Abstract
Enzymes in the Gcn5-related N-acetyltransferase (GNAT) superfamily are widespread and critically involved in multiple cellular processes ranging from antibiotic resistance to histone modification. While acetyl transfer is the most widely catalyzed reaction, recent studies have revealed that these enzymes are also capable of performing succinylation, condensation, decarboxylation, and methylcarbamoylation reactions. The canonical chemical mechanism attributed to GNATs is a general acid/base mechanism; however, mounting evidence has cast doubt on the applicability of this mechanism to all GNATs. This study shows that the Pseudomonas aeruginosa PA3944 enzyme uses a nucleophilic serine residue and a hybrid ping-pong mechanism for catalysis instead of a general acid/base mechanism. To simplify this enzyme’s kinetic characterization, we synthesized a polymyxin B substrate analog and performed molecular docking experiments. We performed site-directed mutagenesis of key active site residues (S148 and E102) and determined the structure of the E102A mutant. We found that the serine residue is essential for catalysis toward the synthetic substrate analog and polymyxin B, but the glutamate residue is more likely important for substrate recognition or stabilization. Our results challenge the current paradigm of GNAT mechanisms and show that this common enzyme scaffold utilizes different active site residues to accomplish a diversity of catalytic reactions.
Highlights
Acetyltransferases are fascinating enzymes found across all domains of life
Based on the crystal structures and kinetic data of PA3944 we have presented, it is clear that PA3944 does not proceed through a general acid/base mechanism as most other characterized Gcn5-related N-acetyltransferase (GNAT)
It has been suggested that serine can act as a general acid in other GNATs (O’Flynn et al, 2018), but its high pKa makes it highly unlikely to act in this capacity
Summary
Acetyltransferases are fascinating enzymes found across all domains of life They are critically important for various cellular functions including those of anabolic and catabolic pathways, cell wall modification, xenobiotic metabolism, and antibiotic drug resistance (Sim et al, 2014; Hentchel and Escalante-Semerena, 2015; Zhang et al, 2017; Burckhardt and Escalante-Semerena, 2020; Sharma et al, 2020). Their seemingly simple ability to catalyze the transfer of an acetyl moiety from a donor molecule to an acceptor molecule is compounded by the diversity of structural. All three of these families of acetyltransferases play intricate roles in bacterial cellular processes, and are worthy of dedicated study
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