In the environment, aromatic compounds are commonly introduced artificially or as natural plant secondary metabolites. A major agricultural pest, Tetranychus urticae, has acquired genes encoding for intradiol ring-cleavage dioxygenases (IDRCDs) that can detoxify such compounds. Studies of one such IDRCD, TuIDRCD930 (encoded by gene tetur07g05930), revealed a dimerization not seen before in enzymes of this class in T. urticae, including a different oligomerization mechanism observed in fungal and bacterial homologs. Through SEC-SAXS and gel filtration experiments, TuIDRCD930 was found to dimerize by utilizing two different interactions in the N-termini: changes in protonation states and the formation of a disulfide bond. Additionally, in vitro studies demonstrate catalytic efficiency of TuIDRCD930 towards polycyclic aromatic compounds, which represents an evolutionary advantage for T. urticae. It was shown that the dimerization process does not obscure access of relatively large substrates to the enzyme active site. We further displayed that the recombinant version of TuIDRCD930 is active in both monomeric and dimeric states. Overall, this work determines oligomerization and activity not previously described in IDRCDs from T. urticae and provides further evidence of how evolution has provided this pest with novel tools to overcome plant defenses.

Fumarate-adding enzymes (FAE) are a subset of the glycyl radical enzyme superfamily involved in anaerobic hydrocarbon degradation. Benzylsuccinate synthase (BSS) catalyzes the enantiospecific formation of R-benzylsuccinate from toluene and fumarate, initiating anaerobic toluene degradation. In this paper, we present a detailed theoretical study of the reaction mechanism using classical molecular dynamics and multiscale modeling (QM/MM). We describe the potential energy surface of the reaction and confirm the previously postulated mechanism. However, the multiscale character of our model allowed us to elucidate the origins of several experimentally observed catalytic phenomena, such as the inversion of the benzylic carbon configuration upon C-C bond formation and the syn addition of the abstracted H atom back to the benzylsuccinyl radical. The obtained model is supported by microkinetic analysis and was able to explain and quantitatively predict the strict R-enantioselectivity of BSS, which is enforced predominantly by the dynamic kinetic behavior of toluene in the active site, leading to over 40-times faster production of the R-enantiomer, not by the binding orientation of the fumarate. Our study contributes to the elucidation of the catalytic processes catalyzed by BSS and its role in the bioremediation of hydrocarbon pollutants.

Abstract Fumarate-adding enzymes (FAE) are a subset of the glycyl radical enzyme superfamily involved in anaerobic hydrocarbon degradation. Benzylsuccinate synthase (BSS) catalyzes the enantiospecific formation of (R)-benzylsuccinate from toluene and fumarate, initiating anaerobic toluene degradation. In this paper, we present the first microkinetic analysis of the full reaction, predicting kinetic isotope effects in the range of 2.587–2.604, close to the experimentally observed value (KIEexp= 2.13 ± 0.1). Our experiments confirmed that KIE values are lower in direct assays with substrate-saturated enzyme, relative to the values obtained for competitive kinetic isotope effects D(V/K) via substrate fractionation (3.69 ± 0.16). We postulate that this apparent KIE suppression originates from preferential binding of unlabeled versus labeled substrates to the enzyme, as well as from the rates of product release. We also show that tunneling effects have only minor influence on the observed KIE and D(V/K) values, although they have been estimated to potentially accelerate the overall reaction rate by approximately 17%. On the other hand, inclusion in the kinetic equation of the barrier recrossing effect, jointly with tunneling correction can result in a lowering of predicted KIE values to the range of 2.38–2.43. Furthermore, we analyze a slowly occurring, experimentally observed H/D exchange process in the product during incubation in D2O, confirming partial reversibility of the reaction. We estimate the rate of this H/D exchange and propose a potential mechanism. Our study contributes to elucidating the processes catalyzed by BSS and its role in the bioremediation of hydrocarbon pollutants.

