Comparative esterase kinetics and hypoxia-induced, 4-hydroxynonenal-mediated regulation of carboxylesterase in three Neotropical Characiform fish.

Functional characterization of 3β-hydroxysteroid dehydrogenases in Asiatic toad reveals an efficient broad substrate specificity of Bg-3βHSD2

Abstract 3β-Hydroxysteroid dehydrogenases (3βHSDs) are key enzymes in steroid metabolism, catalyzing C3 oxidation–reduction and Δ5→Δ4 isomerization reactions that govern metabolic flux across multiple steroidogenic pathways. However, the functional diversity of 3βHSDs involved in bufadienolide metabolism in amphibians remains poorly explored. Here, we systematically characterized the 3βHSD gene family in the Asian toad (Bufo bufo gargarizans) using integrated transcriptomic, biochemical, and metabolomic analyses. Seven Bg-3βHSD genes were identified from multi-tissue transcriptomes generated under control and Pb2+ exposure conditions, and six were heterologously expressed for functional evaluation. In vitro assays revealed pronounced functional divergence among Bg-3βHSD isoforms. Bg-3βHSD1 primarily catalyzed bidirectional C3 redox reactions of C21 steroids and bile acid-related substrates, consistent with canonical steroidogenic roles. In contrast, Bg-3βHSD2 enzyme exhibited broad substrate specificity and high catalytic efficiency toward hormones, bile acids, and bufadienolides. In addition to canonical C3 redox reactions and Δ5→Δ4 isomerization, Bg-3βHSD2 also displayed additional oxidation activity at the C17 position for several steroid substrates. A third homolog, Bg-HSD3B7 (GenBank accession no. XM 044303756.1), selectively converted 7α-hydroxylated sterols, suggesting a potential role in classical bile acid metabolism. Integration of tissue-specific expression profiles with bufadienolide distribution patterns suggests that Bg-3βHSD2 may contribute to connecting classical steroid metabolism with bufadienolide biosynthesis in adrenal tissue. Together, the present study identifies Bg-3βHSD2 as an efficient and versatile steroid-transforming enzyme, expands our knowledge of functional diversity within the amphibian 3βHSD family, and provides insights into the enzymatic basis of steroid and bufadienolide metabolism in B. bufo gargarizans.

Chemical characterization and urate-related activities of constituents of Paeonia lactiflora: 1,2,3,4,6-O-pentagalloylglucose inhibits xanthine oxidase and paeoniflorin modulates urate transporter expression in HK-2 cells.

Hyperuricemia (HUA), a metabolic disorder associated with gout and cardiometabolic diseases, has become an important target for the development of food-derived functional ingredients. Paeonia lactiflora Pall, a widely consumed edible medicinal plant, has attracted attention as a potential source of urate-modulating compounds; however, its key active compounds and their urate-related activities remain unclear. In this study, through comprehensive phytochemical profiling, 20 compounds were isolated from P. lactiflora and systematically evaluated using xanthine oxidase (XO) inhibition and HK-2 cellular models. Among them, 1,2,3,4,6-O-pentagalloylglucose (PGG, 20) exhibited potent XO inhibitory activity, with an IC50 value of 8.11 μM. Molecular docking and 100 ns molecular dynamics simulations further supported the stable binding of PGG to the catalytic pocket of XO, suggesting its role in inhibiting UA production at the enzymatic level. To investigate the urate transport-related activity of the isolates, a UA-induced injury model was established in HK-2 cells. Paeoniflorin (PF, 1) showed the strongest protective effect, improving cell viability by 13.47%. Western blot analysis showed that PF modulated the expression of urate transport-related proteins in HK-2 cells by decreasing the levels of the reabsorption-associated transporters URAT1 and GLUT9 and increasing the levels of the secretion-associated transporters OAT1 and ABCG2. In conclusion, these findings identify PGG and PF as two representative constituents of P. lactiflora with distinct urate-related activity profiles in enzymatic and cellular models, respectively. This study provides a chemical and preliminary functional basis for the future development and quality evaluation of P. lactiflora-derived functional ingredients targeting urate metabolism.

