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

2021 ◽  
Vol 8 ◽  
Author(s):  
Jackson T. Baumgartner ◽  
Thahani S. Habeeb Mohammad ◽  
Mateusz P. Czub ◽  
Karolina A. Majorek ◽  
Xhulio Arolli ◽  
...  
Keyword(s):  
Active Site ◽  
Polymyxin B ◽  
Chemical Mechanism ◽  
Active Site Residues ◽  
Serine Residue ◽  
Acid Base ◽  
Substrate Analog ◽  
General Acid ◽  
Ping Pong ◽  
Base Mechanism

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.

scholarly journals Chiral recognition of prochiral centres and general acid-base catalysis. Necessarily interrelated manifestations of active-site structure

1976 ◽  
Vol 153 (2) ◽  
pp. 491-493 ◽  
Author(s):  
J Südi
Keyword(s):  
Lactate Dehydrogenase ◽  
Active Site ◽  
Chiral Recognition ◽  
Conformational Transition ◽  
Base Catalysis ◽  
Acid Base ◽  
Site Structure ◽  
General Acid ◽  
Active Site Structure

On the basis of Ogston's [(1948) Nature (London) 162, 963] argument, the following conclusions are indicated by the stereochemistry of the reversible oxidation of glycollate by lactate dehydrogenase: (1) general acid-base catalysis is involved in the reaction; (2) the transformation of enzyme-bound (tetrahedral) substrate into enzyme-bound (trigonal) product involves a conformational transition of the enzyme-coenzyme complex.

scholarly journals Chemical feasibility of the general acid/base mechanism ofglmSribozyme self-cleavage

Biopolymers ◽  
2015 ◽  
Vol 103 (10) ◽  
pp. 550-562 ◽  
Author(s):  
Matúš Dubecký ◽  
Nils G. Walter ◽  
Jiří Šponer ◽  
Michal Otyepka ◽  
Pavel Banáš
Keyword(s):  
Acid Base ◽  
General Acid ◽  
Base Mechanism

scholarly journals Characterization of Glycerol Trinitrate Reductase (NerA) and the Catalytic Role of Active-Site Residues

2004 ◽  
Vol 186 (6) ◽  
pp. 1802-1810 ◽  
Author(s):  
Samantha J. Marshall ◽  
Doreen Krause ◽  
Dayle K. Blencowe ◽  
Graham F. White
Keyword(s):  
Active Site ◽  
Structural Model ◽  
Flavin Mononucleotide ◽  
Active Site Residues ◽  
Old Yellow Enzyme ◽  
Ping Pong ◽  
Glycerol Trinitrate ◽  
Catalytic Role

ABSTRACT Glycerol trinitrate reductase (NerA) from Agrobacterium radiobacter, a member of the old yellow enzyme (OYE) family of oxidoreductases, was expressed in and purified from Escherichia coli. Denaturation of pure enzyme liberated flavin mononucleotide (FMN), and spectra of NerA during reduction and reoxidation confirmed its catalytic involvement. Binding of FMN to apoenzyme to form the holoenzyme occurred with a dissociation constant of ca. 10−7 M and with restoration of activity. The NerA-dependent reduction of glycerol trinitrate (GTN; nitroglycerin) by NADH followed ping-pong kinetics. A structural model of NerA based on the known coordinates of OYE showed that His-178, Asn-181, and Tyr-183 were close to FMN in the active site. The NerA mutation H178A produced mutant protein with bound FMN but no activity toward GTN. The N181A mutation produced protein that did not bind FMN and was isolated in partly degraded form. The mutation Y183F produced active protein with the same k cat as that of wild-type enzyme but with altered Km values for GTN and NADH, indicating a role for this residue in substrate binding. Correlation of the ratio of Km GTN to Km NAD(P)H, with sequence differences for NerA and several other members of the OYE family of oxidoreductases that reduce GTN, indicated that Asn-181 and a second Asn-238 that lies close to Tyr-183 in the NerA model structure may influence substrate specificity.

scholarly journals Mechanistic basis for the emergence of EPS1 as a catalyst in plant salicylic acid biosynthesis

2021 ◽  
Author(s):  
Michael P. Torrens-Spence ◽  
Tianjie Li ◽  
Ziqi Wang ◽  
Christopher M. Glinkerman ◽  
Jason O. Matos ◽  
...  
Keyword(s):  
Salicylic Acid ◽  
Active Site ◽  
Active Site Residues ◽  
Substrate Analog ◽  
Lyase Activity ◽  
Mechanistic Basis ◽  
Dynamics Simulations ◽  
Brassicaceae Family ◽  
Bahd Acyltransferase

AbstractUnique to plants in the Brassicaceae family, the production of the plant defense hormone salicylic acid (SA) from isochorismate is accelerated by an evolutionarily young isochorismoyl-glutamate pyruvoyl-glutamate lyase, EPS1, which belongs to the BAHD acyltransferase protein family. Here, we report the crystal structures of apo and substrate-analog-bound EPS1 from Arabidopsis thaliana. Assisted by microsecond molecular dynamics simulations, we uncover a unique pericyclic rearrangement lyase mechanism facilitated by the active site of EPS1. We reconstitute the isochorismate-derived pathway of SA biosynthesis in Saccharomyces cerevisiae, which serves as an in vivo platform that helps identify active-site residues critical for EPS1 activity. This study describes the birth of a new catalyst in plant phytohormone biosynthesis by reconfiguring the ancestral active site of a progenitor enzyme to catalyze alternative reaction.One sentence summaryBy reconfiguring the active site of a progenitor acyltransferase-fold, EPS1 acquired the unique, evolutionarily new lyase activity that accelerates phytohormone salicylic acid production in Brassicaceae plants.

General Acid/Base Catalysis in the Active Site ofEscherichia coliThioredoxin†

Biochemistry ◽  
1997 ◽  
Vol 36 (50) ◽  
pp. 15810-15816 ◽  
Author(s):  
Peter T. Chivers ◽  
Ronald T. Raines
Keyword(s):  
Active Site ◽  
Base Catalysis ◽  
Acid Base ◽  
General Acid

Theoretical Studies of the Acid-Base Equilibria in a Model Active Site of the Human 20S Proteasome

2020 ◽  
Author(s):  
Jon Uranga ◽  
Lukas Hasecke ◽  
Jonny Proppe ◽  
Jan Fingerhut ◽  
Ricardo A. Mata
Keyword(s):  
Active Site ◽  
Boronic Acid ◽  
Computational Study ◽  
Ubiquitin Proteasome System ◽  
Bayesian Optimization ◽  
Inhibition Mechanism ◽  
20S Proteasome ◽  
Acid Base ◽  
Ubiquitin Proteasome ◽  
Infection Cycles

The 20S Proteasome is a macromolecule responsible for the chemical step in the ubiquitin-proteasome system of degrading unnecessary and unused proteins of the cell. It plays a central role both in the rapid growth of cancer cells as well as in viral infection cycles. Herein, we present a computational study of the acid-base equilibria in an active site of the human proteasome, an aspect which is often neglected despite the crucial role protons play in the catalysis. As example substrates, we take the inhibition by epoxy and boronic acid containing warheads. We have combined cluster quantum mechanical calculations, replica exchange molecular dynamics and Bayesian optimization of non-bonded potential terms in the inhibitors. In relation to the latter, we propose an easily scalable approach to the reevaluation of non-bonded potentials making use of QM/MM dynamics information. Our results show that coupled acid-base equilibria need to be considered when modeling the inhibition mechanism. The coupling between a neighboring lysine and the reacting threonine is not affected by the presence of the inhibitor.

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