Molecular analysis of interactions between IgE antibody and allergen allows the structural basis of IgE recognition to be defined. Human IgE (hIgE) epitopes of respiratory lipocalin allergens, including Can f 1, remain elusive due to a lack of IgE-allergen complexes. This study aims to map the structure of allergenic epitopes on Can f 1. The fragment antigen-binding (Fab) regions of Can f 1 specific human IgE monoclonal antibodies (hIgE mAb) were used to determine the structures of IgE epitopes. Epitope mutants were designed to target Can f 1 epitopes. Immunoassays and a human FcεRIα transgenic mouse model of passive anaphylaxis in vivo were used to assess the functional activity of epitope mutants. Crystal structures of natural or recombinant Can f 1 complexed with two hIgE mAb 1J11 and 12F3 Fabs, respectively, were determined. The hIgE mAb bound to two partially overlapping epitopes and recognized two different Can f 1 conformations. The hIgE mAb 12F3 showed an unusual mode of binding by protruding its heavy chain CDR3 inside the Can f 1 calyx. Epitope mutants generated based on the structural analyses displayed a 64%-89% reduction in IgE antibody binding and failed to induce passive anaphylaxis in a human FcεRIα transgenic mouse model. In summary, the structures of Can f 1-hIgE Fab complexes revealed two unique and partially overlapping epitopes on Can f 1. The modification of the identified IgE epitopes provides a pathway for the design of hypoallergens to treat dog allergies.

Ectoine synthase (EctC) catalyses the ultimate step of ectoine biosynthesis, a kosmotropic compound produced as compatible solute by many bacteria and some archaea or eukaryotes. EctC is an Fe2+-dependent homodimeric cytoplasmic protein. Using Mössbauer spectroscopy, molecular dynamics simulations and QM/MM calculations, we determined the most likely coordination number and geometry of the Fe2+ ion and proposed a mechanism of the EctC-catalysed reaction. Most notably, we show that apart from the three amino acids binding to the iron ion (Glu57, Tyr84 and His92), one water molecule and one hydroxide ion are required as additional ligands for the reaction to occur. They fill the first coordination sphere of the Fe2+-cofactor and act as critical proton donors and acceptors during the cyclization reaction.

The present work is another part of our investigation on the pathway of dissimilatory sulfate reduction and covers a theoretical study on the reaction catalyzed by dissimilatory sulfite reductase (dSIR). dSIR is the terminal enzyme involved in this metabolic pathway, which uses the siroheme-[4Fe4S] cofactor for six-electron reduction of sulfite to sulfide. In this study we use a large cluster model containing siroheme-[4Fe4S] cofactor and protein residues involved in the direct interactions with the substrate, to get insight into the most feasible reaction mechanism and to understand the role of each considered active site component. In combination with earlier studies reported in the literature, our results lead to several interesting insights. One of the most important conclusions is that the reaction mechanism consists of three steps of two-electron reduction of sulfur and the probable role of the siroheme-[4Fe4S] cofactor is to ensure the delivery of packages of two electrons to the reactant.

The O2-dependent carbon-carbon (C-C) bond cleavage reactions of the mononuclear Cu(II) chlorodiketonate complexes [(6-Ph2TPA)Cu(PhC(O)CClC(O)Ph)]ClO4 (1-ClO4) and [(bpy)Cu(PhC(O)CClC(O)Ph)(ClO4)] (3-ClO4) have been further examined in terms of their anion and water dependence. The bpy-ligated Cu(II) chlorodiketonate complex 3-ClO4 is inherently more reactive with O2 than the 6-Ph2TPA-ligated analog 1-ClO4. Added chloride is needed to facilitate O2 reactivity for 1-ClO4 but not for 3-ClO4 at 25(1) degrees C. Evaluation of kobs for the reaction of 1-ClO4 with O2 under pseudo first-order conditions as a function of the amount of added chloride ion produced saturation type behavior. The bpy-ligated 3-ClO4 exhibits different behavior, with rate enhancement resulting from both the addition of chloride ion and water. Computational studies indicate that the presence of water lowers the barrier for O2 activation for 3-ClO4 by -12 kcal/mol whereas changing the anion from perchlorate to chloride has a smaller effect (lowering of the barrier by -3 kcal/mol). Notably, the effect of water for 3-ClO4 is of similar magnitude to the barrier-lowering chloride effect found in the O2 activation pathway for 1-ClO4. Thus, both systems involve lower energy O2 activation pathways available, albeit resulting from different ligand effects. Probing the effect of added benzoate anion, it was found that the chloro substituent in the diketonate moiety of 1-ClO4 and 3-ClO4 will undergo displacement upon treatment of each complex with tetrabutyl ammonium benzoate to give Cu(II) benzoyloxydiketonate complexes (4 and 5). Complexes 4 and 5 exhibit slow O2-dependent C-C cleavage in the presence of added chloride ion. These results are discussed in the context of the chemistry identified for various divalent metal chlorodiketonate complexes, which have relevance to catalytic systems and metalloenzymes that mediate O2-dependent C-C cleavage within diketonate substrates.

