Reaction Conformation Research Articles

Significantly enhanced specific activity of Bacillus subtilis (2,3)-butanediol dehydrogenase through computer-aided refinement of its substrate-binding pocket

(2,3)-Butanediol dehydrogenases (BDHs) are widely utilized for the stereoselective interconversion between α-hydroxy ketones and vicinal diols to produce various functional building blocks. In this study, to enhance the specific activity towards (R)-phenyl-1,2-ethanediol (1a) for 2-hydroxyacetophenone (1b), the substrate-binding pocket of a Bacillus subtilis BDH (BsBDHA) was refined through site-directed mutagenesis. Based on molecular docking simulations, 14 residues were identified and subjected to alanine scanning mutagenesis. After screening, two residues, His42 and Gly292, were singled out for partial site-saturation mutagenesis. The results revealed that BsBDHAH42A and BsBDHAG292A displayed high activities of 3.21 and 1.97 U/mg, respectively. Employing combinatorial mutagenesis, a superior mutant, BsBDHAI49L/V266L/G292A, was developed, exhibiting significantly enhanced specific activity and catalytic efficiency towards (R)-1a, achieving 14.81 U/mg and 4.47 mM−1 s−1, respectively, which were 27.4- and 55.9-fold higher than those of BsBDHA. Further substrate spectrum analysis revealed that the superior mutant displayed increased specific activities for (R)-2a–6a by 1.4- to 10.3-fold. The integration of BsBDHAI49L/V266L/G292A into a three-enzymatic cascade for the synthesis of 1b effectively elevated the yield from 58.1 to 82.4%. Molecular mechanism analysis indicated that the mutation-induced changes in intermolecular forces resulted in a higher frequency of reactive conformations for (R)-1a in BsBDHAI49L/V266L/G292A compared to BsBDHA.

Computational Study on the Conformational Flexibility-Mediated Intramolecular Oxidative Spirocyclization of Procyanidin B4.

Procyanidins, found widely in foods and beverages, are prone to oxidation, yet the chemical structures of their oxidation products and the mechanisms involved remain unclear. Herein, we report that the conformation of procyanidin B4 influences its oxidation products and their stereochemistry. Eight spirocyclized oxidation products were obtained from procyanidin B4 and classified as S- or R-forms based on the configuration of the spiro carbons. The ratios of S- and R-forms derived from the compact and extended rotamers of procyanidin B4, respectively, varied with the solvent. DFT calculations suggested that the four lowest-energy conformers of procyanidin B4 are diverged by interflavan bond rotation and heterocyclic ring inversion. Conformations with an axial-oriented B-ring were estimated as reactive conformations showing proximity between reaction sites on the B- and D-rings. Moreover, the extended rotamer bearing the axially oriented B-ring showed greater stabilization by noncovalent interactions (NCIs), such as OH-π interactions, compared to the counterpart of the compact rotamer. This NCI-based stabilization accounts for a higher production of the R-form despite the predominant presence of the compact rotamer in H2O. These findings highlight the conformational effects that bias the stereoselectivity of oxidative spirocyclization in procyanidin B4, advancing our understanding of procyanidin oxidation mechanisms and product stereochemistry.

Pomotrelvir and Nirmatrelvir Binding and Reactivity with SARS‐CoV‐2 Main Protease: Implications for Resistance Mechanisms from Computations

AbstractWe investigate the inhibition mechanism between pomotrelvir and the SARS‐CoV‐2 main protease using molecular mechanics and quantum mechanics/molecular mechanics simulations. Alchemical transformations where each Pi group of pomotrelvir was transformed into its counterpart in nirmatrelvir were performed to unravel the individual contribution of each group to the binding and reaction processes. We have shown that while a γ‐lactam ring is preferred at position P1, a δ‐lactam ring is a good alternative for the design of inhibitors for variants presenting mutations at position 166. For the P2 position, tertiary amines are preferred with respect to secondary amines. Flexible side chains at the P2 position can disrupt the preorganization of the active site, favouring the exploration of non‐reactive conformations. The substitution of the P2 group of pomotrelvir by that of nirmatrelvir resulted in a compound, named as C2, that presents a better binding free energy and a higher population of reactive conformations in the Michaelis complex. Analysis of the chemical reaction to form the covalent complex has shown a similar reaction mechanism and activation free energies for pomotrelvir, nirmatrelvir and C2. We hope that these findings could be useful to design better inhibitors to fight present and future variants of the SARS‐CoV‐2 virus.

