Flavin-containing monooxygenase (FMO) 5 belongs to a family of enzymes that catalyze the oxygenation of nucleophilic N- and S-containing compounds. The FMO enzyme family consists of five forms (FMOs1-5) that share about 50-60% sequence identity to each other. A comparison of FMOs showed that the pH-dependence profile for functional activity of FMO5 differed significantly from that of other FMO enzymes. The objective of this study was to examine the pH-dependence of FMO5 to gain insight into the mechanism of action of FMO5. Recombinant mouse and human FMO5 (mFMO5 and hFMO5, respectively) were expressed as maltose-binding fusion proteins from Escherichia coli, purified with affinity chromatography, and examined for their N-oxygenation functional activity at different pH values. hFMO5 showed a broader range and greater functional activity from pH 6 to 11 compared to mFMO5. mFMO5 lost almost all functional activity at pH 6, while hFMO5 maintained almost normal enzyme activity. In order to identify the amino acid residues involved in the effects of pH on hFMO5 and mFMO5 functional enzyme activity, pH-studies in the range of pH 6-9 were done with chimeras of recombinant mouse and human FMO5 and variants of both. Results of these studies and molecular modeling showed that residues responsible for the differences in the pH profile between mFMO5 and hFMO5 were located at positions 227 and 228 of the enzyme. Further variants were made to investigate the role of these amino acids. The results of this study may help to explain the mechanism of FMO function.
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