wild-type CATIII, Km-value for chloramphenicol is 0.02 mM compared to 0.012 mM for the wild-type CATIII, turnover number is 60% of the wild-type enzyme CAT III
K14/K217E
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Km-value for acetyl-CoA is 0.166 mM compared to 0.093 mM for wild-type CATIII, Km-value for chloramphenicol is 0.017 mM compared to 0.012 mM for the wild-type CATIII, turnover number is 87% of the wild-type enzyme CAT III
L145F
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folding of chloramphenicol acetyltransferase is hampered by deletion of the carboxy-terminal tail including the last residue of the carboxy-terminal alpha-helix. Such truncated CAT polypeptides quantitatively aggregate into cytoplasmic inclusion bodies, which results in absence of chloramphenicol-resistant phenotype for the producing host. Introduction of Phe at amino acid position 145 improves the ability of the protein to fold into a soluble, enzymatically active conformation
L158I
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fluorinated mutant expressed in trifluoroleucine shows enhanced thermostability compared to CAT T (CAT expressed in trifluoroleucine), suggesting that trifluoroleucine at position 158 contributes to a portion of the observed loss in thermostability upon global fluorination. Relative activity: 89% (non-fluorinated mutant), 51.7% (fluorinated mutant)
L208I
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fluorinated mutant expressed in trifluoroleucine shows loss in thermostability
L821I
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fluorinated mutant expressed in trifluoroleucine shows loss in thermostability
[CATI (H195A)]2[CATIII(K14E/K217E)]
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hybrid trimer, Km-value for acetyl-CoA is 0.072 mM compared to 0.093 mM for wild-type CATIII, Km-value for chloramphenicol is 0.018 mM compared to 0.012 mM for the wild-type CATIII, turnover number is 14% of the wild-type enzyme CAT III
[CATIII]2[CATIII(K14E/H195A/K217A)]
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Km-value for acetyl-CoA is 0.143 mM compared to 0.093 mM for wild-type CATIII, Km-value for chloramphenicol is 0.016 mM compared to 0.012 mM for the wild-type CATIII, turnover number is 80% of the wild-type enzyme CAT III
[CATIII][CATIII(K14E/H195A/K217A)]2
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Km-value for acetyl-CoA is 0.198 mM compared to 0.093 mM for wild-type CATIII, Km-value for chloramphenicol is 0.02 mM compared to 0.012 mM for the wild-type CATIII, turnover number is 82% of the wild-type enzyme CAT III
[CATI][CATIII(K14E/H195A/K217E)]2
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hybrid trimer, Km-value for acetyl-CoA is 0.107 mM compared to 0.093 mM for wild-type CATIII, Km-value for chloramphenicol is 0.02 mM compared to 0.012 mM for the wild-type CATIII, turnover number is 50% of the wild-type enzyme CAT III
G61S
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G61S and Y105C contrtibute synergistically to the resistance phenotype of strain PAhcr1
G61S/Y105C
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G61S and Y105C contrtibute synergistically to the resistance phenotype of strain PAhcr1
Y105C
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G61S and Y105C contrtibute synergistically to the resistance phenotype of strain PAhcr1
G61S
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G61S and Y105C contrtibute synergistically to the resistance phenotype of strain PAhcr1
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G61S/Y105C
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G61S and Y105C contrtibute synergistically to the resistance phenotype of strain PAhcr1
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Y105C
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G61S and Y105C contrtibute synergistically to the resistance phenotype of strain PAhcr1
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additional information
APPLICATION
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
analysis
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valuable tool in studies of eukaryotic gene expression, quantitative aspects of the use of bacterial chloramphenicol acetyltransferase as a reporter system in the yeast Saccharomyces cerevisiae
biotechnology
medicine
probiotic mixture for the prevention of gastrointestinal side effects due to oral antibiotic therapy, no resistance transfer to Enterococcus faecalis JH2-2, Enterococcus faecium HM1070 (both resistant to rifampin and fusidic acid) and Bacillus subtilis UCN19 (resistant to ciprofloxacin) in mating experiments
molecular biology
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