Pomotrelvir and Nirmatrelvir Binding and Reactivity with SARS-CoV-2 Main Protease: Implications for Resistance Mechanisms from Computations.

We investigate the inhibition mechanism between pomotrelvir and the SARS-CoV-2 main protease using molecular mechanics and quantum mechanics/molecular mechanics simulations. Alchemical transformations where each Pi group of pomotrelvir was transformed into its counterpart in nirmatrelvir were performed to unravel the individual contribution of each group to the binding and reaction processes. We have shown that while a γ-lactam ring is preferred at position P1, a δ-lactam ring is a good alternative for the design of inhibitors for variants presenting mutations at position 166. For the P2 position, tertiary amines are preferred with respect to secondary amines. Flexible side chains at the P2 position can disrupt the preorganization of the active site, favouring the exploration of non-reactive conformations. The substitution of the P2 group of pomotrelvir by that of nirmatrelvir resulted in a compound, named as C2, that presents a better binding free energy and a higher population of reactive conformations in the Michaelis complex. Analysis of the chemical reaction to form the covalent complex has shown a similar reaction mechanism and activation free energies for pomotrelvir, nirmatrelvir and C2. We hope that these findings could be useful to design better inhibitors to fight present and future variants of the SARS-CoV-2 virus.

Simulation-Guided Conformational Space Exploration to Assess Reactive Conformations of a Ribozyme.

Self-splicing ribozymes are small ribonucleic acid (RNA) enzymes that catalyze their own cleavage through a transphosphoesterification reaction. While this process is involved in some specific steps of viral RNA replication and splicing, it is also of importance in the context of the (putative) first autocatalytic RNA-based systems that could have preceded the emergence of modern life. The uncatalyzed phosphoester bond formation is thermodynamically very unfavorable, and many experimental studies have focused on understanding the molecular features of catalysis in these ribozymes. However, chemical reaction paths are short-lived and not easily characterized by experimental approaches, so molecular simulation approaches appear as an ideal tool to unveil the molecular details of the reaction. Here, we focus on the model hairpin ribozyme. We show that identifying a relevant initial conformation for reactivity studies, which is frequently overlooked in mixed quantum-classical studies that predominantly concentrate on the chemical reaction itself, can be highly challenging. These challenges stem from limitations in both available experimental structures (which are chemically altered to prevent self-cleavage) and the accuracy of force fields, together with the necessity for comprehensive sampling. We show that molecular dynamics simulations, combined with extensive conformational phase space exploration with Hamiltonian replica-exchange simulations, enable us to characterize the relevant conformational basins of the minimal hairpin ribozyme in the ligated state prior to self-cleavage. We find that what is usually considered a canonical reactive conformation with active site geometries and hydrogen-bond patterns that are optimal for the addition-elimination reaction with general acid/general base catalysis is metastable and only marginally populated. The thermodynamically stable conformation appears to be consistent with the expectations of a mechanism that does not require the direct participation of ribozyme residues in the reaction. While these observations may suffer from forcefield inaccuracies, all investigated forcefields lead to the same conclusions upon proper sampling, contrasting with previous investigations on shorter timescales suggesting that at least one reparametrization of the Amber99 forcefield allowed to stabilize aligned active site conformations. Our study demonstrates that identifying the most pertinent reactant state conformation holds equal importance alongside the accurate determination of the thermodynamics and kinetics of the chemical steps of the reaction.

STRUCTURE AND DYNAMICS OF THE ENZYME-SUBSTRATE COMPLEX OF N-ACETYLASPARTYLGLUTAMATE SYNTHASE ACCORDING TO COMPUTER SIMULATION DATA

N-acetylaspartilglutamate is the most common dipeptide in brain cells, which is synthesized using the enzyme N-acetylaspartilglutamate synthase. Herein we utilize bioinformatics methods to predict the protein structure from the primary sequence of the coding gene, classical molecular dynamics to obtain a stable protein complex with N-acetylaspartate and glutamate ligands within the trajectory, as well as machine learning methods to analyze, describe and select potential reactive and non-reactive conformations of the model system describing the enzyme-substrate complex. Molecular dynamics simulations with combined quantum mechanics / molecular mechanics potentials were performed for a set of selected conformations and the potential reaction mechanism were characterized.