The π-extended flavonol Flav-1 (1) undergoes reaction with KOH or KO2 to form 1-, which reacts with O2 at ambient temperature, resulting in CO release and depside formation. Mechanistic and DFT studies support a reaction pathway involving reaction of 1- with O2 on the triplet energy surface in the rate-determining step. Formation of a cyclic peroxide leads to CO extrusion. These studies indicate that if formed in biological environments, 1- will release CO in the absence of light illumination.

TGF-β signaling promotes migration, invasion, and distant colonization of cancer cells in advanced metastatic cancers. TGF-β signaling suppresses the anti-tumor immune response in a tumor microenvironment, allowing sustained tumor growth. TGF-β plays an important role in normal physiology; thus it is no surprise that the clinical development of effective and safe TGF-β inhibitors has been hampered due to their high toxicity. We discovered that increased expression of LY6K in cancer cells led to increased TGF-β signaling and that inhibition of LY6K could lead to reduced TGF-β signaling and reduced in vivo tumor growth. LY6K is a highly cancer-specific protein, and it is not expressed in normal organs except in the testes. Thus, LY6K is a valid target for developing therapeutic strategies to inhibit TGF-β signaling in cancer cells. We employed in vitro pull-down assays and molecular dynamics simulations to understand the structural determinants of the TGF-β receptor complex with LY6K. This combined approach allowed us to identify the critical residues and dynamics of the LY6K interaction with the TGF-β receptor complex. These data are critical in designing novel drugs for the inhibition of TGF-β in LY6K expressing cancer, induction of anti-tumor immune response, and inhibition of tumor growth and metastatic spread.

R-specific 1-(4-hydroxyphenyl)-ethanol dehydrogenase (R-HPED) is a promising biotool for stereoselective synthesis of chiral aromatic alcohols. This work focused on the evaluation of its stability under storage and in-process conditions in the pH range from 5.5 to 8.5. The relationship between the dynamics of aggregation and activity loss under various pH conditions and in the presence of glucose, serving as a stabilizer, was analysed using spectrophotometric techniques and dynamic light scattering. pH 8.5 was indicated as a representative environment in which the enzyme, despite relatively low activity, shows high stability and the highest total product yield. Based on a series of inactivation experiments, the mechanism of thermal inactivation at pH 8.5 was modelled. The irreversible first-order mechanism of R-HPED inactivation in the temperature range of 47.5-60 degrees C was verified by isothermal and multi-temperature evaluation of data, confirming that in the alkaline pH 8.5, R-HPED aggregation is the secondary process occurring at already inactivated protein molecules. The rate constants were from 0.029 min-1 to 0.380 min-1 for a buffer solution but they decreased to 0.011 min-1 and 0.161 min-1, respectively, when 1.5 M glucose was added as a stabilizer. The activation energy was however about 200 kJ mol-1 in both cases.

