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1. Elucidating the metabolic roles of isoflavone synthase-mediated protein-protein interactions in yeast

   https://doi.org/10.1101/2024.10.24.620109  

   Liu, Chang; Han, Jianing; Li, Sijin (October 2024, bioRxiv)

   Abstract Transient plant enzyme complexes formed via protein-protein interactions (PPIs) play crucial regulatory roles in secondary metabolism. Complexes assembled on cytochrome P450s (CYPs) are challenging to characterize metabolically due to difficulties in decoupling the PPIs’ metabolic impacts from the CYPs’ catalytic activities. Here, we developed a yeast-based synthetic biology approach to elucidate the metabolic roles of PPIs between a soybean-derived CYP, isoflavone synthase (GmIFS2), and other enzymes in isoflavonoid metabolism. By reconstructing multiple complex variants with an inactive GmIFS2 in yeast, we found that GmIFS2-mediated PPIs can regulate metabolic flux between two competing pathways producing deoxyisoflavonoids and isoflavonoids. Specifically, GmIFS2 can recruit chalcone synthase (GmCHS7) and chalcone reductase (GmCHR5) to enhance deoxyisoflavonoid production or GmCHS7 and chalcone isomerase (GmCHI1B1) to enhance isoflavonoid production. Additionally, we identified and characterized two novel isoflavoneO-methyltransferases interacting with GmIFS2. This study highlights the potential of yeast synthetic biology for characterizing CYP-mediated complexes. 

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2. Novel Curcumin Inspired Bis-Chalcone Promotes Endoplasmic Reticulum Stress and Glioblastoma Neurosphere Cell Death

   https://doi.org/10.3390/cancers11030357  

   Sansalone, Lorenzo; Veliz, Eduardo; Myrthil, Nadia; Stathias, Vasileios; Walters, Winston; Torrens, Ingrid; Schürer, Stephan; Vanni, Steven; Leblanc, Roger; Graham, Regina (March 2019, Cancers)

   Glioblastoma (GBM) has a dismal prognosis and successful elimination of GBM stem cells (GSCs) is a high-priority as these cells are responsible for tumor regrowth following therapy and ultimately patient relapse. Natural products and their derivatives continue to be a source for the development of effective anticancer drugs and have been shown to effectively target pathways necessary for cancer stem cell self-renewal and proliferation. We generated a series of curcumin inspired bis-chalcones and examined their effect in multiple patient-derived GSC lines. Of the 19 compounds synthesized, four analogs robustly induced GSC death in six separate GSC lines, with a half maximal inhibitory concentration (IC50) ranging from 2.7–5.8 μM and significantly reduced GSC neurosphere formation at sub-cytotoxic levels. Structural analysis indicated that the presence of a methoxy group at position 3 of the lateral phenylic appendages was important for activity. Pathway and drug connectivity analysis of gene expression changes in response to treatment with the most active bis-chalcone 4j (the 3,4,5 trimethoxy substituted analog) suggested that the mechanism of action was the induction of endoplasmic reticulum (ER) stress and unfolded protein response (UPR) mediated cell death. This was confirmed by Western blot analysis in which 4j induced robust increases in CHOP, p-jun and caspase 12. The UPR is believed to play a significant role in GBM pathogenesis and resistance to therapy and as such represents a promising therapeutic target. 

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3. Structural knowledge or X-ray damage? A case study on xylose isomerase illustrating both

   https://doi.org/10.1107/s1600577519005599  

   Taberman, Helena; Bury, Charles S; van_der_Woerd, Mark J; Snell, Edward H; Garman, Elspeth F (July 2019, Journal of Synchrotron Radiation)

   Xylose isomerase (XI) is an industrially important metalloprotein studied for decades. Its reaction mechanism has been postulated to involve movement of the catalytic metal cofactor to several different conformations. Here, a dose-dependent approach was used to investigate the radiation damage effects on XI and their potential influence on the reaction mechanism interpreted from the X-ray derived structures. Radiation damage is still one of the major challenges for X-ray diffraction experiments and causes both global and site-specific damage. In this study, consecutive high-resolution data sets from a single XI crystal from the same wedge were collected at 100 K and the progression of radiation damage was tracked over increasing dose (0.13–3.88 MGy). The catalytic metal and its surrounding amino acid environment experience a build-up of free radicals, and the results show radiation-damage-induced structural perturbations ranging from an absolute metal positional shift to specific residue motions in the active site. The apparent metal movement is an artefact of global damage and the resulting unit-cell expansion, but residue motion appears to be driven by the dose. Understanding and identifying radiation-induced damage is an important factor in accurately interpreting the biological conclusions being drawn. 

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4. Ensemble–function relationships to evaluate catalysis in the ketosteroid isomerase oxyanion hole

   https://doi.org/10.1101/2021.09.29.461692  

   Yabukarski, F.; Doukov, T; Pinney, M.; Biel, J.; Fraser, J.; Herschlag, D. (October 2022, Science advances)

   Following decades of insights from structure–function studies, there is now a need to progress from a static to dynamic view of enzymes. Comparison of prior cryo X-ray structures suggested that deleterious effects from ketosteroid isomerase (KSI) mutants arise from misalignment of the oxyanion hole catalytic residue, Y16. However, multi-conformer models from room temperature X-ray diffraction revealed an ensemble of Y16 conformers indistinguishable from WT for Y32F/Y57F KSI and a distinct, non-native ensemble for Y16 in Y57F KSI. Functional analyses suggested rate effects arise from weakened hydrogen bonding, due to disruption of the Y16/Y57/Y32 hydrogen bond network, and repositioning of the general base. In general, catalytic changes can be deconvoluted into effects on the probability of occupying a state (P-effects) and the reactivity of each state (k-effects). Our results underscore the need for ensemble–function analysis to decipher enzyme function and ultimately manipulate their extraordinary capabilities. 

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5. Catalytic Activity of Water-Soluble Palladium Nanoparticles with Anionic and Cationic Capping Ligands for Reduction, Oxidation, and C-C Coupling Reactions in Water

   https://doi.org/10.3390/nano15050405  

   Farag, Jan W; Khalil, Ragaa; Avila, Edwin; Shon, Young-Seok (March 2025, Nanomaterials)

   The availability of water-soluble nanoparticles allows catalytic reactions to occur in highly desirable green environments. The catalytic activity and selectivity of water-soluble palladium nanoparticles capped with 6-(carboxylate)hexanethiolate (C6-PdNP) and 5-(trimethylammonio)pentanethiolate (C5-PdNP) were investigated for the reduction of 4-nitrophenol, the oxidation of α,β-conjugated aldehydes, and the C-C coupling of phenylboronic acid. The study showed that between the two PdNPs, C6-PdNP exhibits better catalytic activity for the reduction of 4-nitrophenol to 4-aminophenol in the presence of sodium borohydride and the selective oxidation of conjugated aldehydes to conjugated carboxylic acids. For the latter reaction, molecular hydrogen (H2) and H2O act as oxidants for the surface palladium atoms on PdNPs and conjugated aldehyde substrates, respectively. The results indicated that the competing addition activities of Pd-H and H2O toward the π-bond of different unsaturated substrates promote either reduction or oxidation reactions under mild conditions in organic solvent-free environments. In comparison, C5-PdNP exhibited higher catalytic activity for the C-C coupling of phenylboronic acid. Gas chromatography–mass spectrometry (GC-MS) was mainly used as an analytical technique to examine the products of catalytic reactions. 

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