Cloned (Comment) | Organism |
---|---|
expressed in Escherichia coli, wild-type and H351A variant, plasmid pET/codAmg1-H351A | Arthrobacter globiformis |
Crystallization (Comment) | Organism |
---|---|
active site of wild-type choline oxidase, resolution of 1.86 A | Arthrobacter globiformis |
Protein Variants | Comment | Organism |
---|---|---|
H351A | generated by site-directed mutagenesis | Arthrobacter globiformis |
Inhibitors | Comment | Organism | Structure |
---|---|---|---|
glycine betaine | pH dependence of the kcat and kcat/Km values for glycine betaine inhibition shown, inhibition of H351A enzyme maximal at low pH and reduced with increasing pH | Arthrobacter globiformis |
Natural Substrates | Organism | Comment (Nat. Sub.) | Natural Products | Comment (Nat. Pro.) | Rev. | Reac. |
---|---|---|---|---|---|---|
choline + O2 | Arthrobacter globiformis | - |
betaine aldehyde + H2O2 | - |
? |
Organism | UniProt | Comment | Textmining |
---|---|---|---|
Arthrobacter globiformis | Q7X2H8 | strain ATCC 8010 | - |
Purification (Comment) | Organism |
---|---|
wild-type and mutant protein, purified enzymes have 50-70% of the enzyme-bound flavin cofactor as an air-stable anionic flavosemiquinone, converts slowly to oxidized state by extensive dialysis at pH 6 and 4°C | Arthrobacter globiformis |
Specific Activity Minimum [µmol/min/mg] | Specific Activity Maximum [µmol/min/mg] | Comment | Organism |
---|---|---|---|
additional information | - |
pH dependence of substrate deuterium isotope effects and of the kcat and kcat/Km values for choline in the mutant variant H351A determined, steady-state kinetic parameters compared between H351A mutant and wild-type, His351 important but not essential for the reductive half-reaction of choline oxidation, contributes to substrate binding and to the overall polarity of the active site | Arthrobacter globiformis |
Substrates | Comment Substrates | Organism | Products | Comment (Products) | Rev. | Reac. |
---|---|---|---|---|---|---|
choline + O2 | - |
Arthrobacter globiformis | betaine aldehyde + H2O2 | - |
? | |
choline + O2 | choline shown to be a slow substrate for H351A variant, His351 residue important for substrate binding and hydride transfer reaction | Arthrobacter globiformis | betaine aldehyde + H2O2 | - |
? |
Temperature Optimum [°C] | Temperature Optimum Maximum [°C] | Comment | Organism |
---|---|---|---|
25 | - |
activity assay at | Arthrobacter globiformis |
Turnover Number Minimum [1/s] | Turnover Number Maximum [1/s] | Substrate | Comment | Organism | Structure |
---|---|---|---|---|---|
additional information | - |
additional information | overall turnover of mutant variant about 60-fold decreased with choline compared to wild-type enzyme | Arthrobacter globiformis |
pH Optimum Minimum | pH Optimum Maximum | Comment | Organism |
---|---|---|---|
10 | - |
activity assay at, in 50 mM sodium pyrophosphate, neutral hydroquinone species of the flavin stabilized in the H351A enzyme at | Arthrobacter globiformis |
pH Minimum | pH Maximum | Comment | Organism |
---|---|---|---|
5 | 10 | varying concentrations of both choline and oxygen, between pH 7 and pH 10, Km values for oxygen less than 15 microM | Arthrobacter globiformis |
Cofactor | Comment | Organism | Structure |
---|---|---|---|
FAD | His351 stabilizes transition state for hydride transfer reaction to flavin, is not involved in the activation of the reduced flavin for reaction with oxygen | Arthrobacter globiformis |