Roles of acidic residues in SpeG acetyltransferases-insights into importance for kinetic activity and polyamine binding in allosteric and acceptor sites.

The Gcn5-related N-acetyltransferases belong to a massive superfamily of enzymes that perform a wide array of functions in different organisms. This family is comprised of smaller subfamilies, including one called the spermidine/spermine N-acetyltransferases (SSATs). SSATs acetylate positively charged long-chain polyamines to maintain their intracellular concentrations. In bacteria, one primary type of SSAT is the SpeG enzyme, which adopts a homododecameric assembly. In the present study, we sought to detail how polyamines bind to both the allosteric and active sites of SpeG and determine which conserved acidic and polar residues are critical for kinetic activity and polyamine binding. Therefore, we determined a crystal structure of the Vibrio cholerae (VcSpeG) enzyme in complex with spermine in the allosteric site and N1-acetylspermine in the active site. This result clearly defines two distinct and separate polyamine binding sites within the protein. Furthermore, it demonstrates that SpeG is indeed an allosteric enzyme: homotropic in that the ligands are identical and heterotropic in that the allosteric binding sites are distinct from the active sites. We also investigated the kinetic activity of substituted residues in both sites and found several residues are critical for enzyme activity, while some substitutions altered polyamine substrate specificity. These combined structural and functional results begin to illuminate how longer-chain polyamines with terminal aminopropyl groups are recognized and acetylated by SpeG. Finally, we present a hypothetical model for proposed roles of conserved acidic residues in both sites, which provides a framework for subsequent studies of SpeG's intricate allosteric network.