Conformational Analysis of (+)-Germacrene D-4-ol Using the Crystalline Sponge Method to Elucidate the Origin of its Instability.

Unsaturated cyclic terpenes often exhibit instability due to the proximation of C=C bonds in the cyclic skeleton, leading to nonenzymatic degradation. In this study, the crystalline sponge (CS) method was employed for the X-ray conformational analysis of a minute amount of oily and cyclic terpene compound, (+)-germacrene D-4-ol, which was produced by a terpene synthase OILTS under in vitro conditions. The CS method revealed a reactive conformation of the cyclic terpene with proximal double bonds. Under weakly acidic in vivo conditions, OILTS gave four pseudo-natural products or artifacts. The CS method also elucidated the structures of these degraded compounds, proposing a degradation mechanism triggered by the transannular reactions.

Kinetic View of Enzyme Catalysis from Enhanced Sampling QM/MM Simulations.

The rate constants of enzyme-catalyzed reactions (kcat) are often approximated from the barrier height of the reactive step. We introduce an enhanced sampling QM/MM approach that directly calculates the kinetics of enzymatic reactions, without introducing the transition-state theory assumptions, and takes into account the dynamical equilibrium between the reactive and non-reactive conformations of the enzyme/substrate complex. Our computed kcat values are in order-of-magnitude agreement with the experimental data for two representative enzymatic reactions.

Stereocontrol via Propeller Chirality in FLP-Catalyzed Asymmetric Hydrogenation.

Utilization of chiral frustrated Lewis pairs as catalysts in enantioselective hydrogenation of unsaturated molecules represents a promising approach in asymmetric synthesis. In our effort to improve our current understanding of the factors governing the stereoselectivity in these catalytic processes, herein we examined the mechanism of direct hydrogenation of aromatic enamines catalyzed by a binaphthyl-based chiral amino-borane. Our computational analysis reveals that only one particular conformer of the key borohydride reaction intermediate can be regarded as a reactive form of this species. This borohydride conformer has a well-defined chiral propeller shape, which induces facial selectivity in the hydride transfer to pro-chiral iminium intermediates. The propeller chirality of the reactive borohydride conformer is generated by the axially chiral binaphthyl scaffold of the amino-borane catalyst through stabilizing π-π stacking interactions. This new computational insight can be readily used to interpret the high degree of stereoinduction observed for these reactions. We expect that the concept of chirality relay could be further exploited in catalyst design endeavors.

Investing in entropy: the strategy of cucurbit[n]urils to accelerate the intramolecular Diels-Alder cycloaddition reaction of tertiary furfuryl amines.

Cucurbit[n]urils, renowned for their host-guest chemistry, are becoming versatile biomimetic receptors. Herein, we report that cucurbit[7]uril (CB[7]) accelerates the intramolecular Diels-Alder (IMDA) reaction for previously elusive and unreactive tertiary N-methyl-N-(homo)allyl-2-furfurylamines by up to 4 orders of magnitude under mild conditions. Using 1H NMR titrations and ITC experiments, we characterize the dissimilar thermodynamic and kinetic properties of the complexes. We also determine the activation parameters (ΔG ≠, ΔH ≠ and ΔS ≠) leading to the transition state of the IMDA reactions, both in the bulk and included in CB[7], to shed light on the key role of the receptor on the acceleration observed. CB[7] acts as an "entropy trap" utilizing guest binding to primarily pay the entropy penalty for reorganizing the substrate in a high-energy reactive conformation that resembles the geometry of the highly ordered transition state required for the IMDA reaction. This study underscores the potential of cucurbit[n]urils as artificial active sites, emulating specific aspects of enzymatic catalysis.

