Active Site Research Articles

Effect of Dynamic and Preferential Decoration of Pt Catalyst Surfaces by WOx on Hydrodeoxygenation Reactions.

Catalysts containing Pt nanoparticles and reducible transition-metal oxides (WOx, NbOx, TiOx) exhibit remarkable selectivity to aromatic products in hydrodeoxygenation (HDO) reactions for biomass valorization, contrasting the undesired aromatic hydrogenation typically observed for metal catalysts. However, the active site(s) responsible for the high selectivity remains elusive. Here, theoretical and experimental analyses are combined to explain the observed HDO reactivity by interrogating the organization of reduced WOx domains on Pt surfaces at sub-monolayer coverage. The SurfGraph algorithm is used to develop model structures that capture the configurational space (∼1000 configurations) for density functional theory (DFT) calculations of a W3O7 trimer on stepped Pt surfaces. Machine-learning models trained on the DFT calculations identify the preferential occupation of well-coordinated Pt sites (≥8 Pt coordination number) by WOx and structural features governing WOx-Pt stability. WOx/Pt/SiO2 catalysts are synthesized with varying W loadings to test the theoretical predictions and relate them to HDO reactivity. Spectroscopy- and microscopy-based catalyst characterizations identify the dynamic and preferential decoration of well-coordinated sites on Pt nanoparticles by reduced WOx species, consistent with theoretical predictions. The catalytic consequences of this preferential decoration on the HDO of a lignin model compound, dihydroeugenol, are clarified. The effect of WOx decoration on Pt nanoparticles for HDO involves WOx inhibition of aromatic ring hydrogenation by preferentially blocking well-coordinated Pt sites. The identification of preferential decoration on specific sites of late-transition-metal surfaces by reducible metal oxides provides a new perspective for understanding and controlling metal-support interactions in heterogeneous catalysis.

Optimization of Electrochemical Reduction of Biomass Derived 5-Hydroxymethylfurfural (HMF): A Volcano Plot and Bimetallic Catalysts.

2,5-bis(hydroxymethyl)furan (BHMF) derived from 5-Hydroxymethylfurfural (HMF) through a hydrogenation process has extensive applications in the production of resins, polymers, and artificial fibers. However, screening out the candidate and then modulating the active site to optimize the catalyst for high yield of BHMF are currently insufficient. In this study, Gibbs free energy diagrams of the reduction of HMF on 13 metals were presented, along with the identification of the rate-determining step (RDS) with the highest reaction barrier for each metal. We attempted to construct a volcano plot for HMFRR reaction. Additionally, a strategy was proposed to adjust the reaction barriers of RDS by combining two appropriate metals. Further experiments confirmed that Pb with the lowest energy barrier exhibited the highest HMF conversion (BHMF selectivity) among single metals. The modified catalyst by doping Ag on Pb, further boosted the HMF conversion (BHMF selectivity) from 42.1% (59.4%) to 80.8% (80.9%), respectively. These results provide an approach to rationally design and construct the catalyst system for efficient conversion of HMF.

Scanning the active center of formolase to identify key residues for enhanced C1 to C3 bioconversion

BackgroundFormolase (FLS) is a computationally designed enzyme that catalyzes the carboligation of two or three C1 formaldehyde molecules into C2 glycolaldehyde or C3 dihydroxyacetone (DHA). FLS lays the foundation for several artificial carbon fixation and valorization pathways, such as the artificial starch anabolic pathway. However, the application of FLS is limited by its low catalytic activity and product promiscuity.FindingsFLS, designed and engineered based on benzoylformate decarboxylase from Pseudomonas putida, was selected as a candidate for modification. To evaluate its catalytic activity, 25 residues located within an 8 Å distance from the active center were screened using single-point saturation mutagenesis. A screening approach based on the color reaction of the DHA product was applied to identify the desired FLS variants. After screening approximately 5,000 variants (approximately 200 transformants per site), several amino acid sites that were not identified by directed evolution were found to improve DHA formation. The serine-to-phenylalanine substitution at position 236 improved the activity towards DHA formation by 7.6-fold. Molecular dynamics simulations suggested that the mutation increased local hydrophobicity at the active site, predisposing the cofactor-C2 intermediate to nucleophilic attack by the third formaldehyde molecule for subsequent DHA generation.ConclusionsThis study provides improved FLS variants and valuable information into the influence of residues adjacent to the active center affecting catalytic efficiency, which can guide the rational engineering or directed evolution of FLS to optimize its performance in artificial carbon fixation and valorization.

