Abstract
Dopamine N-acetyltransferase (Dat) found in Drosophila melanogaster belongs to arylalkylamine N-acetyltransferase (AANAT, EC 2.3.1.87) family, which is a member of GCN5-related N-acetyltransferase (GNAT) superfamily. Dat catalyzes arylalkylamine N-acetylation which transfers acetyl group of acetyl-CoA (Ac-CoA) to arylalkylamine to generate N-acetyl-arylalkylamine and CoA. AANAT had been reported the ordered bi bi sequential mechanism by enzyme inhibition analysis as well. In our previous study, we had also determined Dat is ordered bi bi sequential mechanism using isothermal titration calorimetry (ITC) and enzyme inhibition kinetics. Dat has to bind cofactor (Ac-CoA) first and then followed by substrate (Arylalkylamine). Nevertheless, the underlying structural mechanism still remains ambiguous. Furthermore, we had found the electron density map of products, N-acetyl-arylalkylamine and CoA, on substrate and cofactor binding site in ternary structure by soaking. It seemed products cannot auto-release. Thus, we interested in how Dat conducts enzymatic recycling and the catalytic process. In this study, we solved 1.20 Å resolution ternary structure (Dat/N-acetyl-arylalkylamine/CoA) of Dat by co-crystallization. Comparing apo form, binary form (tDat/Ac-CoA complex) and ternary form (tDat/CoA/Ac-PEA complex) of tDat, we found conformation of apo form Dat was different from binary and ternary form among them; structures of binary and ternary form were similar with each other. Then, we found the conformational change after Ac-CoA binding with tDat. The conformational change of substrate binding site may decide whether tDat can binding substrate or not. Thus, we elucidated ordered bi bi sequential mechanism of Dat by x-ray structural analysis. Additionally, the overall ternary structure of co-crystallization was similar with soaking except for an additional Ac-PEA outside the protein surface. The phenomenon implied may exist some factors to facilitate product release and enzyme recycle of Dat. Using isothermal titration calorimetry (ITC) and x-ray co-crystallization to carry out the competitive experiments, we found Ac-CoA showed dominantly competitive relation with respect to CoA. The dominantly competitive relation between Ac-CoA and CoA may resulted in enzymatic recycle of Dat. Finally, we based on catalytic triad to generate three variants, E47D, E47Q and E47N, for approaching transition state. All of them lost their substrate binding affinity and led to dramatic catalytic activity decrease. According to our results, we suggested Ac-CoA priorly binds to Dat providing a conformation change which facilitates substrate binding and forms ternary form. Ac-CoA may serve as driving force in catalyzation process of Dat. Our results implied Ac-CoA drives CoA and N-acetyl-arylalkylamine in Dat (ternary form) away, and occupies the cofactor binding site of Dat. Then Dat returns to state of binary form and accomplishes enzymatic recycling.