3 research outputs found
Synthesis of GNAT PA3944 Substrate Analogs
The Gcn5-related N-acetyltransferase (GNAT) superfamily is responsible for diverse biological functions and is critically important in cellular and metabolic processes in all kingdoms of life. GNATs transfer an acetyl-group from an active donor, typically acetyl-coenzyme A (AcCoA), to a primary amine of an acceptor substrate. Members of this family are well known for their roles in aminoglycoside antibiotic resistance, histone modification, protein acetylation, xenobiotic metabolism, and other cellular processes.1, 2 A small subset of bacterial GNAT enzymes have been studied and characterized both structurally and functionally, but the function of the vast majority remains unknown. Most of the reported 3D crystallographic structures of GNATs contain no acceptor substrate bound in their active sites. We previously screened the PA3944 protein against a panel of potential substrates and found the enzyme exhibited the highest activity toward aspartame, polymyxin B and colistin (polymyxin E).3 Our project involves the synthesis of molecular analogs of previously identified functionally relevant acceptor substrates that will be co-crystallized with GNAT-PA3944, in particular simplified derivatives of polymyxin B including NANMO and AAB, and we have shown that NANMO is as efficient as polymyxin B as a substrate. The ligand-bound crystallographic structures will provide insight into the structural features of the active site that are involved in substrate recognition and advance our understanding of types of substrates recognized by this enzyme of unknown function. Syntheses of NANMO and AAB will be described, along with modeling and substrate efficiency
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Gcn5-Related N-Acetyltransferases (GNATs) With a Catalytic Serine Residue Can Play Ping-Pong Too
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
sj-pdf-1-ini-10.1177_17534259231207198 - Supplemental material for Synthesis and validation of click-modified NOD1/2 agonists
Supplemental material, sj-pdf-1-ini-10.1177_17534259231207198 for Synthesis and validation of click-modified NOD1/2 agonists by Ravi Bharadwaj, Madison V. Anonick, Swati Jaiswal, Siavash Mashayekh, Ashley Brown, Kimberly A. Wodzanowski, Kendi Okuda, Neal Silverman and Catherine L. Grimes in Innate Immunity</p