scholarly journals Imaging active site chemistry and protonation states: NMR crystallography of the tryptophan synthase α-aminoacrylate intermediate

2021 ◽  
Author(s):  
Jacob B. Holmes ◽  
Viktoriia Liu ◽  
Bethany G. Caulkins ◽  
Eduardo Hilario ◽  
Rittik K. Ghosh ◽  
...  
Keyword(s):  
Transition State ◽  
Active Site ◽  
Nucleophilic Attack ◽  
Hydroxyl Group ◽  
Tryptophan Synthase ◽  
Structural Water ◽  
Enzyme Active Site ◽  
X Ray Crystallography ◽  
Nmr Crystallography ◽  
And Function

NMR-assisted crystallography – the synergistic combination of solid-state NMR, X-ray crystallography, and first-principles computational chemistry – holds remarkable promise for mechanistic enzymology: by providing atomic-resolution characterization of stable intermediates in the enzyme active site – including hydrogen atom locations and tautomeric equilibria – it offers insight into structure, dynamics, and function. Here, we make use of this combined approach to characterize the α-aminoacrylate intermediate in tryptophan synthase, a defining species for pyridoxal-5'-phosphate-dependent enzymes on the β-elimination and replacement pathway. By uniquely identifying the protonation states of ionizable sites on the cofactor, substrates, and catalytic side chains, as well as the location and orientation of structural waters in the active site, a remarkably clear picture of structure and reactivity emerges. Most incredibly, this intermediate appears to be mere tenths of angstroms away from the preceding transition state in which the β-hydroxyl of the serine substrate is lost. The position and orientation of the structural water immediately adjacent to the substrate β-carbon suggests not only the fate of the hydroxyl group, but also the pathway back to the transition state and the identity of the active site acid-base catalytic residue. Reaction of this intermediate with benzimidazole (BZI), an isostere of the natural substrate, indole, shows BZI bound in the active site and poised for, but unable to initiate, the subsequent bond formation step. When modeled into the BZI position, indole is positioned with C3 in contact with the α-aminoacrylate Cβ and aligned for nucleophilic attack.

X-ray and NMR Crystallography in an Enzyme Active Site: The Indoline Quinonoid Intermediate in Tryptophan Synthase

2011 ◽  
Vol 133 (1) ◽  
pp. 4-7 ◽  
Author(s):  
Jinfeng Lai ◽  
Dimitri Niks ◽  
Yachong Wang ◽  
Tatiana Domratcheva ◽  
Thomas R. M. Barends ◽  
...  
Keyword(s):  
Active Site ◽  
Tryptophan Synthase ◽  
X Ray ◽  
Enzyme Active Site ◽  
Nmr Crystallography

scholarly journals Imaging active site chemistry and protonation states: NMR crystallography of the tryptophan synthase α-aminoacrylate intermediate

2022 ◽  
Vol 119 (2) ◽  
pp. e2109235119
Author(s):  
Jacob B. Holmes ◽  
Viktoriia Liu ◽  
Bethany G. Caulkins ◽  
Eduardo Hilario ◽  
Rittik K. Ghosh ◽  
...  
Keyword(s):  
Active Site ◽  
Active Sites ◽  
Reaction Pathway ◽  
Three Dimensional ◽  
Integrated Approach ◽  
Nucleophilic Attack ◽  
Dimensional Structure ◽  
Tryptophan Synthase ◽  
Hydrogen Bond Donor ◽  
Nmr Crystallography

NMR-assisted crystallography—the integrated application of solid-state NMR, X-ray crystallography, and first-principles computational chemistry—holds significant promise for mechanistic enzymology: by providing atomic-resolution characterization of stable intermediates in enzyme active sites, including hydrogen atom locations and tautomeric equilibria, NMR crystallography offers insight into both structure and chemical dynamics. Here, this integrated approach is used to characterize the tryptophan synthase α-aminoacrylate intermediate, a defining species for pyridoxal-5′-phosphate–dependent enzymes that catalyze β-elimination and replacement reactions. For this intermediate, NMR-assisted crystallography is able to identify the protonation states of the ionizable sites on the cofactor, substrate, and catalytic side chains as well as the location and orientation of crystallographic waters within the active site. Most notable is the water molecule immediately adjacent to the substrate β-carbon, which serves as a hydrogen bond donor to the ε-amino group of the acid–base catalytic residue βLys87. From this analysis, a detailed three-dimensional picture of structure and reactivity emerges, highlighting the fate of the L-serine hydroxyl leaving group and the reaction pathway back to the preceding transition state. Reaction of the α-aminoacrylate intermediate with benzimidazole, an isostere of the natural substrate indole, shows benzimidazole bound in the active site and poised for, but unable to initiate, the subsequent bond formation step. When modeled into the benzimidazole position, indole is positioned with C3 in contact with the α-aminoacrylate Cβ and aligned for nucleophilic attack. Here, the chemically detailed, three-dimensional structure from NMR-assisted crystallography is key to understanding why benzimidazole does not react, while indole does.

