5 research outputs found
Struktur und Funktion der humanen Flavin-haltigen Monooxygenasen 3 und 5
Characterization of flavin-containing monooxygenases 3 and 5 including the characterization of a novel FMO3 variant and its association with trimethylaminuria, the expression and purification of FMO5, crystallisation studies of human FMO5, species-dependent differences in pH-dependent activity profiles of FMO5, and identification of novel FMO5 substrates.Charakterisierung der flavin-haltigen Monooxygenasen 3 and 5. Diese beinhaltet die Charakterisierung einer neuen FMO3 Variante, die mit Trimethylaminurie in Verbindung gebracht wird, die Expression und Reinigung der FMO5, Kristallisationsstudien mit humener FMO5, die Untersuchung von Speziesunterschieden pH-abhängiger Aktivitätsprofile der FMO5 und die Identifizierung neuer FMO5 Substrate
Characterization of Human Flavin-Containing Monooxygenase (FMO) 3 and FMO5 Expressed as Maltose-Binding Protein Fusions
The flavin-containing monooxygenase (FMO) family of enzymes oxygenates nucleophilic xenobiotics and endogenous substances. Human FMO3 and FMO5 are the predominant FMO forms in adult liver. These enzymes are naturally membrane-bound, and recombinant proteins are commercially available as microsomal preparations from insect cells (i.e., Supersome FMO). As an alternative, FMO3 has previously been expressed as a soluble protein, through use of an N-terminal maltose-binding protein (MBP) fusion. In the current study, MBP fusions of both human FMO3 and FMO5 were prepared to >90% purity in the presence of detergent and characterized for biochemical and kinetic parameters, and the parameters were compared with those of Supersome FMO samples. Although MBP-FMO enzymes afforded lower rates of turnover than the corresponding Supersome FMOs, both types of FMO showed identical substrate dependencies and similar responses to changes in assay conditions. Of interest, the FMO3 enzymes showed a 2-fold activation of kcat/Km in the presence of Triton X-100. Oligomeric analysis of MBP-FMO3 also showed disassociation from a high-order oligomeric form to a monomeric status in the presence of Triton X-100. This report serves as the first direct comparison between Supersome FMOs and the corresponding MBP fusions and the first report of a detergent-based activation of kcat/Km that corresponds to changes in oligomerization
Characterization of human FMO3 and FMO5 expressed as maltose binding protein fusions DMD #33639 2 Running Title: Kinetic analysis of MBP-FMO3 and MBP-FMO5
Hepatic Flavin-Containing Monooxygenase Gene Regulation in Different Mouse Inflammation ModelsS⃞
The objective of the study was to investigate the regulation of hepatic
flavin-containing monooxygenases (Fmo) Fmo1, Fmo3,
Fmo4, and Fmo5 in three different mouse models of
inflammation, including treatment with Citrobacter rodentium,
lipopolysaccharide (LPS), and dextran sulfate sodium (DSS). Quantitative
real-time reverse transcription-polymerase chain reaction (RT-PCR) was used to
evaluate the steady-state mRNA levels for the various Fmo isoforms in
these mouse models of inflammation during different treatment time courses.
Fmo3 mRNA was most significantly down-regulated in C.
rodentium-treated female mice. Fmo1, Fmo3, and
Fmo5 mRNAs were also found to be down-regulated in LPS models of
inflammation. The significant down-regulation of hepatic FMO3 protein during
C. rodentium treatment was confirmed with Western blot analysis of
liver microsomes from treated animals. Toll-like receptor (TLR) 4 is known to
be responsible for LPS signaling in association with several proteins. To
investigate whether TLR4 was responsible for regulation of Fmo genes
in both LPS and C. rodentium animal models, Fmo mRNA levels
in female wild-type (C3H/HeOuJ) and TLR4 mutant (C3H/HeJ) mice were compared
in both inflammatory models by real-time RT-PCR. The results showed that
Fmo3 down-regulation during C. rodentium infection is
independent of TLR4. Whereas TLR4 is likely to play only a partial role in
Fmo1 gene regulation in LPS-treated animals, our results show that
the down-regulation of Fmo3 and Fmo5 in this model is
TLR4-dependent. Unlike cytochrome P450 regulation measured in the same mouse
strains, Fmo3 expression was largely refractory to down-regulation in
the DSS model of inflammatory colitis