The team, led by Luping Zhao, Yan Shen, Yuanjin Zhang, and Xin Wang, evaluated juglone against six major CYP activities in both pooled human liver microsomes and microsomes prepared from rat liver. Using a cocktail of probe substrates—phenacetin for CYP1A2, bupropion for CYP2B6, tolbutamide for CYP2C9, dextromethorphan for CYP2D6, chlorzoxazone for CYP2E1, and midazolam for CYP3A4—they quantified metabolite formation by liquid chromatography–tandem mass spectrometry after incubating the microsomes with juglone concentrations ranging from 1 to 100 micromolar. The screening revealed a clear hierarchy of susceptibility in the human preparations, with CYP2C9 and CYP2E1 standing out well ahead of the rest.

In human liver microsomes, CYP2C9 was the most vulnerable activity, with a half-maximal inhibitory concentration of 8.12 micromolar. Kinetic experiments that varied both substrate and inhibitor concentrations pointed to competitive inhibition, with an apparent inhibition constant of 4.97 micromolar—the lowest Ki recorded among the human activities examined. CYP2E1 was the next most susceptible, showing an IC50 of 12.67 micromolar and a mixed inhibition pattern, with apparent Ki and alpha-Ki values of 19.59 and 8.74 micromolar respectively. By contrast, CYP1A2 and CYP2B6 were only weakly inhibited, with IC50 values near 50 micromolar, and CYP2D6 and CYP3A4 were not inhibited by half even at the highest concentration tested.

The rat microsomes told a different story. There, CYP1A2 was the most strongly inhibited activity, with an IC50 of 12.03 micromolar, followed by CYP2E1 at 18.50 micromolar. Follow-up kinetic analysis identified mixed inhibition of rat CYP1A2, with an apparent Ki of 12.31 micromolar, and competitive inhibition of rat CYP2E1, with an apparent Ki of 19.89 micromolar. Notably, rat CYP2C11—the nominal counterpart of human CYP2C9—was considerably less sensitive, with an IC50 of 33.39 micromolar. The authors are careful to note that CYP2C11 is not a one-to-one equivalent of human CYP2C9, and that CYP abundance differs between the two species, so the rat profile should be used to design animal studies rather than as a quantitative surrogate for human risk.

One of the more consequential findings concerns what the study did not detect. Time-dependent inhibition experiments, in which microsomes were preincubated with juglone for up to twenty minutes before the probe reaction was started, showed no consistent progressive loss of enzyme activity for any of the selected CYPs. That argues against mechanism-based inactivation under the tested conditions, though the authors acknowledge important caveats: the preincubation window was short, the concentration range limited, and—critically—no dedicated positive control for time-dependent inhibition was included. Definitive conclusions about reversibility, and any measurement of the inactivation parameters kinact and KI, will require follow-up experiments with appropriate controls and longer preincubation periods.

The species comparison around CYP2E1 is particularly instructive. The apparent Ki values in human and rat microsomes were numerically almost identical—19.59 versus 19.89 micromolar—yet the fitted inhibition models differed, with mixed inhibition in the human system and competitive inhibition in the rat system. The authors resist the temptation to read this as a genuine species difference. The two assays used different microsomal protein concentrations and buffer compositions, factors known to alter apparent inhibitory potency and catalytic activity. Without matched conditions and verified reaction linearity, they write, similar Ki values cannot be taken as evidence of equivalent intrinsic susceptibility, and the observed model differences cannot be attributed solely to species.

To add structural context, the team docked juglone into crystal structures of human CYP2C9 and CYP2E1. The docking produced GlideScores of −6.758 and −7.502 kilocalories per mole respectively, and the top-ranked CYP2C9 pose placed the naphthoquinone ring close to the heme catalytic center, flanked by hydrophobic residues including Leu102, Ala103, Leu208, and Val237, with a predicted hydrogen bond between a juglone carbonyl and Arg108. Interestingly, the docking scores did not reproduce the experimental potency ranking—CYP2E1 scored better yet was inhibited more weakly in human microsomes—a discrepancy the authors attribute to the limits of empirical scoring functions, which cannot be translated directly into inhibition constants and do not model substrate competition or the enzyme–substrate–inhibitor states relevant to mixed inhibition.

The translational question—could juglone-containing products actually perturb the metabolism of prescription drugs?—remains open, and the paper is unusually candid about why. Reliable human plasma data for unchanged juglone under relevant use conditions are simply unavailable. Preclinical evidence suggests partial absorption in rats, formulation-dependent half-lives, and more than 99 percent binding to serum albumin, but none of this establishes the unbound systemic concentration that would determine whether the micromolar inhibition seen in vitro is ever approached in vivo. The authors convert their key numbers for perspective: the CYP2C9 IC50 of 8.12 micromolar corresponds to roughly 1.41 micrograms per milliliter, and the Ki of 4.97 micromolar to about 0.866 micrograms per milliliter—nominal in vitro concentrations, not clinical thresholds. Measuring the unbound, unchanged compound in plasma is flagged as the essential next step.

The study also fits into a broader research program. The same laboratory previously showed that the related naphthoquinones plumbagin, shikonin, and deoxyshikonin inhibited all six tested human CYP activities, whereas juglone’s effects concentrated on CYP2C9 and CYP2E1. That contrast underscores a point the authors emphasize repeatedly: structural relatedness alone does not define a shared inhibition profile, and each compound must be assessed on its own. It also matters clinically, because CYP2C9 clears drugs with narrow therapeutic windows, and inhibitors of this enzyme have well-documented potential to raise exposure to anticoagulants and anticonvulsants.

