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1. An in vitro platform for the enzymatic characterization of the rhomboid protease RHBDL4

   https://doi.org/10.1016/j.jbc.2025.108275  

   Bhaduri, Satarupa; Braza, Mac_Kevin E; Stanchev, Stancho; Tauber, Marina; Al-Bawab, Raghad; Liu, Lawrence J; Trujillo, Diego F; Solorio-Kirpichyan, Kristina; Srivastava, Ambuj; Sanlley-Hernandez, Javier; et al (March 2025, Journal of Biological Chemistry)

   Rhomboid proteases are ubiquitous intramembrane serine proteases that can cleave transmembrane substrates within lipid bilayers. They exhibit many and diverse functions, such as but not limited to, growth factor signaling, immune and inflammatory response, protein quality control, and parasitic invasion. Human rhomboid protease RHBDL4 has been demonstrated to play a critical role in removing misfolded proteins from the endoplasmic reticulum and is implicated in severe diseases such as various cancers and Alzheimer's disease. Therefore, RHBDL4 is expected to constitute an important therapeutic target for such devastating diseases. Despite its critical role in many biological processes, the enzymatic properties of RHBDL4 remain largely unknown. To enable a comprehensive characterization of RHBDL4's kinetics, catalytic parameters, substrate specificity, and binding modality, we expressed and purified recombinant RHBDL4 and employed it in a Förster resonance energy transfer-based cleavage assay. Until now, kinetic studies have been limited mostly to bacterial rhomboid proteases. Our in vitro platform offers a new method for studying RHBDL4's enzymatic function and substrate preferences. Furthermore, we developed and tested potential inhibitors using our assay and successfully identified peptidyl α-ketoamide inhibitors of RHBDL4 that are highly effective against recombinant RHBDL4. We utilize ensemble docking and molecular dynamics simulations to explore the binding modality of substrate-derived peptides bound to RHBDL4. Our analysis focused on key interactions and dynamic movements within RHBDL4's active site that contributed to binding stability, offering valuable insights for optimizing the nonprime side of RHBDL4 ketoamide inhibitors. In summary, our study offers fundamental insights into RHBDL4's catalytic activities and substrate preferences, laying the foundation for downstream applications such as drug inhibitor screenings and structure-function studies, which will enable the identification of lead drug compounds for RHBDL4. 

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2. Prediction and design of protease enzyme specificity using a structure-aware graph convolutional network

   https://doi.org/10.1073/pnas.2303590120  

   Lu, Changpeng; Lubin, Joseph H.; Sarma, Vidur V.; Stentz, Samuel Z.; Wang, Guanyang; Wang, Sijian; Khare, Sagar D. (September 2023, Proceedings of the National Academy of Sciences)

   Site-specific proteolysis by the enzymatic cleavage of small linear sequence motifs is a key posttranslational modification involved in physiology and disease. The ability to robustly and rapidly predict protease–substrate specificity would also enable targeted proteolytic cleavage by designed proteases. Current methods for predicting protease specificity are limited to sequence pattern recognition in experimentally derived cleavage data obtained for libraries of potential substrates and generated separately for each protease variant. We reasoned that a more semantically rich and robust model of protease specificity could be developed by incorporating the energetics of molecular interactions between protease and substrates into machine learning workflows. We present Protein Graph Convolutional Network (PGCN), which develops a physically grounded, structure-based molecular interaction graph representation that describes molecular topology and interaction energetics to predict enzyme specificity. We show that PGCN accurately predicts the specificity landscapes of several variants of two model proteases. Node and edge ablation tests identified key graph elements for specificity prediction, some of which are consistent with known biochemical constraints for protease:substrate recognition. We used a pretrained PGCN model to guide the design of protease libraries for cleaving two noncanonical substrates, and found good agreement with experimental cleavage results. Importantly, the model can accurately assess designs featuring diversity at positions not present in the training data. The described methodology should enable the structure-based prediction of specificity landscapes of a wide variety of proteases and the construction of tailor-made protease editors for site-selectively and irreversibly modifying chosen target proteins. 

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3. Systematic Investigation of Silicon Substitution on Single Macromolecule Mechanics

   https://doi.org/10.1021/acs.macromol.3c01066  

   Wentz, Kelsie E.; Yao, Yunxin; Kevlishvili, Ilia; Kouznetsova, Tatiana B.; Mediavilla, Braden A.; Kulik, Heather J.; Craig, Stephen L.; Klausen, Rebekka S. (August 2023, Macromolecules)

   Four unsaturated poly(carbooligosilane)s (P1–P4) were prepared via acyclic diene metathesis polycondensation of new oligosilane diene monomers (1–4). These novel polymers with varying main-chain Si incorporation have high trans internal olefin stereochemistry (ca. 80%) and molecular weights (9500–21,700 g mol–1). Postpolymerization epoxidation converted all alkene moieties to epoxides and rendered the polymers (P5–P8) more electrophilic, which allowed for single-molecule force spectroscopy studies via a modified atomic force microscope setup with a silicon tip and cantilever. The single-chain elasticity of the polycarbooligosilanes decreased with increasing numbers of Si–Si bonds, a finding reproduced by quantum chemical calculations. 