Improved prediction of reaction kinetics for amine absorbent-based carbon capture using reactive site-based transition state conformer search method

There is no doubt that carbon emissions are one of the greatest challenges facing humanity today. Carbon capture, utilization, and storage is an effective way to achieve carbon neutrality. However, the commonly used commercial absorbents for post-combustion captures still have some limitations such as low chemical absorption rate constants. In this paper, a universal reaction kinetic model is developed for amine-based carbon capture based on the transition state theory, density functional theory, and hybrid solvation model. The developed reaction kinetic model is applicable to a wide range of amine-solvent solutions involving primary/secondary/tertiary amines and aqueous/nonaqueous solvents. The key contribution of this work is developing a reactive site-based search method for the GENeration of Conformers for Transition States (GENConf-TS). GENConf-TS has greatly improved the prediction accuracy of the reaction kinetic model from R2 = 0.882 to R2 = 0.950 based on a dataset of 23 various amine-solvent solutions. The results highlight the critical impacts of the transition state conformational isomers on amine-based CO2 chemical absorption rate constants.

Substrate Positioning Dynamics Involves a Non-Electrostatic Component to Mediate Catalysis.

Substrate positioning dynamics (SPD) orients the substrate in the active site, thereby influencing catalytic efficiency. However, it remains unknown whether SPD effects originate primarily from electrostatic perturbation inside the enzyme or can independently mediate catalysis with a significant non-electrostatic component. In this work, we investigated how the non-electrostatic component of SPD affects transition state (TS) stabilization. Using high-throughput enzyme modeling, we selected Kemp eliminase variants with similar electrostatics inside the enzyme but significantly different SPD. The kinetic parameters of these mutants were experimentally characterized. We observed a valley-shaped, two-segment linear correlation between the TS stabilization free energy (converted from kinetic parameters) and substrate positioning index (a metric to quantify SPD). The energy varies by approximately 2 kcal/mol. Favorable SPD was observed for the distal mutant R154W, increasing the proportion of reactive conformations and leading to the lowest activation free energy. These results indicate the substantial contribution of the non-electrostatic component of SPD to enzyme catalytic efficiency.

Early Steps in the O2 Scavenger Process in the Aqueous Phase: Hydrazine vs DEHA.

We investigate the first direct proton abstraction reactions from reducing agents (RAHs) hydrazine and diethyl hydroxylamine (DEHA), toward dioxygen (O2) in the aqueous phase, spanning ambient to high-temperature conditions. Quantum chemistry methods and molecular dynamics simulations are employed in this study. Quantum chemistry methods are used to analyze the quasi-equilibrium between a reactive conformation and a transition state in the [RAH,O2] cluster. On the other hand, molecular dynamics simulations estimate the probability of observing a reactive conformation of the [RAH,O2] cluster in the solution. In this study, we assume that the energy barrier of the quasi-equilibrium is sufficiently high for the RAH/O2 association process to be at equilibrium. Our findings indicate that the first proton abstraction process from a reactive conformation cluster by DEHA is energetically favored compared to hydrazine. Conversely, the association process of hydrazine and O2 in solution is more favorable than that of DEHA. Consequently, the rate constant for the first proton abstraction process is similar for both hydrazine and DEHA, particularly at high temperatures, with activation energies of approximately 21.5 ± 1.5 kcal mol-1 for both compounds. These results align with recent experiments investigating the complete O2 scavenger process in liquid water with hydrazine and DEHA. Therefore, our findings support the assumption that first proton abstraction reactions are the rate-determining steps in O2 scavenger processes in the aqueous phase.

Computational remodeling of an enzyme conformational landscape for altered substrate selectivity

Structural plasticity of enzymes dictates their function. Yet, our ability to rationally remodel enzyme conformational landscapes to tailor catalytic properties remains limited. Here, we report a computational procedure for tuning conformational landscapes that is based on multistate design of hinge-mediated domain motions. Using this method, we redesign the conformational landscape of a natural aminotransferase to preferentially stabilize a less populated but reactive conformation and thereby increase catalytic efficiency with a non-native substrate, resulting in altered substrate selectivity. Steady-state kinetics of designed variants reveals activity increases with the non-native substrate of approximately 100-fold and selectivity switches of up to 1900-fold. Structural analyses by room-temperature X-ray crystallography and multitemperature nuclear magnetic resonance spectroscopy confirm that conformational equilibria favor the target conformation. Our computational approach opens the door to targeted alterations of conformational states and equilibria, which should facilitate the design of biocatalysts with customized activity and selectivity.