Serpentinite‐hosted chemosynthetic community of South Chamorro Seamount, Mariana Forearc

AbstractDeep‐sea chemosynthetic ecosystems are ‘oases’ of life powered by reducing geofluids, of which serpentinite‐hosted seeps are among the least studied. South Chamorro Seamount, a serpentine mud volcano on the Mariana Arc, has been known to host chemosynthesis‐based assemblages since 1996, but no detailed information on the fauna was published. Here, we revisited South Chamorro to characterise its biodiversity. We located two regions of chemosynthetic communities dominated by bathymodioline mussels, vesicomyid clams, and chaetopterid parchment worms: one on the northwestern flank (‘Fryer Site’) and one on the southern summit (‘Summit Site’). We sampled a total of 20 species including 13 molluscs, five annelids, and two crustaceans – all present on the more active Summit Site but only a subset being found at Fryer Site. A mussel bed surrounding the Fryer Site was drilled by the Ocean Drilling Program in 2001 resulting in six holes, the deepest being 266 m (Hole 1200C). Cuttings ~50 cm deep still cover an approximately 60 m radius around Hole 1200C even 22 years later, and there is no sign of recovery. Low geofluid supply in serpentinite‐hosted seamounts may not allow decadal recovery of animal colonies, unlike a previous drilling site in an Okinawa Trough vent.

Introduction of element Bi in the P-block promotes N2 activation for efficient electrocatalytic nitrogen reduction to produce ammonia

The synthesis of ammonia through electrocatalytic nitrogen reduction reaction (NRR) under ambient conditions is a presumable strategy. However, it still faces the problems of poor activity and low Faradic efficiency, so the development of efficient, stable and highly selective catalysts is of the importance. Molybdenum disulfide (MoS2) materials are widely used in NRR reactions owing to good electrical conductivity and abundant active sites, but the edge active site is the dual active site of NRR and competitive hydrogen evolution reaction (HER). Encouragingly, the strong interaction between elements in the P-block and N 2p orbitals results in excellent performance in adsorbing and activating nitrogen. Meanwhile, the partial occupation of p-orbitals by elements in the P-block also leads to poor HER activity. Herein, we introduced the P-block element Bi into 1 T-MoS2 and prepared Bi2S3/MoS2 composite. Taking advantage of the high conductivity of 1 T-MoS2 and the N2 adsorption and activation advantages of Bi element, an ammonia yield rate of 54.64 µg h−1 mg−1cat. and a high Faradic efficiency of 58.56 % were obtained. In addition, the active site and the evolution pathway of N2 reduction were investigated by in-situ infrared spectroscopy measurements and density functional theory (DFT). The in-situ infrared spectroscopy measurement results manifested the presence of the main intermediate transition state during the reduction of N2 to NH3. The calculation results indicated that N2 had the best adsorption on the introduced P-block element Bi, and the entire NRR reaction process followed an alternating pathway. This study reveals the synergistic effect of P-block elements and transition metal-based electrocatalytic materials on nitrogen reduction to ammonia synthesis.

