Molecular mechanisms of protein disulfide isomerase antagonism by punicalagin.

Molecular insights into the anti-browning effect of protocatechualdehyde.

Polyphenol oxidase (PPO) is a vital polyphenol-metabolizing enzyme responsible for the enzymatic browning of fruits and vegetables. This study assessed the anti-PPO activity of protocatechualdehyde (PCA), yielding an IC50 value of 94 μM (95% CI: 91.8 to 96.4 μM). Kinetic analysis revealed that PCA acted as a reversible and competitive inhibitor of PPO. Molecular docking indicated that hydrogen bonding and hydrophobic interactions were the main driving forces for PCA-PPO binding. Fluorescence spectroscopy further demonstrated that PCA binding altered the microenvironments around tyrosine and tryptophan residues, leading to conformational rearrangements and partial unfolding of the enzyme, evidenced by significant fluorescence quenching. Molecular dynamics simulation demonstrated that PCA perturbed the secondary structure of PPO and caused stretching of its overall structure, thereby inhibiting the catalytic activity of the enzyme. Furthermore, PCA demonstrated excellent anti-browning activity by regulating phenolic metabolism, reducing membrane lipid peroxidation, enhancing the ascorbic acid-glutathione cycle, and modulating cell wall metabolism to delay cellular senescence. Collectively, these findings offer some valuable insights into the development of PPO inhibitor and anti-browning agent, and provide a theoretical foundation for the potential application of PCA in the area of food preservation.

Insights into the enzyme kinetics, structural features, and in silico inhibition of Leishmania donovani prolidase.

Prolidase is a specialized metalloprotease that overcomes the resistance of proline-containing peptide bonds to proteolysis by exclusively hydrolyzing dipeptides with a C-terminal proline. Despite its potential significance in the life cycle of protozoan parasites, knowledge of prolidase in these organisms remains limited. In this study, the complete coding sequence of Leishmania donovani prolidase (LdProl) was cloned into an expression vector. The recombinant protein was purified using affinity chromatography, and its molecular weight was assessed by size-exclusion chromatography. The purified LdProl exhibited enzymatic activity with a strong substrate preference for Trp-Pro-pNA, and manganese ions were identified as essential cofactors for optimal activity. LdProl also stimulated innate immune mechanisms through elevating pro-inflammatory cytokines and nitric oxide levels, suggesting a role in macrophage-mediated immunomodulation. Fluorescence spectroscopy revealed pH-dependent conformational changes, particularly under strongly acidic conditions, whereas circular dichroism spectroscopy indicated a predominantly α-helical structure that was most stable at neutral pH. Computational modeling demonstrated a conserved three-dimensional structure, metal-binding site, and stable dimeric interface similar to prolidases from other organisms. Docking studies identified Trp-Pro-pNA as the substrate with the highest binding affinity, and molecular dynamics simulations confirmed the formation of stable enzyme-substrate complexes. Virtual screening yielded two lead compounds with higher binding affinities for LdProl than for its human counterpart, along with favorable ADME properties. Molecular dynamics simulations further validated these compounds by demonstrating the stability of enzyme-inhibitor complexes. Overall, this study presents the first comprehensive report on the enzyme kinetics, structural characteristics, and in silico inhibition of metal-dependent prolidase from trypanosomatid parasites.

Natural compounds modulating human carbonic anhydrase activity and oxidative stress: a biochemical and molecular study

Elucidating the Antiglycation Potential of Plumbagin Against Methylglyoxal-Induced Glycation of Bovine Liver Catalase.

Non-enzymatic glycosylation of proteins leads to the formation of advanced glycation end products (AGEs), implicated in oxidative stress and the progression of several chronic diseases. Among the reactive carbonyl compounds, methylglyoxal (MGO) is highly potent in inducing glycoxidative damage by modifying proteins. Herein, we investigated the protective effect of plumbagin, a naphthoquinone derived from Plumbago zeylanica, to counteract MGO-induced glycation of catalase, employing integrated biophysical and computational approaches. AGE-specific fluorescence at 335 and 370 nm, ThT dye fluorescence, and microscopic visualizations confirmed that plumbagin markedly reduces AGEs formation and aggregation. Fluorescence quenching experiments revealed a stable catalase-plumbagin complex formation having binding affinity (Ka): 0.205 × 106 M-1 and a stoichiometry of one binding site (n ≈ 1.1), suggesting a single, specific binding pocket. Isothermal titration calorimetry (ITC) confirmed an exothermic, enthalpy-driven interaction. Molecular dynamics simulations (MDS) analysis displayed a more compact and ordered conformation of complex, as evidenced by reduced Rg, SASA, and RMSF values. H-bonds stabilized the complex without altering the catalytic residues (His74, Asn147, Tyr357). PCA and FEL showed complex remained confined to a single deep energy minimum, indicating enhanced thermodynamic stability. These findings highlight plumbagin as a potent multifunctional compound capable of mitigating MGO-induced glycation.

A Peptidase A1 domain protein from Stropharia rugosoannulata: ACE inhibition and multi-pathway vascular modulation.

A Peptidase A1 domain-containing protein (SRP, 43.1 kDa, >95% purity) was isolated from the edible mushroom Stropharia rugosoannulata and characterized for its structural properties, and ACE inhibitory and vascular modulatory activities. This represents the first report of an intact Peptidase A1 family protein exhibiting bioactivities beyond its canonical proteolytic role. SRP inhibited ACE (IC50 = 108.84 ± 3.42 μg mL-1) through strong intermolecular interactions, with Glu264 contributing 45% (-20.76 kcal mol-1) of the total binding energy and additional contributions from conserved active-site residues. Isothermal titration calorimetry confirmed high-affinity binding to ACE (KD = 1.12 × 10-8 M), driven by both enthalpic and entropic contributions with entropy as the dominant thermodynamic force, suggesting a complex binding mode involving both primary and secondary interaction sites. In a zebrafish hypertension model, SRP at 62.5 μg mL-1 decreased cardiac output by 20-24% and increased vessel diameter by 17-18%, with effects comparable to those of captopril at the tested concentration. Integrative transcriptomic and proteomic analyses revealed that SRP reversed the downregulation of core genes and key proteins, modulating multiple vascular homeostasis pathways including calcium signaling, vascular smooth muscle contraction, Toll-like receptor signaling, and neuroactive ligand-receptor interaction. These findings expand the known functional repertoire of Peptidase A1 family proteins and highlight the multi-pathway modulatory potential of food-derived intact proteins.

Structural and biochemical insights into an anthocyanin-related glutathione transferase from bilberry and its inhibition by quercetin.

The anthocyanin biosynthetic pathway (ABP), a branch of the phenylpropanoid pathway, is responsible for the production of a wide range of flavonoid compounds in plants. Anthocyanin-related glutathione transferases (arGSTs) have long been proposed to act as non-catalytic carrier proteins, mediating the vacuolar sequestration of anthocyanins. However, recent structural and biochemical evidence has suggested a potential catalytic role for arGSTs in the ABP, notably in catalyzing the dehydration of flavan-3,3,4-triol to anthocyanidin. Despite their importance, only a limited number of arGSTs have been characterized biochemically and structurally. Here, we investigated the arGST isoform from bilberry, one of the richest fruit sources of anthocyanins. We showed that the expression of arGST gene in bilberry increases during fruit ripening in parallel with other ABP genes and putative transcription factors. Biochemical and structural analyses highlighted the remarkable ability of GSTs to adapt to their substrates and revealed a possible inhibitory effect of quercetin on arGST activity, as quercetin is a by-product of the anthocyanin biosynthetic pathway.