Abstract
To examine the possibility for drug metabolism polymorphism, adult human flavin-containing monooxygenases (form 3) (EC 1.14.13.8) that differ at one amino acid were expressed in Escherichia colias maltose binding protein fusions. The cDNA that was first reported during the cloning of adult human flavin-containing monooxygenase was designated the wild type lys158 enzyme. A second cDNA has been identified as a common polymorphism in some human populations and was designated the glu158enzyme. The cDNA that encodes both enzymes was subcloned into a high yield protein fusion expression system, expressed, and the protein was partially purified by affinity chromatography and characterized for enzyme activity with selective functional substrate probes.N- and S-oxygenation activity of both enzymes was determined with 10-(N,N-dimethylaminopentyl)-2-(trifluoromethyl)phenothiazine and methyl p-tolyl sulfide, respectively. It was found that expression of both lys158 and glu158 enzymes of the human flavin-containing monooxygenase form 3 as fusions with the maltose binding protein resulted in an enzyme that was soluble and greatly stabilized and had a reduced requirement for detergent during enzyme purification and during the assay for activity. Expression of the fusion proteins has allowed the preparation of stable and highly active enzyme at greater purity than was readily possible in the past. With the exception of the stability and solubility characteristics, the physical and chemical properties of lys158 and glu158 maltose binding fusion proteins of human flavin-containing monooxygenase form 3 variants resembled that of flavin-containing monooxygenase enzyme activity associated with human liver microsomes and enzyme isolated from a previous Escherichia coli expression system that lacked the protein fusion. Comparison of the catalytic activity of the two fusion proteins showed that while both forms were active, there were differences in their substrate specificities. Expression of the adult human flavin-containing monooxygenase form 3 as a maltose binding protein has allowed considerable advances over the previously reported cDNA-expressed enzyme systems and may provide the basis for examining the role of the flavin-containing monooxygenase in human xenobiotic or drug metabolism.
Footnotes
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Send reprint requests to: Dr. John Cashman, Seattle Biomedical Research Institute, 4 Nickerson Street, Suite 200, Seattle, WA 98109-1651.
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This work was financially supported by a grant from NIH (GM 36426)
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↵2 Restriction length polymorphism and oligonucleotide sequencing studies showed codon 158 encoded either amino acids glu or lys at approximately equal allele frequencies for the caucasian populations examined: E. Treacy, R. Youil, S. Forest, and M. Knight, unpublished data.
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↵3 The revised full length human FMO3 cDNA sequence was recently reported: Proc. Natl. Acad. Sci. USA 92,9910 (1995) and Eur. J. Biochem. 235, 683 (1996).
- Abbreviations used are::
- TMA
- trimethylamine
- TMANO
- trimethylamine N-oxide
- 5-DPT
- 10-(N,N-dimethylaminopentyl)-2-(trifluoromethyl)phenothiazine
- 5-DPTNO
- 10-(N,N-dimethylaminopentyl)-2-(trifluoromethyl)phenothiazineN-oxide
- MTS
- methyl p-tolyl sulfide
- MTSO
- methyl p-tolyl sulfoxide
- HFMO3
- human flavin-containing monooxygenase (form 3)
- IPTG
- isopropyl-β-D-thiogalactopyranoside
- PMSF
- phenylmethylsulfonylfluoride
- DETAPAC
- diethylenetriaminepentaacetic acid
- MBP
- maltose binding protein
- PCR
- polymerase chain reaction
- HPLC
- high performance liquid chromatography
- DCC
- dicyclohexylcarbodiimide
- THF
- tetrahydrofuran
- TLC
- thin layer chromatography
- SDS PAGE
- sodium dodecylsulfate polyacrylamide gel electrophoresis
- PEG 8000
- polyethylene glycol 8000
- Received December 23, 1996.
- Accepted March 31, 1997.
- The American Society for Pharmacology and Experimental Therapeutics