Bifunctional neuraminidase inhibitory and simultaneously anti-Mycoplasma pneumoniae of flavan-3-ols and flavanones: Combined molecular docking, virtual screening, ADMET, and synthesis prediction

After lifting COVID-19 restrictions, pneumonia caused by influenza A virus coinfection with Mycoplasma pneumoniae bacteria emerges as a matter of concern. Only a few reports have recently proposed natural compounds for treating the coinfection of influenza A virus and M. pneumoniae, especially resistant strains. Hence, this study demonstrated in silico investigation of two natural classes (flavan-3-ol and flavanone) that could become future inhibitors for bifunctional neuraminidase and anti-M. pneumoniae. Combining the molecular docking with based-virtual screening strategies for over 900 ligands with neuraminidase protein of oseltamivir-resistant virus strain at H274Y mutation (PDB ID: 3CL0), 03 flavan-3-ols and 46 flavanones interacted with both the active site and the adjacent 430-cavity, and showed lower binding energies than laninamivir (−8.1 kcal/mol). The four most suitable candidates (FL22, FN166, FN316, and ChEMBL1779463) also exhibited potential characteristics for inhibiting M. pneumoniae through some vital targets (30S Ribosome-binding factor A (PDB ID: 1PA4) and immunodominant protein P40/P90 complex (PDB ID: 6TLZ)). Furthermore, the total-synthesis strategy of ChEMBL1779463 was successfully proposed using commercial reagents and modern techniques. Our results will offer potential insights for future methods to identify biflavonoids, especially biflavanone, to simultaneously suppress the resistant strains of influenza A virus and M. pneumoniae.

Aptameric hirudins as selective and reversible EXosite-ACTive site (EXACT) inhibitors

Potent and selective inhibition of the structurally homologous proteases of coagulation poses challenges for drug development. Hematophagous organisms frequently accomplish this by fashioning peptide inhibitors combining exosite and active site binding motifs. Inspired by this biological strategy, we create several EXACT inhibitors targeting thrombin and factor Xa de novo by linking EXosite-binding aptamers with small molecule ACTive site inhibitors. The aptamer component within the EXACT inhibitor (1) synergizes with and enhances the potency of small-molecule active site inhibitors by many hundred-fold (2) can redirect an active site inhibitor’s selectivity towards a different protease, and (3) enable efficient reversal of inhibition by an antidote that disrupts bivalent binding. One EXACT inhibitor, HD22-7A-DAB, demonstrates extraordinary anticoagulation activity, exhibiting great potential as a potent, rapid onset anticoagulant to support cardiovascular surgeries. Using this generalizable molecular engineering strategy, selective, potent, and rapidly reversible EXACT inhibitors can be created against many enzymes through simple oligonucleotide conjugation for numerous research and therapeutic applications.

Mutational dissection of a hole hopping route in a lytic polysaccharide monooxygenase (LPMO)

Oxidoreductases have evolved tyrosine/tryptophan pathways that channel highly oxidizing holes away from the active site to avoid damage. Here we dissect such a pathway in a bacterial LPMO, member of a widespread family of C-H bond activating enzymes with outstanding industrial potential. We show that a strictly conserved tryptophan is critical for radical formation and hole transference and that holes traverse the protein to reach a tyrosine-histidine pair in the protein’s surface. Real-time monitoring of radical formation reveals a clear correlation between the efficiency of hole transference and enzyme performance under oxidative stress. Residues involved in this pathway vary considerably between natural LPMOs, which could reflect adaptation to different ecological niches. Importantly, we show that enzyme activity is increased in a variant with slower radical transference, providing experimental evidence for a previously postulated trade-off between activity and redox robustness.