scholarly journals Reverse evolution leads to genotypic incompatibility despite functional and active site convergence

eLife ◽  
2015 ◽  
Vol 4 ◽  
Author(s):  
Miriam Kaltenbach ◽  
Colin J Jackson ◽  
Eleanor C Campbell ◽  
Florian Hollfelder ◽  
Nobuhiko Tokuriki
Keyword(s):  
Active Site ◽  
Adaptive Landscape ◽  
Sequence Structure ◽  
Activity Increase ◽  
Enzyme Active Site ◽  
Fundamental Relationship ◽  
And Function ◽  
Alternative Set ◽  
Shed Light ◽  
New Mutations

Understanding the extent to which enzyme evolution is reversible can shed light on the fundamental relationship between protein sequence, structure, and function. Here, we perform an experimental test of evolutionary reversibility using directed evolution from a phosphotriesterase to an arylesterase, and back, and examine the underlying molecular basis. We find that wild-type phosphotriesterase function could be restored (>104-fold activity increase), but via an alternative set of mutations. The enzyme active site converged towards its original state, indicating evolutionary constraints imposed by catalytic requirements. We reveal that extensive epistasis prevents reversions and necessitates fixation of new mutations, leading to a functionally identical sequence. Many amino acid exchanges between the new and original enzyme are not tolerated, implying sequence incompatibility. Therefore, the evolution was phenotypically reversible but genotypically irreversible. Our study illustrates that the enzyme's adaptive landscape is highly rugged, and different functional sequences may constitute separate fitness peaks.

scholarly journals Mechanism of Type IA Topoisomerases

Molecules ◽  
2020 ◽  
Vol 25 (20) ◽  
pp. 4769
Author(s):  
Tumpa Dasgupta ◽  
Shomita Ferdous ◽  
Yuk-Ching Tse-Dinh
Keyword(s):  
Nucleic Acid ◽  
Nucleophilic Attack ◽  
Hydroxyl Group ◽  
Active Site Residues ◽  
Phosphodiester Bond ◽  
X Ray Crystallography ◽  
Dna And Rna ◽  
Catalytic Function ◽  
Universal Common Ancestor ◽  
Type Ia

Topoisomerases in the type IA subfamily can catalyze change in topology for both DNA and RNA substrates. A type IA topoisomerase may have been present in a last universal common ancestor (LUCA) with an RNA genome. Type IA topoisomerases have since evolved to catalyze the resolution of topological barriers encountered by genomes that require the passing of nucleic acid strand(s) through a break on a single DNA or RNA strand. Here, based on available structural and biochemical data, we discuss how a type IA topoisomerase may recognize and bind single-stranded DNA or RNA to initiate its required catalytic function. Active site residues assist in the nucleophilic attack of a phosphodiester bond between two nucleotides to form a covalent intermediate with a 5′-phosphotyrosine linkage to the cleaved nucleic acid. A divalent ion interaction helps to position the 3′-hydroxyl group at the precise location required for the cleaved phosphodiester bond to be rejoined following the passage of another nucleic acid strand through the break. In addition to type IA topoisomerase structures observed by X-ray crystallography, we now have evidence from biophysical studies for the dynamic conformations that are required for type IA topoisomerases to catalyze the change in the topology of the nucleic acid substrates.