On the Role of Temperature in the Depolymerization of PET by FAST-PETase: an Atomistic Point of View on Possible Active-Site Pre-Organization and Substrate-Destabilization Effect.

Enzyme FAST-PETase, recently obtained by machine learning approach, can depolymerize polyethylene terephthalate (PET), a synthetic resin employed in plastics and in clothing fibers. Therefore it represents a promising solution for the recycling of PET-based materials. In the present study, a model of PET was adopted to describe the substrate, and all-atoms classical molecular dynamics (MD) simulations on apo- and substrate bound- FAST-PETase were carried out at 30°C and 50°C, to provide atomistic details on the binding step of the catalytic cycle. Comparative analysis sheds light on the interactions occurring between the FAST-PETase and 4PET at 50 °C, the optimal working condition of the enzyme. Pre-organization of the enzyme active and binding sites has been highlighted while MD of FAST-PETase:4PET pointed out on the occurrence of solvent-inaccessible conformations of the substrate promoted by the enzyme. Indeed, neither of these conformations were observed during MD of substrate alone in solution performed at 30 °C, 50 °C and 150 °C. The analysis led us to propose that, at 50 °C, the FAST-PETase is pre-organized to bind the PET and that the interactions occurring in the binding site can promote more reactive conformation of PET substrate, thus enhancing the catalytic activity of the enzyme.

Computational investigation on the binding modes of PET polymer to PETase

Poly(ethylene terephthalate) (PET) has been widely utilized in daily life, but its non-degradability has induced severe environmental and health problems. Recently, PETase, which has been isolated from bacterium Ideonella sakaiensisis, was reported to have the highest PET degradation activity and specificity under room temperature, but no crystal structure for PET in complex with PETase has been reported. To provide deep insight into the binding mode of PET polymer on PETase and the binding interactions, we employed molecular docking and molecular dynamics simulations to study the substrate binding at the atomic level. Different PET oligomers have been studied with chain lengths varying from 2 to 8. In addition, the binding energies and hot-spot residues were analyzed to gain better insights into the binding mechanism by MM/GBSA approach. The PET oligomers adopt stable and reactive conformations in a shallow cleft on a flat surface of PETase. The binding cleft can only accommodate four moieties, and others beyond the region will be stabilized by the π–stacking interactions with Trp156 at the terephthalic acid terminal. Our studies provide a clear picture of how the binding mode of PET polymer and its interactions with PETase change with the chain length. Those studies would provide useful information for the rational design of catalytically more efficient PETase variants toward plastic degradation. Communicated by Ramaswamy H. Sarma

Regio- and stereoselective steroid hydroxylation by CYP109A2 from Bacillus megaterium explored by X-ray crystallography and computational modeling.

The P450 monooxygenase CYP109A2 from Bacillus megaterium DSM319 was previously found to convert vitamin D3 to 25-hydroxyvitamin D3. Here, we show that this enzyme is also able to convert testosterone in a highly regio- and stereoselective manner to 16β-hydroxytestosterone. To reveal the structural determinants governing the regio- and stereoselective steroid hydroxylation reactions catalyzed by CYP109A2, two crystal structures of CYP109A2 were solved in similar closed conformations, one revealing a bound testosterone in the active site pocket, albeit at a non-productive site away from the heme-iron. To examine if the closed crystal structures nevertheless correspond to a reactive conformation of CYP109A2, docking and molecular dynamics simulations were performed with testosterone and vitamin D3 present in the active site. These molecular dynamics simulations were analyzed for catalytically productive conformations, the relative occurrences of which were in agreement with the experimentally determined stereoselectivities if the predicted stability of each carbon hydrogen bond was taken into account. Overall, the first-time determination and analysis of the catalytically relevant 3D conformation of CYP109A2 will allow for future small molecule ligand screening in silico, as well as enabling site-directed mutagenesis towards improved enzymatic properties of this enzyme.

Structural Analysis of Non-native Peptide-Based Catalysts Using 2D NMR-Guided MD Simulations.

