Structural and functional characterisation of Arabidopsis thaliana and Synechocystis sp. PCC6803 GNAT2 acetyltransferases
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Protein acetylation is a common post-translational modification catalysed by several acetyltransferase families, including GCN5-related N-acetyltransferases (GNATs), which transfer an acetyl group from acetyl coenzyme A (Ac-CoA) to protein N-termini or lysine side chains. Plant GNAT2 and its cyanobacterial homolog have been shown to regulate photosynthesis by acetylating proteins involved in light harvesting in plants and cyclic electron flow in cyanobacteria. However, the molecular details of GNAT2 function remain poorly understood. Thus, this master’s thesis aimed to characterise GNAT2 structure and enzymatic activity.
Recombinant GNAT2 proteins from Arabidopsis thaliana (AtGNAT2) and Synechocystis sp. PCC6803 (SynGNAT2) were produced for crystallisation and activity studies. The apo AtGNAT2 crystal structure was determined by X-ray crystallography at 1.50 Å high resolution, and the Ac-CoA binding motif was analysed using structural comparison and ligand docking. AtGNAT2 adopted the conserved GNAT domain fold and formed a homodimer in the crystallographic asymmetric unit. The structure revealed additional secondary structure elements, an extended polar and charged β2-β3 loop, as well as a conserved Ac-CoA binding motif. Although an experimental SynGNAT2 structure was not obtained, preliminary crystallisation hits were identified, and model-based comparison suggested conservation of the GNAT fold and donor-site architecture between plant and cyanobacterial GNAT2 homologs. DTNB-based activity assays showed that AtGNAT2 acetylated peptides derived from the N-termini of photosynthetic proteins in vitro, with highest activity toward the D1-derived peptide. These results provide new structural and functional insights into GNAT2 acetyltransferases in photosynthetic organisms.