The authors are equally transparent about the study’s statistical and analytical limits. All replicates were technical incubation replicates within a single pooled microsomal preparation, so the reported variability says nothing about inter-individual differences in enzyme activity. The apparent Ki and alpha-Ki values were derived from Lineweaver–Burk plots and secondary replots, now accompanied by 95 percent confidence intervals, and were cross-checked with global nonlinear regression and the corrected Akaike information criterion. For human CYP2C9, the lowest-AICc competitive model agreed with the graphical analysis, yielding a Ki of 5.11 micromolar versus 4.97 micromolar from the replots. For human CYP2E1, the mixed model was only modestly favored over a pure noncompetitive alternative, and some alpha-Ki estimates—particularly for the rat enzymes—were poorly constrained. Comprehensive analytical validation of the LC-MS/MS method under the study’s exact conditions was not performed, leaving quantitative bias incompletely characterized.

Where does this leave juglone? The compound, enriched in Pericarpium Juglandis—the green walnut husk used in Chinese medicine as Qinglongyi—has attracted attention as a scaffold for derivatives targeting cancer, inflammation, and bacterial quorum sensing. This study does not sound an alarm for consumers, but it does something arguably more useful: it converts a vague concern about natural product–drug interactions into a concrete, testable hypothesis centered on CYP2C9. The recommended roadmap includes recombinant human CYP2C9 experiments, measurements of plasma and microsomal protein binding, metabolite identification to check whether juglone’s breakdown products also inhibit CYPs, and in vivo pharmacokinetic studies that quantify unchanged juglone under defined dosing and formulation conditions. Only with that exposure data, combined with binding-adjusted inhibition estimates and knowledge of how much of a co-administered drug’s clearance depends on CYP2C9, can a defensible prediction of interaction risk be made. For now, the message is measured: juglone is a potent CYP2C9 inhibitor in a test tube, and that fact alone makes it a compound worth watching.

Subject of Research: In vitro inhibition of cytochrome P450 enzymes by the natural naphthoquinone juglone in human and rat liver microsomes

Article Title: Inhibition of cytochrome P450 activities by juglone in human and rat liver microsomes: kinetic characterization and implications for natural product–drug interaction assessment

Article References: Zhao, L., Shen, Y., Xu, X., Liu, L., Huang, J., Liang, C., Zhang, Y., & Wang, X. (2026). Inhibition of cytochrome P450 activities by juglone in human and rat liver microsomes: kinetic characterization and implications for natural product–drug interaction assessment. Frontiers in Pharmacology, 17, Article 1968844. https://doi.org/10.3389/fphar.2026.1968844

Image Credits: AI Generated

DOI: 10.3389/fphar.2026.1968844

Keywords: juglone, cytochrome P450, CYP2C9, CYP2E1, liver microsomes, drug metabolism, enzyme kinetics, natural product-drug interactions, naphthoquinone, pharmacokinetics, molecular docking, time-dependent inhibition

Betalains are water-soluble, nitrogen-containing pigments responsible for the yellow-orange to red-violet hues in most species of the plant order Caryophyllales, which includes beetroot, quinoa, and prickly pear. Beyond their visual role, these compounds attract attention for their antioxidant, anti-inflammatory, chemopreventive, and neuroprotective properties, fueling a search for new natural sources and better production methods. Two classes exist: yellow betaxanthins and violet betacyanins, both built around a shared core molecule called betalamic acid. The enzyme that creates this core is 4,5-DOPA-extradiol-dioxygenase, known as DODA, which cleaves the amino acid L-DOPA in a way that lets betalamic acid form spontaneously before condensing with amines or amino acids to make betaxanthins, or with cyclo-DOPA to make betacyanins.

Until recently, betalain biosynthesis was documented almost exclusively in Caryophyllales plants, with scattered reports in a handful of fungi such as Amanita, Hygrocybe, and Hygrophorus, in bacteria including Gluconacetobacter diazotrophicus and Escherichia coli, in the cyanobacterium Anabaena cylindrica, and, strikingly, in the tardigrade Hypsibius henanensis. Neolecta irregularis occupies a uniquely basal position within the Ascomycota, the phylum that contains most known fungi. As one of the very few surviving members of the subphylum Taphrinomycotina and the only genus in the Neolectomycetes, it retains morphological and genomic features considered ancestral, sitting in an intermediate zone between filamentous fungi and yeasts. Its rarity and the inability to cultivate it have long blocked functional studies of its physiology.

The research team, working with two specimens preserved at the Denver Botanic Gardens fungarium, first extracted pigments from the yellow tongue-shaped fruiting bodies and analyzed them by high-performance liquid chromatography coupled to mass spectrometry. They screened for a full panel of fungal betalains, including muscaflavin and numerous amino-acid-derived betaxanthins, but none were detected. Instead, the main pigment turned out to be riboflavin, vitamin B2, identified by its retention time and exact mass of m/z 377.1464, matching the standard at m/z 377.1456. Fluorescence imaging under blue excitation confirmed the assignment, revealing the characteristic yellow-green glow of riboflavin. This finding matters because riboflavin production is a hallmark of certain fungi and bacteria, where it protects spores from ultraviolet radiation and supports flavin cofactor synthesis and redox defense.

