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1.1.3.17: choline oxidase

This is an abbreviated version!
For detailed information about choline oxidase, go to the full flat file.

Word Map on EC 1.1.3.17

Reaction

Betaine aldehyde
+
O2
+
H2O
=
betaine
+
H2O2

Synonyms

alkaliphilic choline oxidase, ANI01nite_22550, An_CodA, APChO-syn, CHO, choline oxidase, choline-oxygen 1-oxidoreductase, choline:oxygen 1-reductase, ChOx, ChOx protein, codA, COX

ECTree

     1 Oxidoreductases
         1.1 Acting on the CH-OH group of donors
             1.1.3 With oxygen as acceptor
                1.1.3.17 choline oxidase

Engineering

Engineering on EC 1.1.3.17 - choline oxidase

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PROTEIN VARIANTS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
E312A
generated for investigation of the negative charge on Glu312, enzyme inactive
E312D
E312Q
generated for investigation of the negative charge on Glu312, Kd value for choline about 500times larger than that of wild-type
F357A
the mutant shows about 240fold reduced catalytic efficiency compared to the wild type enzyme
H351A
generated by site-directed mutagenesis
H351Q
the kcat and kcat/Km values of the H351Q emutant in atmospheric oxygen are 45 and 5000fold lower than those of the wild type enzyme, respectively
H466A
H466D
-
site-directed mutagenesis, the mutation alters the flavin binding to the enzyme, while substrate choline is normally bound, binding og glycine btaine is inhibited, spectrometrical analysis, the mutant shows a different flavin-binding stoichiometry of 0.29:1, compared to 1:1 for the wild-type enzyme, stabilized at pH 6.0-10.0, overview, comparison of midpoint reduction-oxidation potentials of the enzyme-FAD form with mutant H466A and the wild-type enzyme, the mutant shows no catalytic activity
H466Q
H99N
mutant, analysis of kinetic parameters
M359R
mutant displays increased activity with hexan-1-ol, reaction of EC 1.1.3.13
M62A
the mutant shows about 3fold reduced catalytic efficiency compared to the wild type enzyme
M62A/F357A
the enzyme shows a lack of the isomerization detected in wild type choline oxidase, and a lack of saturation with an oxygen concentration as high as 1 mM, while most other kinetic parameters are similar to those of wild type choline oxidase
N510A
-
site-directed mutagenesis of a catalytic residue resulting in low incorporation of FAD into the protein, enzyme kinetics decrease of 4300fold in the kcat/Kcholine, 600fold in the kred, 660fold in the kcat, and 50fold in the kcat/Koxygen values
N510D
-
site-directed mutagenesis of a catalytic residue resulting in low incorporation of FAD into the protein, 75% of the flavin associates noncovalently, inactive mutant
N510H
-
site-directed mutagenesis of a catalytic residue resulting in low incorporation of FAD into the protein, decreases in the kcat/Kcholine, the kred, the kcat, and the kcat/Koxygen values
N510L
-
site-directed mutagenesis of a catalytic residue resulting in low incorporation of FAD into the protein
S101A
S101A/D250G/F253R/V355T/F357R/M359R
mutant displays increased activity with hexan-1-ol, reaction of EC 1.1.3.13, with a 20fold increased kcat compared to that of the wildtype enzyme. This variant enables the oxidation of 10 mM hexanol to hexanal in less than 24h with 100% conversion and catalyzes significantly improved oxidation of saturated, unsaturated, aliphatic, cyclic and benzylic alcohols
S101A/V355T/F357R
mutant displays increased activity with hexan-1-ol, reaction of EC 1.1.3.13
S101A/V355T/F357R/M359R
mutant displays increased activity with hexan-1-ol, reaction of EC 1.1.3.13
S101C
S101T
S101V
contrary to wild-type, stopped-flow traces for the reductive half-reaction are biphasic, corresponding to the reactions of proton abstraction and hydride transfer. The rate constants for proton transfer in the S101T/C/V variants decrease logarithmically with increasing hydrophobicity of residue 101
