Cloned (Comment) | Organism |
---|---|
gene FMO3, recombinant expression of His-tagged wild-type and mutant enzymes in Escherichia coli strain JM109 | Homo sapiens |
Protein Variants | Comment | Organism |
---|---|---|
E158K | naturally occuring polymorphic variant and site-directed mutagenesis, the melting temperature and activation energy of the mutant is nearly unaltered compared to the wild-type enzyme | Homo sapiens |
E308G | naturally occuring polymorphic variant and site-directed mutagenesis, the mutant is unable to bind the NADP+ cofactor, it shows a significantly higher energy of unfolding (Ea) compared to wild-type | Homo sapiens |
additional information | unfolding process of a phase I drug metabolizing enzyme, human flavin-containing monooxygenase 3 (hFMO3) and its single nucleotide polymorphic variants (SNPs) V257M, E158K and E308G are analyzed by differential scanning calorimetry (DSC) indicating that the thermal denaturation of the enzyme is irreversible. The melting temperature (Tm) for the wild-type enzyme and its polymorphic variants is in a range from 46°C to 50°C. Also the activation energies of unfolding (Ea) show no significant differences among all proteins investigated (290-328 KJ/mol), except for the E308G variant that shows a significantly higher Ea of 412 KJ/mol. The presence of the bound NADP+ cofactor stabilizes all the variants by shifting the main Tm by 4-5°C for all the proteins, exception made for E308G where no changes are observed | Homo sapiens |
V257M | naturally occuring polymorphic variant and site-directed mutagenesis, the melting temperature and activation energy of the mutant is nearly unaltered compared to the wild-type enzyme | Homo sapiens |
Natural Substrates | Organism | Comment (Nat. Sub.) | Natural Products | Comment (Nat. Pro.) | Rev. | Reac. |
---|---|---|---|---|---|---|
N,N-dimethylaniline + NADPH + H+ + O2 | Homo sapiens | - |
N,N-dimethylaniline N-oxide + NADP+ + H2O | - |
? |
Organism | UniProt | Comment | Textmining |
---|---|---|---|
Homo sapiens | P31513 | - |
- |
Purification (Comment) | Organism |
---|---|
recombinant His-tagged wild-type and mutant enzymes from Escherichia coli strain JM109 by nickel affinity chromatography and ultrafiltration | Homo sapiens |
Substrates | Comment Substrates | Organism | Products | Comment (Products) | Rev. | Reac. |
---|---|---|---|---|---|---|
N,N-dimethylaniline + NADPH + H+ + O2 | - |
Homo sapiens | N,N-dimethylaniline N-oxide + NADP+ + H2O | - |
? |
Subunits | Comment | Organism |
---|---|---|
More | heat-induced changes in the secondary structure in the presence and absence of NADP+, overview | Homo sapiens |
Synonyms | Comment | Organism |
---|---|---|
flavin-containing monooxygenase | - |
Homo sapiens |
flavin-containing monooxygenase 3 | - |
Homo sapiens |
hFMO3 | - |
Homo sapiens |
Temperature Optimum [°C] | Temperature Optimum Maximum [°C] | Comment | Organism |
---|---|---|---|
25 | - |
assay at | Homo sapiens |
Temperature Stability Minimum [°C] | Temperature Stability Maximum [°C] | Comment | Organism |
---|---|---|---|
46 | 50 | differential scanning calorimetry (DSC) indicates that the thermal denaturation of the enzyme is irreversible in all cases. The melting temperature (Tm) for the wild-type enzyme and its polymorphic variants is in a range from 46°C to 50°C at pH 7.4. Calculation for the activation energy of unfolding is performed using a mathematical model, secondary structures of wild-type and mutant enzymes during heat inactivation, overview | Homo sapiens |
pH Optimum Minimum | pH Optimum Maximum | Comment | Organism |
---|---|---|---|
7.4 | - |
assay at | Homo sapiens |
Cofactor | Comment | Organism | Structure |
---|---|---|---|
FAD | - |
Homo sapiens | |
NADPH | hFMO3 is reduced by its physiological electron donor NADPH. During the enzyme catalytic cycle, NADPH is consumed, then the reduced enzyme binds molecular oxygen to form a C4ahydroperoxy intermediate of the flavin responsible for the oxygenation of the substrate with the concomitant release of a water molecule. During these steps NADP+ remains bound in the active site leaving only when the catalytic cycle is completed. Heat-induced changes in the secondary structure in the presence and absence of NADP+, overview | Homo sapiens |
General Information | Comment | Organism |
---|---|---|
malfunction | the hFMO3 gene contains many naturally occuring single SNPs and these mutations can severely affect the activity of the enzyme resulting in lower or abolished activity | Homo sapiens |
additional information | heat-induced changes in the secondary structure in the presence and absence of NADP+, overview | Homo sapiens |
physiological function | human flavin-containing monooxygenases (hFMOs) comprise a family of five isoenzymes and are the second most important phase 1 drug-metabolizing enzymes after cytochromes P450. Its isoform 3 (hFMO3) is predominantly expressed in the liver where substrates containing nitrogen-, sulphur- and phosphorous-containing soft nucleophiles are transformed into more polar and excretable metabolites. Wild-type hFMO3 contributes to the metabolism of several important drugs such as ranitidine, cimetidine, tamoxifen, clozapine, benzydamine | Homo sapiens |