Abstract
Drosophila dopamine N-acetyltransferase (Dat, EC 2.3.1.87, known as arylalkylamine N-acetyltransferase) has been proved to play an important role in neurotransmitter catabolism, melatonin precursor formation, oviposition and sclerotization. Recently, three dimensional structures of Dat either in the apo or AcCoA-complexed forms have been determined by X-ray crystallography in our lab. On the basis of structural comparison and ternary docking model, side-chain orientations of AcCoA binding-related residues (K192, D46) were flipped by salt bridge and hydrogen bonding, and two aromatic residues (F43, Y64) probably interact with a substrate to trigger the enzyme activity. In order to explore the substrate binding, K192, D46, F43, and Y64 in Dat were replaced with alanine to clarify their roles in enzyme function. Moreover, we compared the biophysical property, enzyme activity of full-length Dat and the designed truncated Dat (ΔDat). The results showed that the chemical stability is similar and the optimal enzyme activity is at 25-30℃ and pH 7.0-8.0. In terms of substrate specificity for Dat, the DTNB-based functional assay showed that the catalytic efficiency of three substrates (tryptamine, serotonin and dopamine) are in the same order, revealing that Dat has no significant substrate specificity. The isothermal titration calorimeter experiment showed that D46A, Y64A and K192A mutations of Dat resulted in a reduction in binding constant (~2 to 10-fold). On the other hand, as compared to wild-type Dat, four subsrate/AcCoA binding-related mutants drametically lose more than 50% enzyme catalytic efficiency, especially the F43A and D46A mutants have only 16.6% and 19.7%. Taken together, this study revealed the four residues of Dat were essential for substrate and AcCoA binding.