Arylalkylamine acetyltransferases (AANAT) belong to the ubiquitous superfamily of GNAT acetyltransferases that catalyze the transfer of acetyl group to a diverse set of acceptor substrates using primarily acetyl-CoA as donors. Kinetic analyses on GNAT family members have revealed that catalysis occurs through an ordered sequential mechanism where acetyl-CoA binds first, followed by the substrate binding. After the chemical step, the acylated substrate is released first, followed by CoA. Despite the wealth of available structural data on GNATs, the molecular events that impose a sequential mechanism remain elusive. Using NMR spectroscopy, in combination with ITC and kinetic experiments, we show that the position of the acetyl group during catalysis regulates protein conformational dynamics. Part of the substrate binding site switches from a stable ordered state in the presence of acetyl-CoA to a conformational labile ensemble of structures in the presence of CoA which may allow for the ordered binding of substrates and release of products. In addition, scanning through available GNAT structures, a conserved salt bridge connecting the cofactor and substrate binding sites was identified, that when broken disrupts the positive binding cooperativity of the substrate.

Acetyl-group sensing through modulation of conformational dynamics in an arylalkylamine N-acetyltransferase

254th National Meeting (2017)

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