Determination of the Inhibitory and Pharmacokinetic Properties of Helipyrone, Norhelipyrone, Italipyrone, Bisnorhelipyrone, and Plicatipyrone by in Silico Methods

Abstract Helichrysum stoechas (L.), commonly known as the eternal flower, has long been used in traditional medicine for the treatment of many diseases. Previous phytochemical studies have reported that H. stoechas contains a variety of secondary metabolites, including the pyrones helipyrone, norhelipyrone, bisnorhelipyrone, plicatipyrone and italipyrone, which have been isolated from the plant. In this study, the inhibitory potential of these compounds against monoamine oxidase A (MAO-A), acetylcholinesterase (AChE), butyrylcholinesterase (BChE), tyrosinase, cyclooxygenase-2 (COX-2), dipeptidyl peptidase-4 (DPP4), angiotensin-converting enzyme (ACE), and stable 5-lipoxygenase (stable-5-LOX) was investigated using in silico approaches, including molecular docking, molecular dynamics (MD) simulations, MM/PBSA analyses, and density functional theory (DFT) calculations. In addition, the pharmacokinetic and toxicity profiles of the compounds were evaluated using ADME/T and PASS prediction tools. The molecular docking results indicated that italipyrone and plicatipyrone exhibited relatively strong binding affinities toward several target enzymes. The MD simulations and MM/PBSA analyses revealed that the ACE-N-domain-plicatipyrone and stable-5-LOX-italipyrone complexes showed binding free energies of -42.79 and -26.32 kcal/mol, respectively. The DFT results showed favorable electronic stability characteristics for the compounds studied, while ADME/T estimates showed relatively high blood-brain barrier permeability for italipyrone and plicatipyrone, and no toxicity was predicted for the tested molecules. Overall, the findings suggest that these compounds may be promising candidates for further experimental evaluation; however, additional in vitro and in vivo studies are needed to confirm their biological activities and pharmacological potential.

Functions of exogenous α-naphthaleneacetic acid and 6-benzylaminopurine in the macroalga Gracilariopsis lemaneiformis revealed by transcriptomic analysis

Abstract Gracilariopsis lemaneiformis is cultivated as feed for abalone and raw material for agar, and its economic benefits are strongly correlated with its yield. Plant growth regulators such as α-naphthaleneacetic acid (NAA) and 6-benzylaminopurine (6-BA), can effectively promote plant growth and biomass accumulation, however, their effects on macroalgae remain less thoroughly investigated. This study aimed to evaluate the roles of NAA and 6-BA on the G. lemaneiformis using physiological and transcriptomic analyses. Results showed that both NAA and 6-BA enhanced the relative growth rate, linear growth rate of main and lateral branches, as well as accumulation of phycobiliprotein, soluble polysaccharide and total lipid. Transcriptomic results corroborated the physiological performances. Both NAA and 6-BA upregulated carbohydrate metabolism, photosynthesis-related metabolism, amino acid biosynthesis and purine metabolism. In addition, NAA specifically increased the expression levels of the genes related to cell division process, including CDC20, CDC42 and expansin-like protein, while 6-BA upregulated the expression levels of ABC transporters, including ABCD3 and ABCG2. These findings will not only deepen our understanding of the NAA and 6-BA roles in promoting algal growth, but also be useful in cultivating the seedlings of G. lemaneiformis.

Glycolytic Inhibition by 3-Bromopyruvic Acid Induces Metabolic Stress and Developmental Impairment in Periplaneta americana

Mechanism-Based Inactivation of Human Ornithine Aminotransferase by Ethynyl- and Nitrile-Substituted Cyclopentene Analogues of γ-Aminobutyric Acids.

Human ornithine aminotransferase (hOAT), a pyridoxal 5'-phosphate (PLP)-dependent enzyme, plays a central role in glutamine, proline, and polyamine metabolism and is increasingly recognized as a metabolic vulnerability in multiple cancers. Previously, we established a second deprotonation strategy to achieve efficient mechanism-based inactivation of hOAT over closely related aminotransferases. Building on this concept, we report the rational design, synthesis, and mechanistic investigation of cyclopentene-based γ-aminobutyric acid analogues bearing alkyne or nitrile warheads as potent hOAT inactivators. These compounds undergo enzyme-catalyzed γ-deprotonation to form ketimine intermediates, priming for a subsequent tautomerization event that leads to irreversible inhibition. Inhibitory activity evaluation revealed pronounced stereochemical effects on binding affinity and partition ratio, with one nitrile analogue (4b) exhibiting an exceptional inactivation efficiency (kinact/KI = 111.8 mM-1·min-1) and ∼400-fold selectivity for hOAT over γ-aminobutyric acid aminotransferase. Intact protein mass spectrometry and X-ray crystallography demonstrated that alkyne-containing analogues form covalent adducts with hOAT, whereas nitrile-containing analogues generate noncovalent but tight-binding species. Kinetic isotope effect studies identified γ-deprotonation as the rate-determining step, and a complementary small-molecule mass and computational study elucidated the inactivation and turnover pathways. Collectively, these results expand the mechanistic repertoire of PLP-dependent enzyme inactivation and provide a generalizable framework for designing highly selective mechanism-based inactivators.