The pigment analysis created a puzzle. RNA sequencing, performed for the first time on this species, showed that the fungus actively expresses the gene encoding a DODA enzyme, along with a cytochrome P450, yet the pathway appears incomplete. The researchers found no expression of prephenate dehydrogenase or tyrosine aminotransferase, the enzymes needed to make L-tyrosine de novo, which is the precursor to L-DOPA. Without this substrate, the betalain machinery cannot run in vivo, explaining why the fungus stays yellow from riboflavin rather than from betalains. The transcriptomic data, deposited in the NCBI Gene Expression Omnibus under accession GSE300630, revealed 24 genes related to riboflavin metabolism, underscoring the pigment’s physiological importance in this lineage.

To test whether the encoded enzyme actually works, the team cloned the NiDODA gene, optimized its sequence for expression in E. coli, and purified the resulting protein using cobalt affinity chromatography. The enzyme emerged as a monomer of 33.4 kilodaltons, confirmed by gel filtration and mass spectrometry. Structural modeling with AlphaFold showed a compact fold resembling the only crystallized betalamic-acid-forming protein, YgiD from E. coli, with which NiDODA shares just 25.2 percent sequence identity. Three conserved histidine residues, His10, His48, and His230, coordinate a metal ion at the active site, mirroring the arrangement in the bacterial enzyme. Inductively coupled plasma mass spectrometry identified iron as the likely cofactor, and supplementing purification buffers with iron boosted activity up to 8.5-fold.

Kinetic characterization revealed remarkable properties. The enzyme operates optimally at pH 6.0, notably more acidic than the pH 8.0 to 8.5 optima reported for DODAs from Amanita muscaria, beetroot, and E. coli. Most strikingly, NiDODA binds L-DOPA with a Km of 0.8 millimolar, the highest affinity ever recorded for any DODA enzyme. For comparison, the Amanita enzyme has a Km of 4.2 millimolar, beetroot 6.9, quinoa 1.5, and the cyanobacterial version a staggering 53,000 millimolar. This makes NiDODA the most efficient L-DOPA converter among all known betalain-forming dioxygenases, capable of producing betalamic acid, muscaflavin, and dopaxanthin from the same substrate.

Phylogenetic analysis placed NiDODA within a distinct fungal branch separate from plant, bacterial, and animal sequences. Ascomycota sequences clustered together, while the tardigrade enzyme branched off before the radiation of plants, fungi, and microorganisms, acting as an outgroup. The team found that Ascomycota genomes contain numerous uncharacterized proteins with similarity to NiDODA, all retaining the conserved histidine triad, suggesting that more fungal betalain-producing enzymes await discovery. This pattern supports convergent evolution, where similar catalytic solutions arise independently in distant lineages, a phenomenon already documented within Caryophyllales plants, where DODA activity emerged at least three times through gene duplication and neofunctionalization.

The broader implication is that betalain biosynthesis may have far deeper roots than previously appreciated. Rather than being a plant-specific innovation, the capacity to cleave L-DOPA into betalamic acid appears scattered across the tree of life, from fungi and bacteria to cyanobacteria and animals. Whether these enzymes represent an ancestral metabolic capacity predating eukaryotic diversification, or the result of horizontal gene transfer and convergent evolution, remains unresolved. The biological role of betalain-producing enzymes in non-plant organisms is also unknown, and the possibility that they serve functions beyond pigment formation, particularly in species that never make betalains, adds another layer of mystery.

For Neolecta irregularis itself, the study provides the first transcriptomic analysis of a species that has resisted cultivation and remains scarce in nature. It confirms that the fungus carries the genetic framework for betalain synthesis but cannot execute the pathway because it lacks the ability to produce L-tyrosine de novo. The identification of NiDODA as the second fungal betalain-producing enzyme characterized to date, and the first monomeric one, expands the known phylogenetic scope of the pathway and opens the door to exploring whether other Ascomycota species harbor similar dormant capacities. As researchers continue to probe these ancient lineages, enzymes like NiDODA may reveal how metabolic innovations emerge, persist, or vanish across evolutionary time, offering a rare glimpse into the biochemical toolkit of early fungal life.

Subject of Research: Betalain biosynthesis enzyme evolution in the early-diverging fungus Neolecta irregularis

Article Title: A functional betalain-producing dioxygenase in Neolecta irregularis reveals an expanded evolutionary origin of the pathway

Article References: Martínez-Rodríguez, P., Guerrero-Rubio, M. A., Henarejos-Escudero, P., Hernández-García, S., & Gandía-Herrero, F. (2026). A functional betalain-producing dioxygenase in Neolecta irregularis reveals an expanded evolutionary origin of the pathway. IMA Fungus, 17, Article e184549. https://doi.org/10.3897/imafungus.17.184549

Image Credits: AI Generated

DOI: 10.3897/imafungus.17.184549

Keywords: betalains, Neolecta irregularis, DODA enzyme, Ascomycota, riboflavin, L-DOPA dioxygenase, betalamic acid, convergent evolution, Taphrinomycotina, fungal pigments, heterologous expression, enzyme kinetics

The new study, published in Frontiers in Chemical Biology, combines enzyme kinetics, thermal stability measurements, analytical ultracentrifugation, and X-ray crystallography into a systematic portrait of the seven disease-associated variants: P157S, T186R, G375del, N379D, G423S, Q435P, and P499A. The findings reveal that there is no single way to break SHMT2. Instead, the mutations attack different layers of the enzyme’s architecture, from its four-subunit assembly to the precise positioning of its vitamin B6-derived cofactor, and even the delicate balance between structural rigidity and the conformational flexibility that catalysis demands.

