0090-9556/97/2508-1001-1007$02.00/0
DRUG METABOLISM AND DISPOSITION
Copyright © 1997 by The American Society for Pharmacology and Experimental Therapeutics
Vol. 25, No. 8

Characterization of Two Human Flavin-Containing Monooxygenase (Form 3) Enzymes Expressed in Escherichia coli as Maltose Binding Protein Fusions

Alan Brunelle, Yi-An Bi, Jing Lin, Brett Russell, Lisa Luy, Clifford Berkman, and John Cashman

Seattle Biomedical Research Institute

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 coli as 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 glu¹⁵⁸ enzyme. 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.