The two-spotted spider mite, Tetranychus urticae, is a major cosmopolitan pest that feeds on more than 1100 plant species. Its genome contains an unprecedentedly large number of genes involved in detoxifying and transporting xenobiotics, including 80 genes that code for UDP glycosyltransferases (UGTs). These enzymes were acquired via horizontal gene transfer from bacteria after loss in the Chelicerata lineage. UGTs are well-known for their role in phase II metabolism; however, their contribution to host adaptation and acaricide resistance in arthropods, such as T. urticae, is not yet resolved. TuUGT202A2 (Tetur22g00270) has been linked to the ability of this pest to adapt to tomato plants. Moreover, it was shown that this enzyme can glycosylate a wide range of flavonoids. To understand this relationship at the molecular level, structural, functional, and computational studies were performed. Structural studies provided specific snapshots of the enzyme in different catalytically relevant stages. The crystal structure of TuUGT202A2 in complex with UDP-glucose was obtained and site-directed mutagenesis paired with molecular dynamic simulations revealed a novel lid-like mechanism involved in the binding of the activated sugar donor. Two additional TuUGT202A2 crystal complexes, UDP-(S)-naringenin and UDP-naringin, demonstrated that this enzyme has a highly plastic and open-ended acceptor-binding site. Overall, this work reveals the molecular basis of substrate promiscuity of TuUGT202A2 and provides novel insights into the structural mechanism of UGTs catalysis.

Abstract Understanding the mechanisms underlying the immune response is crucial for advancing our knowledge of an organism’s defence. One such mechanism is the proteolytic cleavage of the Z-loop in some members of the Toll-like receptor (TLR) family. This process is essential for several reasons: it allows proper receptor dimerisation, facilitates its activation, and introduces a control mechanism by preventing inappropriate or excessive immune reactions. In our study, we focused on investigating the proteolytic cleavage of TLR8 by furin protease, for which the mechanism of this process has not been widely investigated. We employed various computational methods not only to propose the reaction pathway but also to explore the role of water molecules within the reaction site. Those included AI-based structure prediction, molecular dynamics simulations, quantum mechanics and quantum mechanics/molecular mechanics calculations, as well as small-molecule tracking combined with local-distribution methods.

Small molecule NSC243928 binds with LY6K, a potential target for the treatment of triple-negative breast cancer, and induces cancer cell death with an unclear mechanism. We have developed chemical tools to identify the molecular mechanisms of NSC243928-LY6K interaction. Herein, we report on the development and synthesis of biotinylated and fluorophore-tethered derivatives of NSC243928 guided by docking studies and molecular dy-namics. Surface plasmon resonance assay indicates that these derivatives retained a direct binding with LY6K protein. Confocal analysis revealed that nitrobenzoxadiazole (NBD) fluorophore tagged NSC243928 is retained in LY6K expressing cancer cells. These novel modified compounds will be employed in future in vitro and in vivo studies to understand the molecular mechanisms of NSC243928 mediated cancer cell death. These studies will pave the path for developing novel targeted therapeutics and understanding any potential side-effects of these treatments for hard-to-treat cancers such as triple-negative breast cancer or other cancers with high expression of LY6K.

Ectoine is a chemical chaperone synthesised and used by bacteria to defend against osmotic stress. Although it has already gained attention from the pharmaceutical and cosmetic industries, thanks to its hydrating and cell-protecting properties, the reaction mechanism of its final synthesis step is still not fully understood. The ultimate step of ectoine biosynthesis is catalysed by the ectoine synthase enzyme (EctC), which requires an iron ion for substrate binding and overall enzymatic activity. Even though a crystal structure for Paenibacillus lautus EctC—substrate complex is available (PDB: 5ONN), it is not very informative with respect to the geometry of the active site because: (1) the crystal was obtained at a pH value far from the enzyme’s pH optimum, (2) the electron density at the Fe position is weak, and (3) the Fe-ligand distances are too long. To fill this gap, in this work we have used classical molecular dynamics simulations to model the enzyme-substrate (N-gamma-acetyl-L-2,4-diaminobutyric acid) complex of Paenibacillus lautus EctC (PlEctC). Since PlEctC is a homodimeric protein, MD simulations were carried out for a dimer with various plausible occupancies by the substrate and for two plausible coordination geometries around the catalytic Fe ion: tetrahedral and octahedral. MD results revealed that the presence of the ligand has a stabilising effect on the protein structure, most notably on a short helix 112–118, which flanks the entrance to the active site. The most important amino acids for substrate binding are Trp21, Arg25, Asn38, Thr40, and Tyr52, which were also identified in the crystal structure. Importantly, the substrate can easily adopt a conformation suitable for the progress of the catalytic reaction, and it does so spontaneously for the octahedral 6-coordinate geometry of the iron cofactor or with a low energy penalty (ca. 3 kcal/mol) in the case of 4-coordinate tetrahedral geometry. Simulations for different substrate occupancy states did not reveal any signs of cooperativity between the two monomers.