SHMT2 belongs to a large family of pyridoxal 5′-phosphate, or PLP, dependent enzymes. Its canonical job is to catalyze the reversible transfer of a one-carbon unit from serine to tetrahydrofolate, producing glycine and methylenetetrahydrofolate, a versatile carrier that feeds the synthesis of purines and thymidine, the metabolism of amino acids and neurotransmitters, and the maintenance of redox balance in mitochondria. But SHMT2 is no one-trick enzyme. Eukaryotic SHMTs are tetrameric proteins built as dimers of dimers, and this higher-order assembly underpins a suite of moonlighting functions: the enzyme can bind RNA and be regulated by it, participate in inflammatory cytokine signaling through its interaction with the BRISC complex, and even engage with KIRREL3, a synaptic cell-adhesion protein whose own defects are tied to neurological and cognitive disorders.

The enzyme’s conformational landscape is central to all of this. Without PLP bound, the active-site region is largely unstructured, and the dimeric building blocks can adopt either an open, catalytically inert conformation or a closed one. Only closed dimers can assemble into the active tetramer. PLP binding drives a disorder-to-order transition that stabilizes the closed state and, with it, the tetrameric assembly, which is cemented by an extensive network of intersubunit contacts centered on a histidine residue, His158, at the dimer-dimer interface. Mutations that destabilize either interface push the equilibrium back toward the open dimer, which in turn lowers the enzyme’s grip on its cofactor. It is precisely this equilibrium that the disease variants perturb.

When the Roman team expressed and purified the mutant proteins, one variant immediately stood out. Q435P, which replaces a glutamine with a rigid proline in a loop connecting two alpha helices, proved poorly soluble and prone to aggregation, and had to be excluded from further biochemical analysis. The researchers suspect that the proline insertion disrupts a crucial hydrogen bond between Q435 and the backbone of W43 in the partner subunit, destabilizing the dimeric interface and promoting misfolding. The remaining six variants all folded and could be studied, but they fell into three functional classes: P499A remained essentially fully active at about 95 percent of wild-type activity; G375del, N379D, and P157S were mildly impaired, retaining roughly 45 to 65 percent activity; and G423S and T186R were severely compromised, with G423S keeping only about 20 percent activity and T186R showing no measurable catalytic function at all, even at tenfold higher enzyme concentrations.

The structural basis of the two most damaged variants emerged from crystallography. T186R, which swaps a threonine for a bulky, positively charged arginine at the dimeric interface, elutes mainly as a dimer in solution and retains only about a third of the PLP-associated signal seen in the wild-type enzyme. Its cofactor affinity is roughly 42-fold worse than wild-type, with a dissociation constant of 5.50 micromolar versus 0.13 micromolar. In the crystal, the oversized arginine side chain creates steric hindrance that unfolds the entire PLP-interacting loop, residues 165 through 186, leaving the cofactor invisible in the electron density and the active-site surroundings in disarray. The tetramer that does form is a distorted assembly of two semi-open dimers, held together by only half of the normal interface. P157S, located at the tetrameric interface, tells a subtler story. Its mutation flips the carboxyl group of the neighboring His158, dismantling the aromatic stacking and hydrogen-bond cluster that anchors the tetramer, and shifts an adjacent alpha helix by about 1.2 angstroms, enough to force the PLP-binding loop to unfold. Remarkably, the cofactor itself was caught in a never-before-seen relaxed conformation, its pyridine ring tilted by 90 degrees and its phosphate group flipped to the opposite side, where it occupies a position normally reserved for stabilizing the substrate-bound intermediate. The team interprets this as an auto-inhibited state, and notes that such a cofactor tilt had only recently been glimpsed in a cryo-EM structure of a soybean SHMT isoform.

Perhaps the most paradoxical finding comes from G423S, a mutation lying some 20 angstroms away from the PLP-binding region. Despite losing 80 percent of its catalytic activity, this variant is more thermally stable than the wild-type enzyme, with an apparent melting temperature 4 degrees Celsius higher and a folding free energy more favorable by 1.71 kilocalories per mole. The crystal structure, solved at 1.8 angstroms, explains the contradiction: the serine side chain clashes with cysteine 412, nudging its backbone and triggering a domino effect that disorders the folate-binding loop, residues 413 through 420, at the mouth of the active site. In the native enzyme, this loop appears to sit in a frustrated, energetically suboptimal conformation, a common feature of protein regions dedicated to binding and catalysis. By relaxing that frustration, the mutation buys stability at the cost of function, a vivid illustration of the evolutionary trade-off between a protein’s fold and its job. With the loop disordered, the active site becomes more solvent-exposed, PLP affinity drops elevenfold, and tetrahydrofolate can no longer be positioned correctly, crippling the reaction in both directions.

The remaining variants cluster in the enzyme’s C-terminal domain, a region whose dynamics govern allosteric regulation and RNA binding in SHMT enzymes. N379D buries a negative charge in a packed hydrophobic pocket, perturbing a hydrogen-bonding network built around two histidines near the active site, yet the overall damage is mild. G375del shortens a surface loop and breaks the interaction between the carbonyl oxygen of S374 and R230, distorting the geometry of the active-site cavity in much the same way. P499A is the strangest of all: fully active and stable, yet with an eightfold reduction in PLP affinity. Because the mutant does not shed its cofactor during chromatography the way P157S does, the authors propose that its weakened binding reflects a slower association rate, likely caused by restricted access to the active site, rather than faster dissociation, a hypothesis that will require further experiments to confirm.

