Cloned (Comment) | Organism |
---|---|
expressed in Escherichia coli, strain Rosetta(DE3)pLysS, pET/codAmg plasmid, wild-type and Glu312 variants generated by site-directed mutagenesis | Arthrobacter globiformis |
Crystallization (Comment) | Organism |
---|---|
crystal structure determined by synchrotron X-ray radiation, refined to a resolution of 1.86 A, data collected at 100 K | Arthrobacter globiformis |
Protein Variants | Comment | Organism |
---|---|---|
E312A | generated for investigation of the negative charge on Glu312, enzyme inactive | Arthrobacter globiformis |
E312D | generated for investigation of the negative charge on Glu312, kcat values about 230times lower and and kcat/Km values about 35times lower than in the wild-type, solvent viscosity and substrate kinetic isotope effects indicates presence of internal equilibrium prior to the hydride transfer reaction | Arthrobacter globiformis |
E312Q | generated for investigation of the negative charge on Glu312, Kd value for choline about 500times larger than that of wild-type | 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 | - |
- |
Purification (Comment) | Organism |
---|---|
wild-type and mutant variants | Arthrobacter globiformis |
Specific Activity Minimum [µmol/min/mg] | Specific Activity Maximum [µmol/min/mg] | Comment | Organism |
---|---|---|---|
additional information | - |
X-ray data collection and mutant analysis, steady-state kinetic parameters, pH profiles, substrate kinetic isotope effects for mutants and wild-type determined | Arthrobacter globiformis |
Substrates | Comment Substrates | Organism | Products | Comment (Products) | Rev. | Reac. |
---|---|---|---|---|---|---|
choline + O2 | - |
Arthrobacter globiformis | betaine aldehyde + H2O2 | - |
? | |
choline + O2 | spatial location of the negative charge on residue 312 important for oxidation of alcohol substrate | Arthrobacter globiformis | betaine aldehyde + H2O2 | - |
? |
Temperature Optimum [°C] | Temperature Optimum Maximum [°C] | Comment | Organism |
---|---|---|---|
25 | - |
activity assay at, rate of oxygen consumption | Arthrobacter globiformis |
pH Minimum | pH Maximum | Comment | Organism |
---|---|---|---|
5 | 10 | pH profiles of kinetic parameters with choline as a substrate | Arthrobacter globiformis |
Cofactor | Comment | Organism | Structure |
---|---|---|---|
FAD | covalent binding of FAD to purified proteins ascertained, rates of flavin reduction determined in wild-type and mutant variants | Arthrobacter globiformis |