Pharmacological inhibition of LY6K induced cell cycle arrest and DNA damage by disrupting the LY6K-Histone-Aurora B signaling axis Benson C. Selvanesan1,2, Sheelu Varghese1,2, Justyna Andrys5, Ricardo H. Arriaza6, Rahul Prakash6, Purushottam B Tiwari7, Cara Olsen8, Daniel Hupalo2,4, Yuriy Gusev5, Megha N. Patel6, Sara Contente1, Miloslav Sanda9, Aykut Uren7, Matthew D. Wilkerson3,4, Clifton L. Dalgard3,4, Linda S. Shimizu6, Maksymilian Chruszcz6, Tomasz Borowski5, Geeta Upadhyay 1,3,7. Affiliations 1 Department of Pathology, 2 Henry M. Jackson Foundation, 3 Murtha Cancer Center, 4 Department of Anatomy, Physiology, and Genetics 8 Department of Preventive Medicine and Biostatistics Uniformed Services University of the Health Sciences, Bethesda, MD, USA. 5 Jerzy Haber Institute of Catalysis and Surface Chemistry Polish Academy of Sciences, Cracow, Poland. 6 Department of Chemistry and Biochemistry, University of South Carolina, Columbia, SC, USA. 7 Department of Oncology, Georgetown University Medical Center, Washington, DC, USA. 9 Max Planck Institute for Heart and Lung Research, Ludwigstrasse, 43, 61231 Bad Nauheim, Germany. Correspond Disclaimer The opinions expressed herein are those of the authors and are not necessarily representative of the official policies of the Uniformed Services University of the Health Sciences (USUHS), the Department of Defense (DOD), the United States Army/Navy/Air Force, the U.S. Government, or any other funding agencies Conflict of Interest None Acknowledgments NIH, NCI, R01 CA227694. NIH, NCI, R21CA256424. DOD, USUHS, VPR-NFP-74-9824. Biomedical Instrumentation Center, USUHS. The American Genome Center, USUHS. Antibody Characterization Program, Clinical Proteomics Tumor Analysis Consortium (CPTAC), National Cancer Institute, National Institute of Health. The Polish Grid Infrastructure, Cracow, Poland. NIH P30CA51008 and 1S10OD019982-01 to Biacore Molecular Interaction Shared Resource (BMISR), Georgetown University. ABSTRACT Increased expression of LY6K is significantly associated with poor survival outcomes in many solid cancers, including triple-negative and estrogen receptor-positive breast, ovarian, gastric, head and neck, neuroblastoma, bladder, and lung cancers. Inhibition of LY6K signaling is an ideal therapeutic approach for cancer, since the LY6K protein is not involved in vital organ function. Previously, we identified the small molecule NSC243928 as a binder of LY6K using surface plasmon resonance screening and showed that its activity was dependent on LY6K expression in triple-negative breast cancer cells. Here, we demonstrate the structural basis of the molecular interaction of NSC243928 with LY6K protein and the subsequent inhibition of LY6K function in mitosis and cell division via Aurora B-histone pathway. We observed that LY6K interacts with phosphorylated histones and Aurora B kinases during mitosis and that this interaction was disrupted in the presence of NSC243928. Disruption of LY6K function in mitosis/cytokinesis leads to DNA damage, senescence, and apoptosis of cancer cells. We observed that NSC243928 led to increased binding of LY6K to phosphorylated gammaH2X at S139, which was dependent on NSC243928 interaction with LY6K on phenylalanine 79. Furthermore, we observed increased levels of phosphorylated gammaH2X at S139 and increased caspase-3 activation in the tumor isografts of 4T1 and E0771 mammary tumors treated with NSC243928. These data reveal that LY6K is a novel cell cycle target for therapeutic development in triple-negative breast cancer and other solid cancers with high expression of LY6K, such as bladder cancer, head and neck, and lung cancer. Citation Format: Benson Selvanesan, Sheelu Varghese, Justyna Andrys, Ricardo Arriaza, Rahul Prakash, Purushottam Tiwari, Cara Olsen, Daniel Huplo, yuriy Gusev, Megha Patel, Sara Contente, Miloslav Sanda, Matthew Wilkerson, Clifton Dalgard, Linda S. Shimizu, Maksymilian Chruszcz, Tomasz Borowski, Geeta Upadhyay. Pharmacological inhibition of LY6K induced cell cycle arrest and DNA damage by disrupting the LY6K-Histone-Aurora B signaling axis [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr P2-17-04.