Delineating organizational principles of the endogenous L-A virus by cryo-EM and computational analysis of native cell extracts

The high abundance of most viruses in infected host cells benefits their structural characterization. However, endogenous viruses are present in low copy numbers and are therefore challenging to investigate. Here, we retrieve cell extracts enriched with an endogenous virus, the yeast L-A virus. The determined cryo-EM structure discloses capsid-stabilizing cation-π stacking, widespread across viruses and within the Totiviridae, and an interplay of non-covalent interactions from ten distinct capsomere interfaces. The capsid-embedded mRNA decapping active site trench is supported by a constricting movement of two flexible opposite-facing loops. tRNA-loaded polysomes and other biomacromolecules, presumably mRNA, are found in virus proximity within the cell extract. Mature viruses participate in larger viral communities resembling their rare in-cell equivalents in terms of size, composition, and inter-virus distances. Our results collectively describe a 3D-architecture of a viral milieu, opening the door to cell-extract-based high-resolution structural virology.

A Combined Molecular Dynamics and Hydropathic INTeraction (HINT) Approach to Investigate Protein Flexibility: The PPARγ Case Study

Molecular Dynamics (MD) is a computational technique widely used to evaluate a molecular system’s thermodynamic properties and conformational behavior over time. In particular, the energy analysis of a protein conformation ensemble produced though MD simulations plays a crucial role in explaining the relationship between protein dynamics and its mechanism of action. In this research work, the HINT (Hydropathic INTeractions) LogP-based scoring function was first used to handle MD trajectories and investigate the molecular basis behind the intricate PPARγ mechanism of activation. The Peroxisome Proliferator-Activated Receptor γ (PPARγ) is an emblematic example of a highly flexible protein due to the extended ω-loop delimiting the active site, and it is responsible for the receptor’s ability to bind chemically different compounds. In this work, we focused on the PPARγ complex with Rosiglitazone, a common anti-diabetic compound and analyzed the molecular basis of the flexible ω-loop stabilization effect produced by the Oleic Acid co-binding. The HINT-based analysis of the produced MD trajectories allowed us to account for all of the energetic contributions involved in interconverting between conformational states and describe the intramolecular interactions between the flexible ω-loop and the helix H3 triggered by the allosteric binding mechanism.

Thiadiazole/Thiadiazine Derivatives as Insecticidal Agent: Design, Synthesis, and Biological Assessment of 1,3,4-(Thiadiazine/Thiadiazole)-Benzenesulfonamide Derivatives as IGRs Analogues against Spodoptera littoralis.

In keeping with our investigation, a simple and practical synthesis of novel heterocyclic compounds with a sulfamoyl moiety that can be employed as insecticidal agents was reported. The compound 2-hydrazinyl-N-(4-sulfamoylphenyl)-2-thioxoacetamide 1 was coupled smoothly with triethylorthoformate or a variety of halo compounds, namely phenacyl chloride, chloroacetyl chloride, chloroacetaldehyde, chloroacetone, 1,3-dichloropropane, 1,2-dichloroethane, ethyl chloroformate, 2,3-dichloro-1,4-naphthoquinone, and chloroanil respectively, which afforded the 1,3,4-thiadiazole and 1,3,4-thiadiazine derivatives. The new products structure was determined using elemental and spectral analysis. Under laboratory conditions, the biological and toxicological effects of the synthetic compounds were also evaluated as insecticides against Spodoptera littoralis (Boisd.). Compounds 3 and 5 had LC50 values of 6.42 and 6.90 mg/L, respectively. The investigated compounds (from 2 to 11) had been undergoing molecular docking investigation for prediction of the optimal arrangement and strength of binding between the ligand (herein, the investigated compounds (from 2 to 11)) and a receptor (herein, the 2CH5) molecule. The binding affinity within docking score (S, kcal/mol) ranged between -8.23 (for compound 5), -8.12 (for compound 3) and -8.03 (for compound 9) to -6.01 (for compound 8). These compounds were shown to have a variety of binding interactions within the 2CH5 active site, as evidenced by protein-ligand docking configurations. This study gives evidence that those compounds have 2CH5-inhibitory capabilities and hence may be used for 2CH5-targeting development. Furthermore, the three top-ranked compounds (5, 3, and 9) and the standard buprofezin were subjected to density functional theory (DFT) analysis. The highest occupied molecular orbital-lowest unoccupied molecular orbital (HOMO-LUMO) energy difference (ΔE) of compounds 5, 3, and 9 was found to be comparable to that of buprofezin. These findings highlighted the potential and relevance of charge transfer at the molecular level.

