Structural characterization of a Gcn5-related N-acetyltransferase from Staphylococcus aureus

Abstract

The Gcn5-related N-acetyltransferases (GNATs) are ubiquitously expressed in nature and perform a diverse range of cellular functions through the acetylation of small molecules and protein substrates. Using activated acetyl coenzyme A as a common acetyl donor, GNATs catalyse the transfer of an acetyl group to acceptor molecules including aminoglycoside antibiotics, glucosamine-6-phosphate, histones, serotonin and spermidine. There is often only very limited sequence conservation between members of the GNAT superfamily, in part, reflecting their capacity to bind a diverse array of substrates. In contrast, the secondary and tertiary structures are highly conserved, but then at the quaternary level there is further diversity, with GNATs shown to exist in monomeric, dimeric, or tetrameric states. Here we describe the X-ray crystallographic structure of a GNAT enzyme from Staphylococcus aureus with only low sequence identity to previously solved GNAT proteins. It contains many of the classical GNAT motifs, but lacks other hallmarks of the GNAT fold including the classic β-bulge splayed at the β-sheet interface. The protein is likely to be a dimer in solution based on analysis of the asymmetric unit within the crystal structure, homology with related GNAT family members, and size exclusion chromatography. The study provides the first high resolution structure of this enzyme, providing a strong platform for substrate and cofactor modelling, and structural/functional comparisons within this diverse enzyme superfamily.

Figure 1. Protein purification profile of SaGNAT.

(A) FPLC profile of the affinity purification, with SDS-PAGE insert showing lane 1 - whole bacterial cell lysate; lane 2 - soluble protein fraction of the bacterial cell lysate; lane 3 – flow-through from the affinity column; lane 4 – affinity elution. (B) Size exclusion purification indicating the theoretical elution volumes for monomer, dimer, and trimer, and an SDS PAGE insert lane 5 – showing the final purity of the protein.

Figure 2. Tertiary structure of SaGNAT in cartoon format, with α-helices, β-strands, and loops colored in red, yellow, and green respectively.

Figure 3. Structure based alignment of the SaGNAT (green) with 3 acyl-transferases and RMSD less than 1 Å. 1YR0 (orange), 1BL1 (blue), and 2JLM (magenta) are crystal structure of phosphinothricin acetyltransferase from Agrobacterium tumefaciens, Pseudomonas aeruginosa, and Acinetobacter baylyi respectively.

Blue and red boxes depict conserved CoA binding and active site residues respectively, yellow box indicate residues involved in dimer formation, and unfilled boxes represent strictly conserved residues.

Figure 4. Quaternary structure of SaGNAT showing interacting residues at the dimer interface.