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1. Derivatives of the Fungal Natural Product Illudalic Acid Inhibit the Activity of Protein Histidine Phosphatase PHPT1

   https://doi.org/10.1002/cmdc.202300187  

   Wang, Hanfei; Gaston, Jr., Robert; Ahmed, Kh_Tanvir; Dudley, Gregory_B; Barrios, Amy_M (June 2023, ChemMedChem)

   Abstract PHPT1 is a protein histidine phosphatase that has been implicated in several disease pathways, but the chemical tools necessary to study the biological roles of this enzyme and investigate its utility as a therapeutic target have yet to be developed. To this end, the discovery of PHPT1 inhibitors is an area of significant interest. Here, we report an investigation of illudalic acid and illudalic acid analog‐based inhibition of PHPT1 activity. Four of the seven analogs investigated had IC₅₀values below 5 μM, with the most potent compound (IA1‐8H2) exhibiting an IC₅₀value of 3.4±0.7 μM. Interestingly, these compounds appear to be non‐covalent, non‐competitive inhibitors of PHPT1 activity, in contrast to other recently reported PHPT1 inhibitors. Mutating the three cysteine residues to alanine has no effect on inhibition, indicating that cysteine is not critical for interactions between inhibitor and enzyme. 

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2. A Model-Based 13C-Sucrose Breath Test Diagnostic for Gut Function Disorders Characterized by a Loss of Sucrase-Isomaltase Enzymatic Activity

   https://doi.org/10.1016/j.tjnut.2023.11.017  

   Brouwer, Andrew F; Lee, Gwenyth O; Van_Wyk, Hannah; Schillinger, Robert J; Edwards, Christine A; Morrison, Douglas J (March 2024, The Journal of Nutrition)

   Background: Environmental enteric dysfunction (EED) causes malnutrition in children in low-resource settings. Stable isotope breath tests have been proposed as non-invasive tests of altered nutrient metabolism and absorption in EED, but uncertainty over interpreting the breath curves has limited their use. The activity of sucrase-isomaltase, the glucosidase enzyme responsible for sucrose hydrolysis, may be reduced in EED. We previously developed a mechanistic model describing the dynamics of the 13C-sucrose breath test (13C-SBT) as a function of underlying metabolic processes. Objective: 1) To determine which breath test curve dynamics are associated with sucrose hydrolysis and with the transport and metabolism of the fructose and glucose moieties, and 2) to propose and evaluate a model-based diagnostic for the loss of activity of sucrase-isomaltase. Methods: We applied the mechanistic model to two sets of exploratory 13C-SBT experiments in healthy adult participants. First, 19 participants received differently labeled sucrose tracers (U-13C fructose, U-13C glucose, and U-13C sucrose) in a cross-over study. Second, 16 participants received a sucrose tracer accompanied by 0 mg, 100 mg, and 750 mg of Reducose®, a sucrase-isomaltase inhibitor. We evaluated a model-based diagnostic distinguishing between inhibitor concentrations using receiver operator curves, comparing to conventional statistics. Results: Sucrose hydrolysis and the transport and metabolism of the fructose and glucose moieties were reflected in the same mechanistic process. The model distinguishes these processes from the fraction of tracer exhaled and an exponential metabolic process. The model-based diagnostic performed as well as the conventional summary statistics in distinguishing between no and low inhibition (AUC 0.77 vs 0.66–0.79) and for low vs high inhibition (AUC 0.92 vs 0.91–0.99). Conclusions: Current summary approaches to interpreting 13C breath test curves may be limited to identifying only gross gut dysfunction. A mechanistic model-based approach improved interpretation of breath test curves characterizing sucrose metabolism. 

