Abstract
The Gcn5-related N-acetyltransferase (GNAT) superfamily is a large group of evolutionarily related acetyltransferases, with multiple paralogs in organisms from all kingdoms of life. The functionally characterized GNATs have been shown to catalyze the transfer of an acetyl group from acetyl-coenzyme A (Ac-CoA) to the amine of a wide range of substrates, including small molecules and proteins. GNATs are prevalent and implicated in a myriad of aspects of eukaryotic and prokaryotic physiology, but functions of many GNATs remain unknown. In this work, we used a multi-pronged approach of x-ray crystallography and biochemical characterization to elucidate the sequence-structure-function relationship of the GNAT superfamily member PA4794 from Pseudomonas aeruginosa. We determined that PA4794 acetylates the Nε amine of a C-terminal lysine residue of a peptide, suggesting it is a protein acetyltransferase specific for a C-terminal lysine of a substrate protein or proteins. Furthermore, we identified a number of molecules, including cephalosporin antibiotics, which are inhibitors of PA4794 and bind in its substrate-binding site. Often, these molecules mimic the conformation of the acetylated peptide product. We have determined structures of PA4794 in the apo-form, in complexes with Ac-CoA, CoA, several antibiotics and other small molecules, and a ternary complex with the products of the reaction: CoA and acetylated peptide. Also, we analyzed PA4794 mutants to identify residues important for substrate binding and catalysis.
Highlights
Gcn5-related N-acetyltransferases (GNATs) are involved in small molecule and protein acetylation in all organisms
PA4794 is a member of the GNAT superfamily of acyltransferases, with a fold comprised of an N-terminal -strand followed by three ␣-helices ( two helices are generally more common in GNATs [4]), three antiparallel -strands, followed by an ␣-helix, a fifth -strand, a fifth ␣-helix, and two -strands
The discovery that GNATs are involved in protein acetylation in bacteria, not just in eukaryotes, suggests that acetylation-based regulation may be an important process in prokaryotes as well
Summary
Gcn5-related N-acetyltransferases (GNATs) are involved in small molecule and protein acetylation in all organisms. Members of the GNAT superfamily have been identified in all kingdoms of life, with multiple paralogs found in many organisms These enzymes have been shown to catalyze the transfer of an acetyl group from acetyl-coenzyme A (Ac-CoA) to the primary amine of a wide range of substrates, including glucosamine 6-phosphate, aminoglycoside antibiotics, spermine, spermidine, and other small molecules [3, 4]. We used semi-high throughput crystallographic and biochemical screening experiments to characterize the structure and activity of this protein and found that it selectively acetylates the N⑀ group of the C-terminal lysine of peptides. This suggests that PA4794 functions as a C-terminal lysine protein acetyltransferase. We kinetically characterized the enzyme, which included inhibition studies for the identified complexes and determining the influence of selected point mutations on kinetic activity
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