Electrochemical Imaging of Catalytically Active Site by Scanning Electrochemical Cell Microscopy

Electrochemical Imaging of Catalytically Active Site by Scanning Electrochemical Cell Microscopy

Eugenol hydrodeoxygenation over mixed Mo-W carbides.

The modification of molybdenum carbide catalysts by another transition metal has raised an increasing research interest due to the significant improvement of catalyst activity in hydrodeoxygenation of lignin derivatives. At par with the commonly used Co and Ni that add a strong hydrogenation functionality, it was found that the addition of the more oxophilic W restricts ring hydrogenation while allowing the deoxygenation of oxygenated compounds and thus yielding higher selectivity toward the formation of non-oxygenated aromatic compounds. The coexistence of Mo2C with W2C along with metallic W altered the electronic properties of Mo2C which resulted in an increase of catalyst active site density and facilitated further total eugenol deoxygenation. Propyl-benzene selectivity of up to 83% was reached at close to 100% eugenol conversion. These findings will allow a better overview of the effect of different metal phases of mixed carbides on the catalyst performance and raise the prospect of optimizing catalyst design for a hydrodeoxygenation processing of lignins depolymerization products.

Oxygen Vacancy-Laden Confinement Impact on Degradation of Metal Complexes.

Oxygen vacancies (Vo) have been recognized as the superior active site for PS-mediated environmental remediation; however, the formation and activation of Vo associated with the effects of chemical and spatial environments remain ambiguous. Herein, attributing to the low defect-formation energy of Vo in the presence of sulfonate groups, an in situ nucleating Vo-laden CuO nanosheet was deliberately fabricated inside the phase of a sulfonated mesoporous polystyrene substrate (Vo-CuO@SPM). The as-prepared nanocomposite demonstrated an excellent treatment efficiency toward metal complexes [Cu-EDTA as a case] with ignorable Cu(II) leaching, and it can be repeatedly employed for 25 recycles (not limited). Mechanistically, the electron transfer and the mass transport for PDS nonradical activation were proved to be substantially enhanced by the delocalized electrons and with the assistance of the microchannel environment. This work not only establishes insight into the formation of oxygen vacancies but also reveals the PS activation mechanism in the spatially confined sites.

Catalytic mechanism and kinetics of malate dehydrogenase.

Malate dehydrogenase (MDH) is a ubiquitous and central enzyme in cellular metabolism, found in all kingdoms of life, where it plays vital roles in the cytoplasm and various organelles. It catalyzes the reversible NAD+-dependent reduction of L-malate to oxaloacetate. This review describes the reaction mechanism for MDH and the effects of mutations in and around the active site on catalytic activity and substrate specificity, with a particular focus on the loop that encloses the active site after the substrates have bound. While MDH exhibits selectivity for its preferred substrates, mutations can alter the specificity of MDH for each cosubstrate. The kinetic characteristics and similarities of a variety of MDH isozymes are summarized, and they illustrate that the KM values are consistent with the relative concentrations of the substrates in cells. As a result of its existence in different cellular environments, MDH properties vary, making it an attractive model enzyme for studying enzyme activity and structure under different conditions.

Insights of potential trypanocidal effect of the synthetic derivative (2E)-1-(4-aminophenyl)-3-(2,4-dichlorophenyl)prop-2-en-1-one: in vitro assay, MEV analysis, quantum study, molecular docking, molecular dynamics, MPO analysis, and predictive ADMET.

