Abstract
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.
Footnotes
This work was supported by the National Institutes of Health National Institute of Diabetes and Digestive and Kidney Diseases [Grant DK59618].
Article, publication date, and citation information can be found at http://dmd.aspetjournals.org.
doi:10.1124/dmd.110.033639.
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ABBREVIATIONS:
- FMO
- flavin-containing monooxygenases
- P450
- cytochrome P450
- MMI
- methimazole
- PAGE
- polyacrylamide gel electrophoresis
- 8-DPT
- 10-[(N,N-dimethylaminooctyl)-2-(trifluoromethyl)]phenothiazine
- CHAPS
- 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid
- DDM
- n-dodecyl-β-d-maltoside
- QFF
- Q Fast-Flow
- DC
- detergent-compatible colorimetric
- HPLC
- high-performance liquid chromatography
- SEC
- size exclusion chromatography
- CMC
- critical micelle concentration.
- Received April 2, 2010.
- Accepted August 31, 2010.
- Copyright © 2010 by The American Society for Pharmacology and Experimental Therapeutics
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