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Characterization of human flavin-containing monooxygenase (FMO) 3 and FMO5 expressed as maltose-binding protein

Robert R Reddy1, Erik C Ralph, Meike S Motika

  • 1Human BioMolecular Research Institute, 5310 Eastgate Mall, San Diego, CA 92121, USA.

Drug Metabolism and Disposition: the Biological Fate of Chemicals
|September 3, 2010
PubMed

Insights

Recombinant human flavin-containing monooxygenase (FMO) enzymes, expressed as maltose-binding protein (MBP) fusions, were characterized. These MBP-FMOs showed similar substrate dependencies to insect cell-derived Supersome FMOs, with detergent activating FMO3 activity.

Area of Science:

  • Biochemistry
  • Enzymology
  • Drug Metabolism

Background:

  • Flavin-containing monooxygenase (FMO) enzymes metabolize xenobiotics and endogenous compounds.
  • Human FMO3 and FMO5 are key FMO isoforms in adult liver.
  • Recombinant FMOs are typically available as membrane-bound insect cell microsomes (Supersome FMOs).

Purpose of the Study:

  • To prepare and characterize soluble N-terminal maltose-binding protein (MBP) fusions of human FMO3 and FMO5.
  • To compare the biochemical and kinetic parameters of MBP-FMOs with Supersome FMOs.
  • To investigate the effect of detergent on FMO activity and oligomerization.

Main Methods:

  • Expression and purification of MBP-FMO3 and MBP-FMO5 fusions (>90% purity) in the presence of detergent.
  • Biochemical and kinetic characterization of recombinant FMO enzymes.
  • Oligomeric analysis of MBP-FMO3 using Triton X-100.

Main Results:

  • MBP-FMO enzymes exhibited lower turnover rates than Supersome FMOs but identical substrate dependencies.
  • Both MBP-FMO and Supersome FMO preparations showed similar responses to assay condition changes.
  • Triton X-100 activated MBP-FMO3's catalytic efficiency (kcat/Km) and induced dissociation from higher-order oligomers to monomers.

Conclusions:

  • MBP fusions provide a soluble alternative for studying human FMO3 and FMO5.
  • Detergent-mediated activation of FMO3 activity correlates with a shift in oligomeric state.
  • This study presents the first direct comparison of MBP-FMOs and Supersome FMOs, revealing detergent-dependent effects on enzyme kinetics and structure.

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