Structure, dynamics and function of the evolutionarily changing biliverdin reductase B family

2020 ◽  
Vol 168 (2) ◽  
pp. 191-202
Author(s):  
Michael R Duff ◽  
Jasmina S Redzic ◽  
Lucas P Ryan ◽  
Natasia Paukovich ◽  
Rui Zhao ◽  
...  
Keyword(s):  
Active Site ◽  
Active Sites ◽  
Biliverdin Reductase ◽  
X Ray Crystallography ◽  
Functional Consequences ◽  
Dynamics And Function ◽  
And Function ◽  
Slow Turnover ◽  
Binding Substrate

Abstract Biliverdin reductase B (BLVRB) family members are general flavin reductases critical in maintaining cellular redox with recent findings revealing that BLVRB alone can dictate cellular fate. However, as opposed to most enzymes, the BLVRB family remains enigmatic with an evolutionarily changing active site and unknown structural and functional consequences. Here, we applied a multi-faceted approach that combines X-ray crystallography, NMR and kinetics methods to elucidate the structural and functional basis of the evolutionarily changing BLVRB active site. Using a panel of three BLVRB isoforms (human, lemur and hyrax) and multiple human BLVRB mutants, our studies reveal a novel evolutionary mechanism where coenzyme ‘clamps’ formed by arginine side chains at two co-evolving positions within the active site serve to slow coenzyme release (Positions 14 and 78). We find that coenzyme release is further slowed by the weaker binding substrate, resulting in relatively slow turnover numbers. However, different BLVRB active sites imposed by either evolution or mutagenesis exhibit a surprising inverse relationship between coenzyme release and substrate turnover that is independent of the faster chemical step of hydride transfer also measured here. Collectively, our studies have elucidated the role of the evolutionarily changing BLVRB active site that serves to modulate coenzyme release and has revealed that coenzyme release is coupled to substrate turnover.

scholarly journals Assessment of enzyme active site positioning and tests of catalytic mechanisms through X-ray–derived conformational ensembles

2020 ◽  
Vol 117 (52) ◽  
pp. 33204-33215
Author(s):  
Filip Yabukarski ◽  
Justin T. Biel ◽  
Margaux M. Pinney ◽  
Tzanko Doukov ◽  
Alexander S. Powers ◽  
...  
Keyword(s):  
Active Site ◽  
Active Sites ◽  
Enzyme Design ◽  
Reaction Cycle ◽  
New Era ◽  
X Ray ◽  
Enzyme Active Site ◽  
Conformational Ensembles ◽  
X Ray Crystallography ◽  
General Base

How enzymes achieve their enormous rate enhancements remains a central question in biology, and our understanding to date has impacted drug development, influenced enzyme design, and deepened our appreciation of evolutionary processes. While enzymes position catalytic and reactant groups in active sites, physics requires that atoms undergo constant motion. Numerous proposals have invoked positioning or motions as central for enzyme function, but a scarcity of experimental data has limited our understanding of positioning and motion, their relative importance, and their changes through the enzyme’s reaction cycle. To examine positioning and motions and test catalytic proposals, we collected “room temperature” X-ray crystallography data for Pseudomonas putida ketosteroid isomerase (KSI), and we obtained conformational ensembles for this and a homologous KSI from multiple PDB crystal structures. Ensemble analyses indicated limited change through KSI’s reaction cycle. Active site positioning was on the 1- to 1.5-Å scale, and was not exceptional compared to noncatalytic groups. The KSI ensembles provided evidence against catalytic proposals invoking oxyanion hole geometric discrimination between the ground state and transition state or highly precise general base positioning. Instead, increasing or decreasing positioning of KSI’s general base reduced catalysis, suggesting optimized Ångstrom-scale conformational heterogeneity that allows KSI to efficiently catalyze multiple reaction steps. Ensemble analyses of surrounding groups for WT and mutant KSIs provided insights into the forces and interactions that allow and limit active-site motions. Most generally, this ensemble perspective extends traditional structure–function relationships, providing the basis for a new era of “ensemble–function” interrogation of enzymes.

scholarly journals A Trojan horse transition state analogue generated by MgF3- formation in an enzyme active site

2006 ◽  
Vol 103 (40) ◽  
pp. 14732-14737 ◽  
Author(s):  
N. J. Baxter ◽  
L. F. Olguin ◽  
M. Golicnik ◽  
G. Feng ◽  
A. M. Hounslow ◽  
...  
Keyword(s):  
Transition State ◽  
Active Site ◽  
Trojan Horse ◽  
Enzyme Active Site ◽  
Transition State Analogue
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