Abstract
Acetyl coenzyme A (Ac-CoA)-dependent N-acetylation is performed by arylalkylamine N-acetyltransferase (AANAT) and is important in many biofunctions. AANAT catalyzes N-acetylation through an ordered sequential mechanism in which cofactor (Ac-CoA) binds first, with substrate binding afterward. No ternary structure containing AANAT, cofactor, and substrate was determined, meaning the details of substrate binding and product release remain unclear. Here, two ternary complexes of dopamine N-acetyltransferase (Dat) before and after N-acetylation were solved at 1.28 Å and 1.36 Å resolution, respectively. Combined with the structures of Dat in apo form and Ac-CoA bound form, we addressed each stage in the catalytic cycle. Isothermal titration calorimetry (ITC), crystallography, and nuclear magnetic resonance spectroscopy (NMR) were utilized to analyze the product release. Our data revealed that Ac-CoA regulates the conformational properties of Dat to form the catalytic site and substrate binding pocket, while the release of products is facilitated by the binding of new Ac-CoA.
Highlights
Acetyl coenzyme A (Ac-CoA)-dependent N-acetylation is performed by arylalkylamine Nacetyltransferase (AANAT) and is important in many biofunctions
Overall structures revealed that dopamine N-acetyltransferase (Dat) was a globular protein containing a tunnel-like cavity, a substrate located in the middle of the protein, a CoA group located at the bottom, and an acetyl group between the substrate and CoA (Fig. 2a)
According to the above data, we propose a complete catalytic cycle of Dat (Fig. 7)
Summary
Acetyl coenzyme A (Ac-CoA)-dependent N-acetylation is performed by arylalkylamine Nacetyltransferase (AANAT) and is important in many biofunctions. AANAT catalyzes Nacetylation through an ordered sequential mechanism in which cofactor (Ac-CoA) binds first, with substrate binding afterward. No ternary structure containing AANAT, cofactor, and substrate was determined, meaning the details of substrate binding and product release remain unclear. Our data revealed that Ac-CoA regulates the conformational properties of Dat to form the catalytic site and substrate binding pocket, while the release of products is facilitated by the binding of new Ac-CoA. The complex structure of SNAT with bisubstrate analog reveals that the conserved histidines do not directly participate in the deprotonation of the substrate amine group, but the water molecules between the conserved histidines and substrate could act as mediators for the exchangeable alkylamine proton and facilitate the nucleophilic attack of deprotonated amine on Ac-CoA13. Other reports indicated that the rate-determining step in the entire reaction of Nacetylation is the diffusion of the released product[3], in which the acetyl-substrate is released first followed by the release of CoA
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