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3. Mix-and-inject XFEL crystallography reveals gated conformational dynamics during enzyme catalysis

   https://doi.org/10.1073/pnas.1901864116  

   Dasgupta, Medhanjali; Budday, Dominik; de_Oliveira, Saulo_H P; Madzelan, Peter; Marchany-Rivera, Darya; Seravalli, Javier; Hayes, Brandon; Sierra, Raymond G; Boutet, Sébastien; Hunter, Mark S; et al (December 2019, Proceedings of the National Academy of Sciences)

   How changes in enzyme structure and dynamics facilitate passage along the reaction coordinate is a fundamental unanswered question. Here, we use time-resolved mix-and-inject serial crystallography (MISC) at an X-ray free electron laser (XFEL), ambient-temperature X-ray crystallography, computer simulations, and enzyme kinetics to characterize how covalent catalysis modulates isocyanide hydratase (ICH) conformational dynamics throughout its catalytic cycle. We visualize this previously hypothetical reaction mechanism, directly observing formation of a thioimidate covalent intermediate in ICH microcrystals during catalysis. ICH exhibits a concerted helical displacement upon active-site cysteine modification that is gated by changes in hydrogen bond strength between the cysteine thiolate and the backbone amide of the highly strained Ile152 residue. These catalysis-activated motions permit water entry into the ICH active site for intermediate hydrolysis. Mutations at a Gly residue (Gly150) that modulate helical mobility reduce ICH catalytic turnover and alter its pre-steady-state kinetic behavior, establishing that helical mobility is important for ICH catalytic efficiency. These results demonstrate that MISC can capture otherwise elusive aspects of enzyme mechanism and dynamics in microcrystalline samples, resolving long-standing questions about the connection between nonequilibrium protein motions and enzyme catalysis. 

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4. Molecular interactions and inhibition of the SARS‐CoV‐2 main protease by a thiadiazolidinone derivative

   https://doi.org/10.1002/prot.26385  

   Andrzejczyk, Jacob; Jovic, Katarina; Brown, Logan M.; Pascetta, Valerie G.; Varga, Krisztina; Vashisth, Harish (May 2022, Proteins: Structure, Function, and Bioinformatics)

   We report molecular interactions and inhibition of the main protease (MPro) of SARS-CoV-2, a key enzyme involved in the viral life cycle. By using a thiadiazolidinone (TDZD) derivative as a chemical probe, we explore the conformational dynamics of MPro via docking protocols and molecular dynamics simulations in all-atom detail. We reveal the local and global dynamics of MPro in the presence of this inhibitor and confirm the inhibition of the enzyme with an IC50 value of 1.39 ± 0.22 μM, which is comparable to other known inhibitors of this enzyme. 

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5. Biochemical Consequences of a Leucine-to-Cysteine Clamp Substitution in Lipoxygenases

   https://doi.org/10.3390/biom15081153  

   Hill, Samuel G; DeFeo, Katherine; Offenbacher, Adam R (August 2025, Biomolecules)

   Lipoxygenases (LOXs) are a family of metalloenzymes that oxidize polyunsaturated fatty acids producing cell-signaling hydroperoxides. Fungal LOXs have drawn interest because of their roles in plant and animal pathogenesis. A new subfamily of annotated fungal LOXs has been predicted. One of its unique structural features is the presence of a cysteine amino acid encoded at the invariant leucine clamp. Herein, we isolate three representatives of this LOX subfamily from recombinant expressions in both yeast and bacterial cultures. Metal analysis indicates that the proteins accommodate a mononuclear manganese ion center, similar to other eukaryotic LOXs, but have nominal LOX activity. The functional consequence of the non-conservative mutation is further explored using a Leu-to-Cys (L546C) variant of soybean lipoxygenase, a model plant orthologue. While this L546C variant has comparable structural integrity and metal content to the native enzyme, the variant is associated with a 50-fold decrease in the first-order rate constant. The presence of cysteine at 546, compared to leucine, alanine, or serine, also results in a distinctive kinetic lag phase and product inhibition. The collective data highlight that Cys encoded at the Leu clamp is detrimental to LOX activity. Potential biological functions of these annotated fungal LOXs are discussed. 

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