V355T/F357R
mutant displays increased activity with hexan-1-ol, reaction of EC 1.1.3.13
V464A
V464T
A21V
-
site-directed mutagenesis in the FAD binding site
A21V/G62D
-
site-directed mutagenesis, the mutant shows 1.93fold increased activity with tris-(2-hydroxyethyl)-methylammonium methylsulfate and slightly reduced activity with choline compared to the wild-type enzyme
A21V/G62D/I69V
-
site-directed mutagenesis, the mutant shows 1.68fold increased activity with tris-(2-hydroxyethyl)-methylammonium methylsulfate compared to the wild-type enzyme
A21V/G62D/I69V/S348L
-
site-directed mutagenesis, the mutant shows 3.45fold increased activity with tris-(2-hydroxyethyl)-methylammonium methylsulfate and highly reduced activity with choline compared to the wild-type enzyme
A21V/G62D/S348C
-
site-directed mutagenesis, the mutant shows 5.18fold increased activity with tris-(2-hydroxyethyl)-methylammonium methylsulfate and reduced activity with choline compared to the wild-type enzyme
A21V/G62D/S348L
-
site-directed mutagenesis, the mutant shows 3.72fold increased activity with tris-(2-hydroxyethyl)-methylammonium methylsulfate and highly reduced activity with choline compared to the wild-type enzyme
A21V/G62D/S348L/V349L
-
site-directed mutagenesis, the mutant shows 5.75fold increased activity with tris-(2-hydroxyethyl)-methylammonium methylsulfate and highly reduced activity with choline compared to the wild-type enzyme
A21V/K394R
-
site-directed mutagenesis, the mutant shows 85% activity with tris-(2-hydroxyethyl)-methylammonium methylsulfate compared to the wild-type enzyme
F351Y
-
site-directed mutagenesis in the substrate binding site, the mutant shows reduced activity with choline compared to the wild-type enzyme
G62D
-
site-directed mutagenesis in the FAD binding site, the mutant shows 2fold increased activity with tris-(2-hydroxyethyl)-methylammonium methylsulfate and reduced activity with choline compared to the wild-type enzyme
G62D/F351Y
-
site-directed mutagenesis, the mutant shows 2.14fold increased activity with tris-(2-hydroxyethyl)-methylammonium methylsulfate compared to the wild-type enzyme
G62D/R249H/F351Y
-
site-directed mutagenesis, the mutant shows 2.7fold increased activity with tris-(2-hydroxyethyl)-methylammonium methylsulfate and reduced activity with choline compared to the wild-type enzyme
I69V
-
site-directed mutagenesis in the FAD binding site, the mutant shows93% activity with tris-(2-hydroxyethyl)-methylammonium methylsulfate compared to the wild-type enzyme
P393Q/S530G
-
site-directed mutagenesis, the mutant shows 1.5fold increased activity with choline compared to the wild-type enzyme
S348L
-
site-directed mutagenesis in the substrate binding site
T116I/K128M
-
site-directed mutagenesis, the mutant shows unaltered activity with choline compared to the wild-type enzyme
T116I/K128M/P393Q/S530G
-
site-directed mutagenesis, the mutant shows 2.32fold increased activity with choline compared to the wild-type enzyme
V349L
-
site-directed mutagenesis in the substrate binding site
A21V
-
site-directed mutagenesis in the FAD binding site
-
G62D
-
site-directed mutagenesis in the FAD binding site, the mutant shows 2fold increased activity with tris-(2-hydroxyethyl)-methylammonium methylsulfate and reduced activity with choline compared to the wild-type enzyme
-
I69V
-
site-directed mutagenesis in the FAD binding site, the mutant shows93% activity with tris-(2-hydroxyethyl)-methylammonium methylsulfate compared to the wild-type enzyme
-
S348L
-
site-directed mutagenesis in the substrate binding site
-
V349L
-
site-directed mutagenesis in the substrate binding site
-
additional information