Gcn5-related N-acetyltransferases (GNATs) are part of an extensive superfamily of enzymes that catalyze the transfer of an acetyl group from acetyl-coenzyme A to an amino group of an array of substrates. GNATs are abundant in all organisms, yet the functions for many of these enzymes remain unknown. To improve the functional annotation of bacterial GNATs of unknown function, our laboratory screens them against a library of potential substrates and then methodically kinetically characterizes each enzyme based on the substrates identified. Our recent studies have focused on previously uncharacterized proteins SACOL1063 from Staphylococcus aureus and CD1211 from Clostridium difficile. Our initial “hits” from this screen and then further kinetic characterization showed both enzymes preferentially N-acetylate L-threonine. Additionally, we determined the 3D structure of the SACOL1063 enzyme in its apo- and CoA-complex forms. The apo-form exhibited substantial movement of a loop important for substrate specificity and the liganded structure revealed two CoA molecules in the acceptor and canonical AcCoA-binding sites. Here, we present a structural comparison and genomic context evaluation of these two enzymes as the next step toward exploring their potential native functions.

Evaluation of potential functions of two previously uncharacterized bacterial GNAT threonine N-acetyltransferases from Clostridium difficile and Staphylococcus aureus

253rd National Meeting (2017)

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