This study aims to evaluate the antitrypanosomiasis activity of a synthetic dichloro-substituted aminochalcone via in vitro assays against infected cell cultures, as well as a theoretical characterization of pharmacokinetics and pharmacodynamics against the protein targets of the evolutionary cycle of T. cruzi. The in vitro evaluation of parasite proliferation inhibition was performed via cytotoxicity analysis on mammalian host cells, effect on epimastigote and trypomastigote forms, and cell death analysis, while computer simulations characterized the electronic structure of (2E)-1-(4-aminophenyl)-3-(2,4-dichlorophenyl)prop-2-en-1-one (DCl), the mechanism of action against the proteins of the evolutionary cycle of T. cruzi: Cruzain, Trypanothione reductase, TcGAPDH, and CYP51 by molecular docking and dynamics and predictive pharmacokinetics by MPO-based ADMET. The in vitro tests showed that the DCl LC50 in order of 178.9 ± 23.9 was similar to the BZN, evidencing the effectiveness of chalcone against Trypomastigotes. Molecular docking and dynamics simulations suggest that DCl acts on the active site of the CYP51 receptor, with hydrogen interactions that showed a high degree of occupation, establishing a stable complex with the target. MPO analysis and ADMET prediction tests suggest that the compound presents an alignment between permeability and hepatic clearance, although it presents low metabolic stability. Chalcone showed stable pharmacodynamics against the CYP51 target, but can form reactive metabolites from N-conjugation and C = C epoxidation, as an indication of controlled oral dose, although the estimated LD50 rate > 500mg/kg is a indicative of low incidence of lethality by ingestion, constituting a promising therapeutic strategy.

Curriculum Making Across Sites of Activity in Upper Secondary School Vocational Education and Training: A Review of the Research in Sweden

Purpose: This paper presents a qualitative systematic review of Swedish research on vocational education and training (VET) at the upper secondary school level over the past 20 years. The review is based on a theoretical model on curriculum making as social practice that may serve as model for comparative studies between countries. By introducing the model, the ambition is to open for new perspectives on VET curriculum in policy and practice. Questions regarding key themes and the interplay of discourses and processes across multiple sites in the education system have not been addressed in previous systematic reviews of Swedish VET research. Methods: The methodological approach in the present paper is a qualitative systematic research review with an integrative and interpretative purpose and research design. The qualitative review is based on the conceptual model of curriculum making as social practice, seeking to capture the inherent complexity and porous boundaries of education systems and movements of ideas, discourses and actors between sites of activity. The model is used for mapping the research, and a content analysis for identifying main themes and emphases and exploring and discussing the potential gaps that may inform future international research studies. Findings: The results show that the research is focused on the micro and nano sites of curriculum making, with connections to macro site activities of national curriculum policy enactment. Research focusing on the macro site of activity has an emphasis on national policy and policymaking regarding the relationship between academic and vocational knowledge/programmes and apprenticeship and employability. In the micro and nano sites of activity – which comprise the majority of the research – the main themes are vocational knowing and identity, teaching, learning and assessment practices and work-based learning. Conclusion: An observation is the absence of principals and middle leaders as actors and informants in the studies. There is little evidence of actors moving between sites of activity and the meso site of activity only comprise a very small part of the research. In this respect, there is a potential gap to be explored, not least regarding how local curricula and syllabi are made and shaped in terms of the influence of representatives from local authorities, companies, trade unions, employer associations, universities and regional agencies. Curriculum making as social practice has the potential to be used for comparative international studies and as a framework that takes national differences in VET education systems into account.

Enhanced tetracycline degradation through nonradical processes by modulating the exposure of active sites on the iron nanoconfined catalysts