Beyond cataloguing molecular defects, the study delivers a broader message about how disease emerges from protein architecture. Rather than converging on a single failure mode, the seven SHMT2 variants impair the enzyme through distinct mechanisms: broken oligomerization, destabilized active-site loops, weakened cofactor binding, disrupted folate positioning, and outright misfolding. The work also provides the first direct crystallographic visualization of a frustrated cofactor conformation in SHMT, reinforcing the idea that the enzyme’s catalytic power depends on holding PLP in a precisely tuned, energetically strained position. As clinical sequencing continues to surface new SHMT2 variants in patients, this structural framework offers a rational starting point for interpreting each mutation’s pathogenic potential, and underscores how subtle perturbations of a protein’s conformational landscape can cascade into profound developmental disease.

Subject of Research: Structural and biochemical characterization of SHMT2 genetic variants linked to a neurodevelopmental syndrome

Article Title: Structural and functional defects of mitochondrial serine hydroxymethyltransferase genetic variants responsible for a novel neurodevelopmental syndrome

Article References: Boumis, G., Breccia, S., Pistoia, G., Spizzichino, S., Zamparelli, C., Troilo, F., Di Matteo, A., Travaglini Allocatelli, C., Contestabile, R., Tramonti, A., Cutruzzolà, F., & Giardina, G. (2026). Structural and functional defects of mitochondrial serine hydroxymethyltransferase genetic variants responsible for a novel neurodevelopmental syndrome. Frontiers in Chemical Biology, 5, Article 1941005. https://doi.org/10.3389/fchbi.2026.1941005

Image Credits: AI Generated

DOI: 10.3389/fchbi.2026.1941005

Keywords: SHMT2, one-carbon metabolism, pyridoxal phosphate, mitochondria, neurodevelopmental syndrome, X-ray crystallography, protein oligomerization, enzyme kinetics, conformational frustration, genetic variants, tetrahydrofolate, structural biology

Mycophenolic acid, usually given as the prodrug mycophenolate mofetil, is a cornerstone of immunosuppressive therapy for kidney, heart, lung and liver transplant recipients. It works by inhibiting inosine monophosphate dehydrogenase, blocking the de novo synthesis of guanosine nucleotides that activated lymphocytes need to proliferate. But the drug is notorious for its pharmacokinetic variability. Blood concentrations swing widely between patients and even within the same patient over time, driven by factors ranging from age, sex and albumin levels to co-administered drugs and comorbidities. Overexposure raises the risk of adverse effects; underexposure raises the risk of acute organ rejection. Identifying previously unrecognized factors that shape this variability is therefore a genuine clinical priority.

The liver disposes of mycophenolic acid through glucuronidation, a reaction catalyzed by UDP-glucuronosyltransferase enzymes that attach glucuronic acid to the drug. The dominant product, MPA glucuronide or MPAG, is pharmacologically inactive and represents the main clearance route. A much smaller fraction is converted to the acyl glucuronide, AcMPAG, which is the problem child: it can stimulate the release of pro-inflammatory cytokines, forms protein adducts in rat liver and colon tissue, and has been linked to the gastrointestinal and hematological toxicities seen with mycophenolate therapy. In humans the two reactions are handled mainly by UGT1A9 and UGT2B7 respectively; in rats, the acyl pathway is attributed largely to UGT2B1, the functional ortholog of the human enzyme.

Music, meanwhile, has well-documented physiological reach. Tempo, rhythm and harmony have been associated in humans with shifts in sympathetic and parasympathetic activity, heart rate, blood pressure, cognition and mood. In rodents, exposure to music has been reported to alter neurochemistry, corticosterone, prolactin, gastric emptying, immune modulators and even sex hormone production. Because those hormones and inflammatory mediators intersect with the same enzyme systems that metabolize xenobiotics, the Alberta team hypothesized that music could act as an extrinsic modifier of drug metabolism, and set out to test the idea systematically rather than anecdotally.

The experimental design was unusually rigorous for a music study. Student composers wrote original pieces in which tempo (fast or slow), rhythm (regular or irregular) and later harmony (tonal or atonal) were varied independently, with the percentages of continuous legato and detached staccato articulation held constant during the initial screening. Groups of eight rats, four of each sex, were exposed for 24 hours in a sound-monitored room at levels between roughly 60 and 80 decibels, played on a loop through a digital speaker. Livers were then removed, microsomes isolated, and the formation of MPAG and AcMPAG quantified under initial velocity conditions using liquid chromatography-tandem mass spectrometry, with enzyme kinetics fitted to the Michaelis-Menten model.

The headline finding concerned the acyl metabolite. Fast tempo combined with irregular rhythm reduced AcMPAG formation by 42.5 percent on average compared with silence, and adding atonal harmony deepened the suppression to 74.4 percent, an effect significant in both sexes. Kinetic analysis showed the mechanism was a collapse in catalytic capacity rather than a change in enzyme affinity: for AcMPAG formation, maximum velocity fell by 89.7 percent and intrinsic clearance by 91.5 percent, while the Km, the substrate concentration at half-maximal rate, was unchanged. Crucially, MPAG formation was unaffected by any music combination, with Vmax and intrinsic clearance comparable to controls and only a modest rise in Km, confirming that the major clearance pathway was preserved. Niflumic acid, a positive-control inhibitor, cut MPAG formation by about 67 percent, demonstrating that the assay could have detected such an effect.