Proteins and enzymes generally achieve their functions by creating well-defined 3D architectures that pre-organize reactive functionalities. Mimicking this approach to supramolecular pre-organization is leading to the development of highly versatile artificial chemical environments, including new biomaterials, medicines, artificial enzymes, and enzyme-like catalysts. The use of β-turn and α-helical motifs is one approach that enables the precise placement of reactive functional groups to enable selective substrate activation and reactivity/selectivity that approaches natural enzymes. Our recent work has demonstrated that helical peptides can serve as scaffolds for pre-organizing two reactive groups to achieve enzyme-like catalysis. In this study, we used CYANA and AmberTools to develop a computational approach for determining how the structure of our peptide catalysts can lead to enhancements in reactivity. These results support our hypothesis that the bifunctional nature of the peptide enables catalysis by pre-organizing the two catalysts in reactive conformations that accelerate catalysis by proximity. We also present evidence that the low reactivity of monofunctional peptides can be attributed to interactions between the peptide-bound catalyst and the helical backbone, which are not observed in the bifunctional peptide.

How and When Does an Enzyme React? Unraveling α-Amylase Catalytic Activity with Enhanced Sampling Techniques

Enzymatic catalysis is a complex process that can involve multiple conformations of the enzyme:substrate complex and several competitive reaction pathways, resulting in a multi-dimensional free energy landscape. The study of enzymatic activity often requires deep knowledge of the system to establish the catalytic mechanism and identify the possible reactive conformations of the complex. Here, we present an enhanced sampling and machine learning-based approach to explore the catalytic reaction space and characterize the transformation from reactive to non-reactive conformations with minimal a priori knowledge of the system. We applied this approach to study the rate-determining step of the glycolysis reaction of maltopentose catalyzed by human pancreatic α-amylase, an important enzyme in glucose production as well as a major drug target for the treatment of type-II diabetes. We unravel the complexity of the enzymatic reaction, reveal three binding modes of the substrate within the active site, and highlight the role of water in the catalytic process and in the stepwise conversion of reaction-ready to non-reactive conformations. Overall, these insights offer atomistic details on the catalytic mechanism and dynamics of the active site, allowing one to shed light on two fundamental questions in enzymatic catalysis, that is how and when does an enzyme react?

Extending the competitive threshold collision-induced dissociation of Zn(II) ternary complexes using traveling-wave ion mobility-mass spectrometry

The competitive threshold collision-induced dissociation (TCID) of the ternary complex [amb5+Zn(II)+NTA]− → [amb5+Zn(II)]− + NTA or → [NTA+Zn(II)]− + amb5, where amb5 = His-Cys-Gly-Pro-Gly-Gly-Cys or Asp-His-Gly-Pro-Gly-Gly-Cys and NTA = nitrilotriacetic acid, were studied using traveling-wave ion mobility-mass spectrometry (TWIMMS). The analyses included calculating the reaction cross sections as a function of center-of-mass collision energy at three pressures of the argon collision gas, which covers the pressure range available in the TWIMMS instrument. The ternary complex contained one of the amb5 peptides, whose His, Cys and/or Asp substituent groups could compete with the NTA carboxylate sites for binding Zn(II). A new molecular modeling procedure was developed, initially using PM6 geometry-optimizations and thermochemical analyses to locate low-energy conformers of the ternary complex and its products that also had collision cross sections (ΩHe), measured using the ion size scaled Lennard-Jones (L-J) method, that were close to the TWIMMS measured ΩHe. A selection of these conformers were further geometry-optimized at the DFTB/3OB_D4 level of theory and using these as starting structures, molecular dynamics (MD) simulations were conducted to sample the ensemble of structures L-J ΩHe at 300 K. From these analyses, a selection of reactant and product conformers whose L-J ΩHe best matched the TWIMMS ΩHe were used in the TCID fitting of the center-of-mass energy-dependent, reaction cross sections extrapolated to zero pressure. The TCID fitting determined the threshold energies of the two product channels which in the absence of an activation barrier in excess of the energy of the products equates to the 0 K dissociation enthalpies (ΔH0). The difference in ΔH0 for the two competing reactions allowed for a relative gas-phase Zn(II) affinity scale to be constructed for the amb5 peptides studied here and from our previous published amb5 research.