Journal
FRONTIERS IN MOLECULAR BIOSCIENCES
Volume 8, Issue -, Pages -Publisher
FRONTIERS MEDIA SA
DOI: 10.3389/fmolb.2021.646046
Keywords
Gcn5-related N-acetyltransferase; ping-pong kinetic mechanism; acetylation; acetyltransferase; docking; enzyme mechanism; Pseudomonas aeruginosa
Categories
Funding
- National Science Foundation [CHE-1708863, CHE-1708927, MCB-1616851]
- NIGMS [GM117325, GM132595]
- National Institute of Allergy and Infectious Diseases, National Institutes of Health, Department of Health and Human Services [HHSN272201200026C, HHSN272201700060C]
- Facilitating Research and Creative Work at SFSU grant
- Robert R. Wagner Fellowship at the University of Virginia
- Soft and Hybrid Nanotechnology Experimental (SHyNE) Resource [NSF ECCS-1542205]
- State of Illinois
- International Institute for Nanotechnology (IIN)
- DOE Office of Science [DE-AC02-06CH11357]
- Michigan Economic Development Corporation
- Michigan Technology Tri-Corridor [085P1000817]
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Enzymes in the GNAT superfamily play crucial roles in cellular processes, with recent studies showing their capability of catalyzing various reactions beyond acetyl transfer. This study on the Pseudomonas aeruginosa PA3944 enzyme reveals the use of a nucleophilic serine residue and a hybrid ping-pong mechanism for catalysis, challenging the traditional acid/base mechanism attributed to GNATs. The findings suggest that different active site residues are utilized by these enzymes to achieve a range of catalytic reactions, questioning the current paradigm of GNAT mechanisms.
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.
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