Information on EC 2.3.1.28 - chloramphenicol O-acetyltransferase and Organism(s) Escherichia coli and UniProt Accession P62577

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Escherichia coli
UNIPROT: P62577
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The expected taxonomic range for this enzyme is: Bacteria, Eukaryota


The taxonomic range for the selected organisms is: Escherichia coli

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
ternary complex mechanism with a rapid equilibrium and essentially random order of addition of substrates
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REACTION TYPE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
Acyl group transfer
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-
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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
-
UniProt
Manually annotated by BRENDA team
GENERAL INFORMATION
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
additional information
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method development for a sensitive model system for analyzing the rapid delivery of active enzymes into various regions of the brain of Rattus norvegicus with therapeutic bioavailability, intranasal delivery of chloramphenicol acetyltransferase from Escherichia coli, a relatively large enzyme, in its active form into different regions of the brain
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 3-acetate
show the reaction diagram
acetyl-CoA + chloramphenicol 1-acetate
CoA + chloramphenicol 1,3-diacetate
show the reaction diagram
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the enzyme acetylates specifically at the 3-hydroxy position. The diacetylation is possible only because of non-enzymatic interconversion of chloramphenical 3-acetate to chloramphenicol 1-acetate at higher pH values
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?
acetyl-CoA + D-threo-1-p-nitrophenyl-2-bromoacetamido-1,3-propanediol
CoA + ?
show the reaction diagram
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-
-
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?
acetyl-CoA + D-threo-1-phenyl-2-dichloroacetamido-1,3-propanediol
CoA + ?
show the reaction diagram
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-
-
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?
additional information
?
-
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a CAP derivative on sulfidic monolayers on gold chips can still serve as a substrate for the enzyme
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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
COFACTOR
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
acetyl-CoA
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METALS and IONS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
Mg2+
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required
INHIBITORS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
4,4'-dithiodipyridine
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5,5'-dithiobis(2-nitrobenzoic acid)
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Crystal violet
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strong, competitive for chloramphenicol, non-competitive for acetyl-CoA
ethyl violet
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strong, competitive for chloramphenicol, non-competitive for acetyl-CoA
methyl methanethiolsulfonate
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KM VALUE [mM]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
0.055 - 0.093
acetyl-CoA
0.0061 - 0.033
Chloramphenicol
0.0037
D-threo-1-p-nitrophenyl-2-bromoacetamido-1,3-propanediol
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0.021
D-threo-1-phenyl-2-dichloroacetamido-1,3-propanediol
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-
additional information
additional information
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kinetic analysis and profiling using substrate chloramphenicol bound on monolayers on gold chips, recombinant enzyme
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TURNOVER NUMBER [1/s]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
additional information
additional information
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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
6
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assay at
7.4
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in vivo assay in brain tissue
TEMPERATURE OPTIMUM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
additional information
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enzyme expression is increased at 42°C. At 42°C the enzyme is still soluble and active
SOURCE TISSUE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
SOURCE
PDB
SCOP
CATH
UNIPROT
ORGANISM
Escherichia coli;
MOLECULAR WEIGHT
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
78000
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sucrose density gradient centrifugation
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
STORAGE STABILITY
ORGANISM
UNIPROT
LITERATURE
-20 C, activity in elution buffer is stable for at least 1 year
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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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Cloned/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
expressed in Escherichia coli
recombinant expression in Escherichia coli strain BL21(DE3)
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ENGINEERING
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
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 65°C 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 65°C 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 65°C 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 65°C 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 65°C 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 65°C 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 65°C 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 65°C 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 65°C 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 65°C 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 65°C 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 65°C 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 65°C 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
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
medicine
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method development for a sensitive model system for analyzing the rapid delivery of active enzymes into various regions of the brain of Rattus norvegicus with therapeutic bioavailability, intranasal delivery of chloramphenicol acetyltransferase from Escherichia coli, a relatively large enzyme, in its active form into different regions of the brain, overview
molecular biology
pharmacology
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method development for a sensitive model system for analyzing the rapid delivery of active enzymes into various regions of the brain of Rattus norvegicus with therapeutic bioavailability, intranasal delivery of chloramphenicol acetyltransferase from Escherichia coli, a relatively large enzyme, in its active form into different regions of the brain, overview