Structural and Biochemical Characterisation of GCN5-Related N-Acetyltransferases from Staphylococcusaureus

Gcn5-related N-acetyltransferases (GNATs) are members of a very large superfamily that catalyse the transfer of an acetyl group from acetyl coenzyme A to a wide range of acceptor molecules, releasing the acetylated product and free CoA. The superfamily represents more than 10,000 members, and is present within all forms of life. Though GNATs are ubiquitous and involved in diverse functions of prokaryotes and eukaryotes, most of them still remain structurally and functionally uncharacterised. This PhD study investigates the structural and functional characterisation of five uncharacterised GNATs from the pathogenic Staphylococcus aureus (SaGNAT-1, 2, 3, 4 and SaSpeG). Moreover, this study reveals novel structures and strong substrates of GNATs through x-ray crystallography, broad-substrate screening and functional assays.
This study illustrates the structure and function of SaGNAT-1, a structure exhibiting a core GNAT fold, and has high structural homology with phosphinothricin acetyltransferases. Putative residues involved in acetyl-CoA and have been identified based on structural homology within the GNAT family. We also confirm the SaGNAT-1 activity against cadaverine, putrescine, L-glutamine and agmatine.
In this thesis we also report first high resolution structure of SaGNAT-2, diffracting to 1.6 Å. The structural characterisation of SaGNAT-2 confirms it belongs to the GNAT superfamily, and is likely to exist in the monomeric form in solution. SaGNAT-2 has high structural homology with RimI protein that acetylates ribosomal S18 peptide.
The study of two other GNATs in this thesis, SaGNAT-3 and 4, also reveal the presence of a GNAT-fold, however SaGNAT-3 possesses five helices rather than four, a unique extra helix present between !3 and !4. Through broad substrate screening we confirm SaGNAT-3 has strong activity with substrates cadaverine, putrescine, and agmatine.
whereas SaGNAT-4 shows activity with poly-L-lysine and pterine. These structures were also crystallised in the absence and presence of CoA to provide insights into the substrate- binding pocket.
Finally, structural and biochemical investigation of SaSpeG protein confirms it forms of a dodecameric biological assembly. SaSpeG shows activity against spermine, spermidine, cadaverine, putrescine, agmatine, and N-acetyl spermine, among which greatest activity noted with putrescine. Overall, this thesis reports novel structural and functional aspects of GNATs that will assist to understand their importance in Staphylococcus aureus and potential as drug targets.