Acetyl group coordinated progression through the catalytic cycle of an arylalkylamine N-acetyltransferase.

Adam A Aboalroub,David J Merkler,Ziming Zhang,Ioannis Gelis,Dimitra Keramisanou,Ashleigh B Bachman,Viswanathan V Krishnan

doi:10.1371/journal.pone.0177270

Abstract

The transfer of an acetyl group from acetyl-CoA to an acceptor amine is a ubiquitous biochemical transformation catalyzed by Gcn5-related N-acetyltransferases (GNATs). Although it is established that the reaction proceeds through a sequential ordered mechanism, the role of the acetyl group in driving the ordered formation of binary and ternary complexes remains elusive. Herein, we show that CoA and acetyl-CoA alter the conformation of the substrate binding site of an arylalkylamine N-acetyltransferase (AANAT) to facilitate interaction with acceptor substrates. However, it is the presence of the acetyl group within the catalytic funnel that triggers high affinity binding. Acetyl group occupancy is relayed through a conserved salt bridge between the P-loop and the acceptor binding site, and is manifested as differential dynamics in the CoA and acetyl-CoA-bound states. The capacity of the acetyl group carried by an acceptor to promote its tight binding even in the absence of CoA, but also its mutually exclusive position to the acetyl group of acetyl-CoA underscore its importance in coordinating the progression of the catalytic cycle.

Highlights

Gcn5-related N-acetyltransferases (GNATs) enzymes catalyze the acetylation of a wide range of primary amine acceptors from histone and ribosomal proteins, to small molecules like serotonin, dopamine and aminoglycoside antibiotics [1,2,3]
To characterize the mode of donor binding, we followed the titration of bmAANAT3 with acetyl-CoA and CoA by nuclear magnetic resonance spectroscopy (NMR)
Changes in the 15N-HSQC spectrum of bmAANAT3 as a function of added acetyl-CoA occur on the slow-exchange regime of the NMR timescale, causing progressive disappearance of the signals of the free-state and the concomitant appearance of a new set of signals corresponding to the bound-state (Fig 1B)

Summary

Introduction

GNAT enzymes catalyze the acetylation of a wide range of primary amine acceptors from histone and ribosomal proteins, to small molecules like serotonin, dopamine and aminoglycoside antibiotics [1,2,3]. They are central regulators of diverse cellular processes such as gene transcription [4], time and seasonal adaptation [5,6,7] and metabolism [8, 9]. Strands β4 and β5, from motifs A and B, respectively, form the only parallel stretch of the mixed β sheet. The amide nitrogen of the residue downstream of the β bulge forms a conserved hydrogen bond with the carbonyl of the acetyl

Methods

Results

Conclusion