What was happening inside the enzyme? The answer, intriguingly, was not altered expression. Real-time quantitative PCR showed no significant changes in hepatic Ugt2b1, Ugt1a1, Ugt1a5, Ugt1a6, Ugt1a7 or Ugt2b12 mRNA after exposure to the fast-tempo, irregular-rhythm, atonal-harmony music, and an ELISA confirmed that UGT2B1 protein levels in the microsomes were essentially identical to controls. That points the investigators toward other explanations: direct or indirect enzyme inhibition, post-translational modifications such as phosphorylation or acylation, or competition at the active site from endogenous substrates. Music is known to shift rodent sex hormones, and testosterone and estradiol are themselves UGT2B1 substrates, so hormonally driven competition for enzyme binding is one plausible mediator the team intends to test by measuring circulating hormones during exposure.

Perhaps the most curious result was that the effect depended on who composed the music. Pieces with the same fast-tempo, irregular-rhythm, atonal-harmony recipe from two of three student composers reduced AcMPAG formation by roughly 73 to 78 percent, while the third composer’s piece produced only a small, non-significant drop. Among published works, Arnold Schoenberg’s Violin Concerto Op. 36 reduced AcMPAG formation by 32 percent, whereas John Cage’s First Construction (In Metal) had no significant effect. Spectrogram analysis offered a clue: the effective compositions were overwhelmingly legato, with smooth, connected notes, while the weaker ones were dominated by staccato, with short, detached notes and silent gaps. Since staccato music is associated with altered respiratory patterns and higher skin conductance, while legato music tends to lower heart rate and blood pressure, the researchers speculate that articulation may act through autonomic nervous system changes that influence hepatic perfusion or enzyme efficiency.

The authors are careful about scope. The study used a single 24-hour exposure, an in vitro microsomal system that cannot capture drug transporters such as OAT3 or MRP2, and a modest sample size for the sex-stratified analyses, which showed no significant male-female differences. Whether the effect translates to living animals, to humans, or to actual clinical outcomes such as neutropenia or graft rejection remains untested; the team proposes in vivo pharmacokinetic studies with frequent sampling and biomarker monitoring, and eventually preclinical transplant models. Still, the concept is tantalizing: if a precisely defined acoustic environment could selectively dampen formation of a drug’s toxic metabolite without touching its therapeutic clearance, music would graduate from adjunctive comfort therapy to a genuine pharmacokinetic intervention. For now, the study stands as the first quantified demonstration that specific musical elements can reshape the metabolism of a critical immunosuppressant, and as a vivid reminder that the boundaries between sound, physiology and pharmacology are far blurrier than convention assumes.

Subject of Research: Effects of music elements on mycophenolic acid glucuronidation in rat liver microsomes

Article Title: Effects of music elements on mycophenolate glucuronidation in rat liver microsomes

Article References: Adhiya, J., Shalaby, A. H., Al-Dajani, A. R., El-Mahrouk, S. R., Olympus, C., Ahonen, H., El-Kadi, A. O. S., & Kiang, T. K. L. (2026). Effects of music elements on mycophenolate glucuronidation in rat liver microsomes. Journal of Pharmacy & Pharmaceutical Sciences, 29, Article 17260. https://doi.org/10.3389/jpps.2026.17260

Image Credits: AI Generated

DOI: 10.3389/jpps.2026.17260

Keywords: mycophenolic acid, glucuronidation, UGT2B1, music therapy, enzyme kinetics, transplantation, immunosuppressants, rat liver microsomes, tempo, rhythm, harmony, pharmacokinetics

Alpha-amylases are among the most heavily used enzymes on the planet. These catalysts chop long starch molecules into shorter sugars, a deceptively simple reaction that underpins the production of glucose syrups, high-fructose sweeteners, ethanol, laundry detergents, paper coatings, and textiles. The trouble is that many industrial processes run at temperatures and pH values that would denature most proteins within minutes. Starch gelatinization, for example, typically requires heating starch slurries well above the boiling point of water, forcing manufacturers to either cool their process streams before adding enzymes or pay a premium for thermostable variants. Enzymes that naturally thrive under these brutal conditions are therefore worth their weight in gold, and the search for new ones has increasingly turned to the most extreme environments Earth has to offer.

That is precisely where the Turkish team went looking. The researchers collected samples from four geothermal springs, environments where thermophilic bacteria have evolved proteins with unusual structural rigidity, and screened the isolates for their ability to secrete alpha-amylase into the surrounding medium. One isolate, designated SO7, stood out. Through a combination of phenotypic testing, biochemical profiling, enzymatic assays, and sequencing of the 16S rRNA gene, the standard molecular barcode for bacterial identification, the team confirmed that SO7 belonged to Bacillus licheniformis, a species with a long and distinguished history in industrial biotechnology. The identification was methodical rather than dramatic, but it laid the groundwork for everything that followed: a rigorous biochemical dissection of the enzyme the bacterium produces.

