Generating enzyme and radical‐mediated bisubstrates as tools for investigating Gcn5‐related <i>N‐</i>acetyltransferases

Reidl, Cory T.; Majorek, K.A.; Dang, Joseph; Tran, David; Jew, Kristen; Law, Melissa; Payne, Yasmine; Minor, W.; Becker, Daniel P.; Kuhn, Misty L.

doi:10.1002/1873-3468.12753

Cited by 6 publications

(8 citation statements)

References 43 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Of the GNATs that have had their kinetic mechanism experimentally determined, these enzymes overwhelmingly favor a direct transfer/sequential mechanism regardless of the identity of their acceptor substrate classification. Indeed, a large number of kinetic studies have shown that Gcn5/pCAF histone N- acetyltransferases (HATs) ( Jiang et al, 2012 ), arylalkylamine N- acetyltransferases (AANATs) ( Dempsey et al, 2014 ; Battistini et al, 2019 ), aminoglycoside N- acetyltransferases (AACs) ( Vetting et al, 2008b ), spermidine/spermine N- acetyltransferases (SSATs) ( Bewley et al, 2006 ; Filippova et al, 2015 ), and GNATs with unknown native substrates ( Majorek et al, 2017 ; Reidl et al, 2017 ) all utilize some form of a direct transfer mechanism.…”

Section: Discussionmentioning

confidence: 99%

Gcn5-Related N-Acetyltransferases (GNATs) With a Catalytic Serine Residue Can Play Ping-Pong Too

Baumgartner

Mohammad

Czub

et al. 2021

Front. Mol. Biosci.

Self Cite

View full text Add to dashboard Cite

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.

show abstract

Section: Discussionmentioning

confidence: 99%

Gcn5-Related N-Acetyltransferases (GNATs) With a Catalytic Serine Residue Can Play Ping-Pong Too

Baumgartner

Mohammad

Czub

et al. 2021

Front. Mol. Biosci.

Self Cite

View full text Add to dashboard Cite

show abstract

“…This new insight into the enzymatic mechanism also explains the observed inactivity of monomeric DapE constructs [ 10 ]. This products-bound DapE structure has enabled further refinement of a mechanistic hypothesis for amide bond cleavage by DapE enzymes, supported by our products-bound transition state modeling (PBTSM) approach [ 14 , 15 ], which in turn will facilitate inhibitor identification.…”

Section: Introductionmentioning

confidence: 99%

Indoline-6-Sulfonamide Inhibitors of the Bacterial Enzyme DapE

et al. 2020

Self Cite

View full text Add to dashboard Cite

Inhibitors of the bacterial enzyme dapE-encoded N-succinyl-L,L-diaminopimelic acid desuccinylase (DapE; EC 3.5.1.18) hold promise as antibiotics with a new mechanism of action. Herein we describe the discovery of a new series of indoline sulfonamide DapE inhibitors from a high-throughput screen and the synthesis of a series of analogs. Inhibitory potency was measured by a ninhydrin-based DapE assay recently developed by our group. Molecular docking experiments suggest active site binding with the sulfonamide acting as a zinc-binding group (ZBG).

show abstract

“…12 The requirement of this His residue explains the observed inactivity of a truncated, monomeric construct of DapE. 6 The product-bound structure, aided by our product-bound transition state modeling (PBTSM) approach, 12,13 enabled further refinement of the proposed reaction mechanism of DapE that will facilitate inhibitor identification 14−16 for the discovery of new antibiotics that inhibit DapE. We report herein a new atomic-resolution (1.3 Å) X-ray crystal structure of NmDapE (PDB entry 5UEJ) with sulfate ions bound in the substrate recognition pocket.…”

mentioning

confidence: 99%

Atomic-Resolution 1.3 Å Crystal Structure, Inhibition by Sulfate, and Molecular Dynamics of the Bacterial Enzyme DapE

et al. 2021

Self Cite

View full text Add to dashboard Cite

We report the atomic-resolution (1.3 Å) X-ray crystal structure of an open conformation of the dapE-encoded N-succinyl-l,l-diaminopimelic acid desuccinylase (DapE, EC 3.5.1.18) from Neisseria meningitidis. This structure [Protein Data Bank (PDB) entry 5UEJ] contains two bound sulfate ions in the active site that mimic the binding of the terminal carboxylates of the N-succinyl-l,l-diaminopimelic acid (l,l-SDAP) substrate. We demonstrated inhibition of DapE by sulfate (IC50 = 13.8 ± 2.8 mM). Comparison with other DapE structures in the PDB demonstrates the flexibility of the interdomain connections of this protein. This high-resolution structure was then utilized as the starting point for targeted molecular dynamics experiments revealing the conformational change from the open form to the closed form that occurs when DapE binds l,l-SDAP and cleaves the amide bond. These simulations demonstrated closure from the open to the closed conformation, the change in RMS throughout the closure, and the independence in the movement of the two DapE subunits. This conformational change occurred in two phases with the catalytic domains moving toward the dimerization domains first, followed by a rotation of catalytic domains relative to the dimerization domains. Although there were no targeting forces, the substrate moved closer to the active site and bound more tightly during the closure event.

show abstract

Generating enzyme and radical‐mediated bisubstrates as tools for investigating Gcn5‐related N‐acetyltransferases

Cited by 6 publications

References 43 publications

Gcn5-Related N-Acetyltransferases (GNATs) With a Catalytic Serine Residue Can Play Ping-Pong Too

Gcn5-Related N-Acetyltransferases (GNATs) With a Catalytic Serine Residue Can Play Ping-Pong Too

Indoline-6-Sulfonamide Inhibitors of the Bacterial Enzyme DapE

Atomic-Resolution 1.3 Å Crystal Structure, Inhibition by Sulfate, and Molecular Dynamics of the Bacterial Enzyme DapE

Contact Info

Product

Resources

About