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4. Characterization of the substrate specificity and regioselectivity of ring-cleavage of Pseudomonas putida DLL-E4 hydroquinone 1,2-dioxygenase (PnpC1C2)

   https://doi.org/10.1007/s00775-025-02101-4  

   Machonkin, Timothy E; Maker, Madeleine S; Ganjoloo, Nandin; Conkin, Drew F (February 2025, JBIC Journal of Biological Inorganic Chemistry)

   PnpC1C2 is an enzyme from the soil bacterium Pseudomonas putida DLL-E4 that is in the pathway for the oxidative catabolism of 4-nitrophenol. PnpC1C2 oxidatively cleaves hydroquinone into -hydroxymuconic semialdehyde. It belongs to the type II hydroquinone dioxygenase family, a relatively uncharacterized group of mononuclear nonheme Fe(II)-dependent enzymes that catalyze oxidative ring-cleavage reactions, which includes the well-studied catechol extradiol dioxygenases as well as the structurally unrelated 2,6-dichlorohydro-quinone dioxygenase (PcpA). Steady-state kinetics studies using UV/Vis spectroscopy were performed to characterize the enzyme specificity towards various substituted hydroquinones. In addition to its native substrate, PnpC1C2 was active towards a variety of monosubstituted hydroquinones. Methyl- and methoxyhydroquinone showed a moderately higher , and chloro- and bromohydroquinone showed a moderately lower , but all had a within an order of magnitude of unsubstituted hydroquinone. Likewise, only small differences in the rates of mechanism-based inactivation were observed among these substrates. Among disubstituted hydroquinones, only 2,6- and 2,5-dimethylhydroquinone showed any activity, with the latter only barely detectable. A variety of para-substituted phenols were found to be good inhibitors of PnpC1C2. NMR studies were performed to determine the regioselectivity of ring-cleavage with monosubstituted hydroquinones. All monosubstituted hydroquinones tested (methyl-, chloro-, bromo-, and methoxyhydroquinone) yielded exclusively the 1,6-cleavage product. Thus, PnpC1C2 shows notable differences in both its substrate specificity and the ring-cleavage regioselectivity compared to that of PcpA. These results provide an important basis for future comparison of structure-function correlations among the hydroquinone ring-cleaving dioxygenases. 

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5. Spectroscopy and crystallography define carotenoid oxygenases as a new subclass of mononuclear non-heme FeII enzymes

   https://doi.org/10.1016/j.jbc.2025.108444  

   DeWeese, Dory E; Everett, Michael P; Babicz, Jeffrey T; Daruwalla, Anahita; Solomon, Edward I; Kiser, Philip D (March 2025, Journal of Biological Chemistry)

   NA (Ed.)

   Carotenoid cleavage dioxygenases (CCDs) are non-heme FeII enzymes that catalyze the oxidative cleavage of alkene bonds in carotenoids, stilbenoids, and related compounds. How these enzymes control the reaction of O2 with their alkene substrates is unclear. Here, we apply spectroscopy in conjunction with X-ray crystallography to define the iron coordination geometry of a model CCD, CAO1, in its resting state and following substrate binding and coordination sphere substitutions. Resting CAO1 exhibits a five-coordinate (5C), square pyramidal FeII center that undergoes steric distortion towards a trigonal bipyramidal geometry in the presence of piceatannol. Titrations with the O2-analog, nitric oxide (NO), show a >100-fold increase in iron-NO affinity upon substrate binding, defining a crucial role for the substrate in activating the FeII site for O2 reactivity. The importance of the 5C FeII structure for reactivity was probed through mutagenesis of the second-sphere Thr151 residue of CAO1, which occludes ligand binding at the sixth coordination position. A T151G substitution resulted in the conversion of the iron center to a six-coordinate (6C) state and a 135-fold reduction in apparent catalytic efficiency towards piceatannol compared to the wild-type enzyme. Substrate complexation resulted in partial 6C to 5C conversion, indicating solvent dissociation from the iron center. Additional substitutions at this site demonstrated a general functional importance of the occluding residue within the CCD superfamily. Taken together, these data suggest an ordered mechanism of CCD catalysis occurring via substrate-promoted solvent replacement by O2. CCDs thus represent a new class of mononuclear non-heme FeII enzymes. 

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