Lymphocyte antigen 6K (LY6K) is a small GPI-linked protein that is normally expressed in testes. Increased expression of LY6K is significantly associated with poor survival outcomes in many solid cancers, including cancers of the breast, ovary, gastrointestinal tract, head and neck, brain, bladder, and lung. LY6K is required for ERK-AKT and TGF-β pathways in cancer cells and is required for in vivo tumor growth. In this report, we describe a novel role for LY6K in mitosis and cytokinesis through aurora B kinase and its substrate histone H3 signaling axis. Further, we describe the structural basis of the molecular interaction of small molecule NSC243928 with LY6K protein and the disruption of LY6K-aurora B signaling in cell cycle progression due to LY6K-NSC243928 interaction. Overall, disruption of LY6K function via NSC243928 led to failed cytokinesis, multinucleated cells, DNA damage, senescence, and apoptosis of cancer cells. LY6K is not required for vital organ function, thus inhibition of LY6K signaling is an ideal therapeutic approach for hard-to-treat cancers that lack targeted therapy such as triple-negative breast cancer.

Organic compounds that can be triggered using light to release CO in biological environments are of significant current interest to probe the role of CO in biology and as potential therapeutics. We recently reported that a 3-hydroxybenzo[g]quinolone (5) can be used as a CO delivery molecule to produce anticancer and potent anti-inflammatory effects. Herein we report mechanistic studies of the visible light-induced CO release reaction of this compound. In wet CH3CN under aerobic conditions, 5 releases 0.90(2) equivalents of CO upon illumination with visible light (419 nm) to give a single depside product. Performing the same reaction under an 18O2 atmosphere results in quantitative incorporation of two labeled oxygen atoms in the depside product. Monitoring via1H NMR and UV-vis during the illumination of 5 in CH3CN using 419 nm light revealed the substoichiometric formation of a diketone (6) in the reaction mixture. H2O2 formation was detected in the same reaction mixtures. DFT studies indicate that upon light absorption an efficient pathway exists for the formation of a triplet excited state species (5b) that can undergo reaction with 3O2 resulting in CO release. DFT investigations also provide insight into diketone (6) and H2O2 formation and subsequent reactivity. The presence of water and exposure to visible light play an important role in lowering activation barriers in the reaction between 6 and H2O2 to give CO. Overall, two reaction pathways have been identified for CO release from a 3-hydroxybenzo[g]quinolone.

Fe(II)/2-oxoglutarate dependent dioxygenases (ODDs) share a double stranded beta helix (DSBH) fold and utilise a common reactive intermediate, ferryl species, to catalyse oxidative transformations of substrates. Despite the structural similarities, ODDs accept a variety of substrates and facilitate a wide range of reactions, that is hydroxylations, desaturations, (oxa)cyclisations and ring rearrangements. In this review we present and discuss the factors contributing to the observed (regio)selectivities of ODDs. They span from inherent properties of the reactants, that is, substrate molecule and iron cofactor, to the interactions between the substrate and the enzyme's binding cavity; the latter can counterbalance the effect of the former. Based on results of both experimental and computational studies dedicated to ODDs, we also line out the properties of the reactants which promote reaction outcomes other than the "default" hydroxylation. It turns out that the reaction selectivity depends on a delicate balance of interactions between the components of the investigated system.