Abstract
Flavin-containing monooxygenases (FMO) are membrane-associated enzymes contributing to oxidative metabolism of drugs and other chemicals. There are no known structures similar enough to FMO to provide accurate insights into the structural basis for differences in metabolism observed among FMOs. To develop an FMO amenable to crystallization, we introduced mutations into rabbit FMO2 (rF2) to increase solubility, decrease aggregation, and simplify isolation. Alterations included removal of 26 AA (Delta26) from the carboxyl-terminus, His(6)-fusion to the amino-terminus and a double Ser substitution designed to reduce local hydrophobicity. Only Delta26 FMO variants retained normal activity, increased the yield of cytosolic rF2 and decreased protein aggregation. Delta26 constructs increased rF2 in cytosol in low (from 2 to 13%), and high salt (from 24 to 62%) conditions. His-fusion proteins, while active and useful for purification, did not affect solubility. Delta26 variants should prove useful for identifying conditions suitable for production of an FMO crystal.
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