Expression and characterization of recombinant tyramine β-monooxygenase from Drosophila: A monomeric copper-containing hydroxylase

Abstract

We report here the development of a robust recombinant expression system for Drosophila melanogaster tyramine β-monooxygenase (TβM), the insect analog of mammalian dopamine β-monooxygenase. Recombinant TβM is rapidly purified from the host cell media in three chromatographic steps. The expression system produces ∼3–10 mg of highly purified, active protein per liter of culture. Recombinant TβM requires copper for activity and has a typical type 2 copper EPR spectrum. While TβM efficiently hydroxylates the aliphatic carbon of phenolic amines such as tyramine (the physiological substrate) and dopamine, phenethylamine is a poor substrate. TβM is most likely a monomer under physiological conditions, although under conditions of high pH and low ionic strength the dimeric form predominates. The lower oligomeric state of TβM may provide an advantage for structural studies over DβM, which exists as a mixture of dimer and tetramer.