Acetylating Polymyxin Antibiotics: Clues Toward Substrate Specificity of Pa3944 Gcn5-Related N-Acetyltransferase of Unknown Function

Although Gcn5‐related N‐acetyltransferases (GNATs) have been studied for several decades in the context of aminoglycoside resistance and histone acetylation, the majority of their functions across all kingdoms are unknown. Members of this family typically catalyze the transfer of an acetyl group from acetyl coenzyme A (AcCoA) to an acceptor substrate, which may include a protein, antibiotic, polyamine, or other metabolite. The goal of our laboratory is to determine the structure/function relationships of bacterial GNATs of unknown function. To achieve this, we use a broad‐substrate screening assay to identify initial substrates to kinetically characterize the enzymes and to provide ligands for crystallization trials. We selected the uncharacterized GNAT PA3944 enzyme from Pseudomonas aeruginosa, determined its three‐dimensional structure, and found that it acetylates polymyxin antibiotics (polymyxin B and E). Polymyxin antibiotics are cyclized antimicrobial peptides with multiple L‐2,4‐diaminobutyric acid residues that carry a positive charge and can disrupt the cell membrane. Since the rate of developing new antibiotics lags considerably behind the rise in bacterial antibiotic drug resistance, polymyxin antibiotics are being revisited as viable candidates for treating P. aeruginosa infections. It is not known if the PA3944 enzyme is important for polymyxin resistance and if it specifically acetylates the antibiotic at one position or can multiply acetylate the antibiotics. Here, we present the kinetic characterization of the PA3944 enzyme and provide insight into its substrate specificity toward polymyxin antibiotics, which will be important for understanding the necessary requirements for potential adaptive drug resistance mechanisms. Support or Funding Information Support for this project was provided to MLK through San Francisco State University in the form of an ORSP Small Grant and Startup funds.