There is an urgent need to develop effective and sustainable methods for reducing water pollution. Non-homogeneous Fenton catalysts are commonly used to eliminate pollutants from water. However, the effectiveness of these catalysts is limited by the restricted availability and accessibility of the active site (AS). In this work, we presented a strategy for modulating the exposure of active nanometallic particles to fully expose the AS at the nanoscale. We also confined the AS in carbon frameworks with micropores and mesopores to enhance the effectiveness of AS utilization in Fenton-like reactions. The experimental results indicate that the Fe-out@N-C/PMS system achieved complete degradation of tetracycline within 10 min and maintained an efficient degradation rate of over 91% after five consecutive cycles. The degradation of contaminants was due to an effective non-radical mechanism, as shown by free radical quenching experiments, EPR reactive species capture tests, in situ Raman, and PMSO probe experiments. Furthermore, density-functional theory (DFT) calculations revealed that iron nanoparticles supported on nitrogen-doped carbon acted as active sites, enhancing electron transfer capacity (N-C (0.24 e) vs. Fe-out@N-C (0.69 e)). Finally, the intermediates have a limited environmental impact because of their extremely low concentrations, despite having a high relative toxicity. The exposed metal nanoparticle catalysts provide a new approach for effectively activating PMS to efficiently degrade organic contaminants with a high mineralization rate via the non-radical pathway.

Noble metal modified copper-exchanged mordenite zeolite (Cu-ex-MOR) catalysts for catalyzing the methane efficient gas-phase synthesis methanol

Copper mordenite catalysts are key for methane oxidation to methanol, yet lack sufficient activity. In this paper, noble metal (Au, Ru, Pt and Pd) modified copper ion-exchanged mordenite catalysts were prepared by the ion-exchange method for further improving the methanol yield in the gas-phase continuous catalytic direct partial oxidation of methane to methanol reaction, and the role of noble metal doping on Cu-ex-MOR catalysts was investigated. Experimental results showed that the doping of Ru resulted in a significant increase in the methanol yield of Ru/Cu-ex-MOR catalyst to 157.36 μmol/gcat/h compared to that of Cu-ex-MOR catalyst (12.89 μmol/gcat/h). SEM, XRD, FT-IR, N2 adsorption-desorption, XPS, NH3-TPD and H2-TPR results showed that Ru/Cu-ex-MOR had uniformly dispersed Ru elements and the largest number of surface acidic and oxidation sites, which facilitated the adsorption and activation of methane. Additionally, it was found by TEM, in situ FT-IR and DFT characterization that Ru played a role in stabilizing the Cu active sites, the adsorptive activation of water on the Ru site and the H-transfer process reduced the energy required for breaking C–H bond of CH4 at the Cu active site, which significantly improved the methane activation capacity of the Ru/Cu-ex-MOR catalysts, resulting in higher methanol yields.

Effects of the Y432S Cancer-Associated Variant on the Reaction Mechanism of Human DNA Polymerase κ.

Human DNA polymerases are vital for genetic information management. Their function involves catalyzing the synthesis of DNA strands with unparalleled accuracy, which ensures the fidelity and stability of the human genomic blueprint. Several disease-associated mutations and their functional impact on DNA polymerases have been reported. One particular polymerase, human DNA polymerase kappa (Pol κ), has been reported to be susceptible to several cancer-associated mutations. The Y432S mutation in Pol κ, associated with various cancers, is of interest due to its impact on polymerization activity and markedly reduced thermal stability. Here, we have used computational simulations to investigate the functional consequences of the Y432S using classical molecular dynamics (MD) and coupled quantum mechanics/molecular mechanics (QM/MM) methods. Our findings suggest that Y432S induces structural alterations in domains responsible for nucleotide addition and ternary complex stabilization while retaining structural features consistent with possible catalysis in the active site. Calculations of the minimum energy path associated with the reaction mechanism of the wild type (WT) and Y432S Pol κ indicate that, while both enzymes are catalytically competent (in terms of energetics and the active site's geometries), the cancer mutation results in an endoergic reaction and an increase in the catalytic barrier. Interactions with a third magnesium ion and environmental effects on nonbonded interactions, particularly involving key residues, contribute to the kinetic and thermodynamic distinctions between the WT and mutant during the catalytic reaction. The energetics and electronic findings suggest that active site residues favor the catalytic reaction with dCTP3- over dCTP4-.