Abstract
We report here the development of a robust recombinant expression system for Drosophila melanogaster tyramine beta-monooxygenase (TbetaM), the insect analog of mammalian dopamine beta-monooxygenase. Recombinant TbetaM is rapidly purified from the host cell media in three chromatographic steps. The expression system produces approximately 3-10 mg of highly purified, active protein per liter of culture. Recombinant TbetaM requires copper for activity and has a typical type 2 copper EPR spectrum. While TbetaM efficiently hydroxylates the aliphatic carbon of phenolic amines such as tyramine (the physiological substrate) and dopamine, phenethylamine is a poor substrate. TbetaM 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 TbetaM may provide an advantage for structural studies over DbetaM, which exists as a mixture of dimer and tetramer.