Purifying enzymes from crude bacterial cultures is traditionally a laborious, multi-step affair involving ammonium sulfate precipitation, ion-exchange chromatography, and gel filtration, each step bleeding away precious protein. The researchers compared several purification strategies, including starch affinity purification, precipitation with ethanol and acetone, and a technique called three-phase partitioning, or TPP. The comparison produced a striking result. TPP, which uses t-butanol and ammonium sulfate to drive proteins into a concentrated interfacial layer between the aqueous and organic phases, delivered a 71-fold purification with an extraordinary 98.6 percent recovery of enzymatic activity. The aqueous phase after TPP treatment showed an activity of 0.1482 units per milliliter and a specific activity of 592.8 units per milligram, figures that compare favorably with published amylase purifications that required far more handling and suffered far greater losses.

The elegance of TPP matters beyond mere laboratory convenience. Because the method concentrates and purifies the enzyme in a single operation, it dramatically reduces the cost and complexity of scaling up production, a persistent bottleneck in translating laboratory enzyme discoveries into commercial products. For an enzyme intended for industrial use, where kilograms rather than milligrams are needed, a purification scheme that recovers nearly all activity in one step is not just a technical nicety; it is the difference between a publishable curiosity and a viable product candidate. The Turkish team’s results suggest that SO7’s amylase could clear that hurdle more easily than most of its competitors.

Once purified, the enzyme revealed its true character. Sodium dodecyl sulfate polyacrylamide gel electrophoresis pinned its apparent molecular mass at approximately 83.17 kilodaltons, consistent with the size range of bacterial alpha-amylases. More importantly, the enzyme’s activity peaked at pH 7.0 and a scorching 90 degrees Celsius, a temperature at which most ordinary proteins would have long since unfolded into useless tangles. The enzyme also retained substantial activity after being pre-incubated at alkaline pH values and elevated temperatures, indicating that its thermal and pH stability is not merely a transient optimum but a durable property of the protein’s folded structure. This combination of near-neutral pH optimum with alkaline tolerance and extreme heat resistance is exactly the profile that starch liquefaction processes demand.

The enzyme’s resilience extended into chemical territory that typically destroys catalytic proteins. After short-term exposure to selected surfactants, inhibitors, metal ions, and organic solvents, the amylase retained meaningful activity, a property that earned it the label of organic solvent-stable. Solvent tolerance is rare among amylases and highly prized, because many industrial reactions and product formulations involve organic compounds that would strip water from a protein’s surface and collapse its structure. Enzymes that can operate in the presence of solvents open the door to novel biocatalytic processes, including the synthesis of chemicals in non-aqueous media and the processing of solvent-containing waste streams. The researchers were careful to note, however, that this tolerance was assessed only under short-term laboratory conditions, and that performance under real industrial process conditions remains to be validated, an honest caveat that distinguishes careful science from promotional hype.

Kinetic analysis added further depth to the enzyme’s profile. When tested against a panel of starch substrates, rice starch produced the highest relative activity, with a Michaelis constant of 3.24 milligrams per milliliter and a maximum velocity of 204.08 millimoles per milliliter per minute. The Km value indicates a reasonable affinity for the substrate, while the Vmax reflects a brisk catalytic rate under saturating conditions. All measurements were obtained from three independent experiments, giving the numbers a statistical footing that single-run assays often lack. Substrate preference matters in practice: starches from different botanical sources vary in granule structure, amylose content, and susceptibility to enzymatic attack, and an enzyme that performs well on rice starch may behave differently on corn, wheat, or potato starch, the workhorses of the industrial starch sector.

What makes this discovery resonate beyond the enzymology community is the broader story it tells about where biotechnology’s raw materials come from. Extremophiles, organisms that flourish in conditions lethal to most life, have become one of the richest sources of industrially useful enzymes, from DNA polymerases that power the polymerase chain reaction to proteases that clean clothes in hot washes. Every geothermal spring, deep-sea vent, and acidic hot pool sampled by microbiologists yields new candidates, and each one carries the evolutionary fingerprints of proteins stabilized by subtle changes in amino acid composition, salt bridges, and hydrophobic packing. The SO7 amylase is a fresh addition to this catalog, and its combination of thermostability, alkaline tolerance, and solvent resistance suggests that Bacillus licheniformis strains in Türkiye’s hot springs have solved, through billions of years of natural selection, engineering problems that synthetic protein designers still struggle with.

The path from a purified enzyme in a Turkish laboratory to an enzyme working inside a starch plant or detergent factory is long, and the authors themselves flag the limitations of their in vitro study. Scale-up of TPP purification, testing against industrially relevant substrate concentrations, long-term stability trials, and cost analysis all lie ahead. Yet the core achievement stands: a thermostable, solvent-tolerant alpha-amylase purified to near homogeneity with nearly complete activity recovery, characterized with quantitative rigor, and sourced from a bacterium that required nothing more than a trip to a hot spring to find. As industries push toward greener, enzyme-driven processes that replace harsh chemical treatments, discoveries like this one quietly expand the toolkit. The next time a glucose syrup or a biofuel ethanol batch is produced at 90 degrees Celsius in an alkaline reactor, the enzyme doing the work may well trace its ancestry to a steaming pool in the Anatolian countryside.

