Flavin-dependent monooxygenase from Methylophaga sp. strain sk1 (mFMO) catalyzes the NADPH and oxygen dependent hydroxylation of several xenobiotics. The active site of mFMO contains a conserved tryptophan residue that is in the opposite site of the substrate binding pocket. This residue is conserved in all known FMO sequences and in other related flavin-monooxygenases. Here, we report the role of W47 in mFMO. While substitution of this residue to alanine results in an inactive enzyme, the W47F mutant is active. Kinetic characterization shows a decrease of 5-8 fold on kcat and kcat/Km values for NADPH, trimethylamine, and methimazole as substrates. The rate of flavin reduction decreases and primary kinetic isotope effect values increase. These results show that W47F is important for hydride transfer in mFMO. This work was supported in part by a grant from the National Science Foundation MCB-1021384 and by a grant from the Virginia Academy of Sciences. 

Role of conserved tryptophan 47 in the active site of flavin-monooxygenase from <i>Methylophaga sp.</i> strain sk1

245th National Meeting (2013)

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Role of conserved tryptophan 47 in the active site of flavin-monooxygenase from Methylophaga sp. strain sk1