Abstract
Melatonin is a major hormonal mediator of light-induced photoperiodic changes in circadian biological events and is found in bacteria, protozoa, macroalgae, plants, fungi, invertebrates, and vertebrates. Despite of the structural characterization of melatonin, there has been increasing interest in this “molecular pacemaker” hormone. The physiological roles of melatonin have been widely reported on sleep, mood, immune response, cardiovascular fitness and aging. Research on melatonin biosynthesis could help improve our knowledge of circadian rhythm. The daily cycle of melatonin biosynthesis in mammals is regulated by AANAT (arylalkylamine N-acetyltransferase; EC 2.3.1.87), making it an attractive target for therapeutic control of abnormal melatonin production in mood and sleep disorders. Drosophila melanogaster Dat (dopamine N-acetyltransferase) is an arylalkylamine N-acetyltransferase, which is involved in melatonin formation, sclerotization, and neurotransmitter inactivation and has been found in the head, the eyes, the optic lobe and the brain of Drosophila melanogaster. Moreover, Dat belongs to the GCN5-related N-acetyltransferase (GNAT) superfamily. Dat catalyzes the transfer of the acetyl group in acetyl coenzyme A (AcCoA, cofactor) to various arylalkylamines (substrate). In order to unravel the detail molecular mechanism for Dat activity, we worked on the structural and functional studies of Dat. We have determined high-resolution crystal structure of D. melanogaster Dat in apo form, binary complex form (AcCoA-bound), and ternary complex form (acetylarylalkylamine/CoA-bound). A binding study using isothermal titration calorimetry suggested that the cofactor bound to Dat first before substrate. Examination of the binary complex structure and a substrate-docked model indicated that Dat contains a novel AANAT catalytic triad. Site-directed mutagenesis, kinetic studies and pH-rate profiles confirmed that Glu47, Ser182 and Ser186 were critical for catalysis. Collectively, the results of the present study suggest that Dat possesses a specialized active site structure dedicated to a catalytic mechanism where nucleophilic attack and leaving group protonation occur in a coordinated manner dependent on catalytic triad. The molecular basis of substrate recognition and the kinetic mechanism by which Dat interacts with substrate and cofactor are unclear. Here, two-substrate kinetic analysis and dead end analog inhibition studies with the tryptophol and palmitoyl CoA indicated that Dat utilizes an ordered sequential mechanism requiring binding of acetyl-CoA first. Furthermore, we presented the first crystal structure of ternary complex in this AANAT family. Detailed analyses of ternary complexes of Dat revealed a hydrophobic substrate-binding pocket near the acetylation active site. The shape and size of the pocket dictate substrate selectivity and specificity. We have mapped two key aromatic residues in the protein-substrate interface essential for substrate binding and selection between phenylalkylamines (PAAs), indoalkylamines (IAAs), and arylamines substrates. The Dat has higher activity with the PAAs than with the IAAs. It appears that the aromatic ring and alkyl chain length on arylalkylamine molecule greatly define the Dat extended substrate specificity profile. By analyzing ternary complex structure as well as site-directed mutagenesis, we demonstrated that Phe43 significantly influence the substrate binding and the activity of Dat, while Tyr64 was an important determinant of substrate preference. Kinetic studies confirmed that the Y64W mutation is sufficient to increase the activity of the enzyme toward IAAs being the preferred substrate for the Y64W mutant, which indicated that this residue modulates the substrate preference of Dat between PAAs and IAAs. These results confirmed that these residues are critical for aromatic interaction between Dat and substrate. This work provides a structural foundation for the detailed understanding of the structural and biological properties of arylalkylamine N-acetyltransferases and of GCN5-related N-acetyltransferase superfamily proteins in general.