Insight into the 3D structure and substrate specificity of previously uncharacterized GNAT superfamily acetyltransferases from pathogenic bacteria

• We characterized SACOL1063 and CD1211 GNATs of unknown function.
• SACOL1063 preferentially acetylates the amino acid l-threonine.
• CD1211 prefers l-Thr, but can also acetylate l-Ser, O-acetyl-l-Ser, and l-Met.
• The SACOL1063 structure shows substantial conformational changes of an important loop.
• Two molecules of CoA were bound in donor and acceptor sites of SACOL1063.

Members of the Gcn5-related N-acetyltransferase (GNAT) superfamily catalyze the acetylation of a wide range of small molecule and protein substrates. Due to their abundance in all kingdoms of life and diversity of their functions, they are implicated in many aspects of eukaryotic and prokaryotic physiology. Although numerous GNATs have been identified thus far, many remain structurally and functionally uncharacterized. The elucidation of their structures and functions is critical for broadening our knowledge of this diverse and important superfamily. In this work, we present the structural and kinetic analyses of two previously uncharacterized bacterial acetyltransferases - SACOL1063 from Staphylococcus aureus strain COL and CD1211 from Clostridium difficile strain 630. Our structures of SACOL1063 show substantial flexibility of a loop that is likely responsible for substrate recognition and binding compared to structures of other homologs. In the CoA complex structure, we found two CoA molecules bound in both the canonical AcCoA/CoA-binding site and the acceptor-substrate-binding site. Our work also provides initial clues regarding the substrate specificity of these two enzymes; however, their native function(s) remain unknown. We found both proteins act as N- rather than O-acetyltransferases and preferentially acetylate l-threonine. The combination of structural and kinetic analyses of these two previously uncharacterized GNATs provides fundamental knowledge and a framework on which future studies can be built to elucidate their native functions.