Subject of Research: Purification and biochemical characterization of a thermostable, organic solvent-stable alpha-amylase from Bacillus licheniformis SO7 isolated from geothermal springs in Türkiye

Article Title: Efficient purification and biochemical characterization of a thermostable and organic solvent-stable α-amylase from Bacillus licheniformis SO7

Article References: Sevinc, S., Akbulut, S., Aygun, E., Saoula, S., Sisecioglu, M., & Adiguzel, A. (2026). Efficient purification and biochemical characterization of a thermostable and organic solvent-stable α-amylase from Bacillus licheniformis SO7. 3 Biotech, 16(11), Article 460. https://doi.org/10.1007/s13205-026-05056-8

Image Credits: AI Generated

DOI: 10.1007/s13205-026-05056-8

Keywords: alpha-amylase, Bacillus licheniformis, thermostable enzyme, three-phase partitioning, enzyme purification, organic solvent tolerance, thermophilic bacteria, geothermal springs, biocatalysis, starch hydrolysis, enzyme kinetics, industrial biotechnology

The research, published in Angewandte Chemie International Edition, focused on phosphofructokinase-1, or PFKP, the platelet isoform of an enzyme that controls a pivotal and rate-limiting step in glycolysis. Glycolysis is the ancient metabolic pathway through which cells break down glucose to generate energy in the form of ATP and to produce molecular building blocks needed for growth and repair. Because PFKP sits at such a critical control point, even modest changes in its behavior can ripple through the entire metabolic network of a cell, altering how efficiently glucose is consumed and how energy production is balanced against biosynthesis.

Enzymes, however, never work in isolation. As Simon Ebbinghaus, one of the lead authors of the study, explains, the molecular environment surrounding an enzyme changes constantly. Cells experience stress, reorganize their metabolism in response to nutrient availability or temperature shifts, and form temporary assemblies in which enzymes cluster together with other biomolecules. These dynamic conditions raise a fundamental question that has remained open in biochemistry: is it mainly the limited physical space around an enzyme that determines how it functions, or is the chemical nature of the molecules sharing that space equally important? The Bochum team designed a series of experiments to disentangle these two effects for the first time in a rigorous, controlled way.

To do so, the researchers studied PFKP in solutions containing so-called crowding agents, molecules added deliberately to mimic the dense interior of a cell. Crucially, they selected crowders that varied widely in size and chemical identity, ranging from small sugar molecules such as glucose to large glucose polymers such as dextran. This design allowed the team to separate the purely physical effect of molecular crowding, in which available volume is reduced, from the chemical effects that arise from specific, weak interactions between the crowder and the enzyme itself. The team then measured how enzyme activity changed with temperature, assessed the structural stability of the protein, and ran molecular dynamics simulations to examine interactions that cannot be observed directly in experiments.

The first result was consistent across all tested environments: at lower temperatures, every crowded solution reduced PFKP activity. This outcome fits the classical picture of macromolecular crowding, in which reduced volume and increased viscosity slow the diffusion of substrates and constrain the conformational movements an enzyme needs to catalyze its reaction. If the story had ended there, it would have confirmed a familiar principle. But when the researchers raised the temperature, the results diverged in a way that no simple volume-exclusion model could explain.

At higher temperatures, a striking difference emerged. Sugar-based crowding molecules, including glucose and the far larger dextran polymer, helped PFKP retain its activity across a broader temperature range than other crowders did. The fact that glucose, a small molecule, and dextran, a polymer many times its size, produced similar protective effects was decisive. If molecular size alone were responsible for the behavior of crowded enzymes, these two agents should have behaved very differently. Instead, their shared chemical identity as sugars pointed to a different mechanism altogether, one rooted in what chemists call soft interactions.

Soft interactions are weak, reversible, non-covalent contacts among the surrounding molecules, the enzyme, and its substrates. Unlike the strong bonds that hold a protein’s structure together, soft interactions flicker on and off constantly, forming a transient chemical cloud around the enzyme. The molecular simulations performed by the team showed that sugar-based crowders can influence regions near the enzyme’s active site, the precisely shaped pocket where the catalytic reaction takes place. By doing so, they affected both the flexibility of the protein and the positioning of its substrates, two factors that together determine how efficiently and how reliably the enzyme can do its job.

This balance between flexibility and positioning lies at the heart of enzyme function. An enzyme must be supple enough to undergo the conformational changes required for catalysis, yet rigid enough to hold its substrates in the correct orientation long enough for the reaction to proceed. The study suggests that sugar-rich environments tune this delicate equilibrium, making PFKP more robust at elevated temperatures precisely when many proteins begin to lose their functional shape. Importantly, the enzyme is not simply protected from unfolding in a passive sense. Its dynamics and catalytic behavior are actively shaped by its local chemical surroundings, meaning the environment participates in defining the enzyme’s working properties rather than merely preserving them.

The implications reach well beyond the test tube. If the chemical composition of the molecules surrounding an enzyme can determine how it responds to thermal stress, then the cell’s interior may be far more chemically sophisticated than the traditional picture of crowding as a purely physical traffic jam suggests. As Ebbinghaus puts it, the chemical composition of the surrounding molecules may actively influence how metabolic enzymes respond to stress. During heat shock, nutrient deprivation, or metabolic reprogramming, the mix of sugars, polyols, and other small solutes inside a cell changes, and this study raises the possibility that such shifts are not incidental byproducts but part of a regulatory strategy that helps keep energy production running under difficult conditions.

Future research will need to determine whether living cells actually exploit these local chemical environments to preserve metabolic activity when challenged, a question the Bochum team identifies as the next step. If confirmed, the findings could reshape how scientists think about metabolic regulation, offering a layer of control that operates through the physical chemistry of the cellular interior rather than through genetic switches alone. The work may also inform biotechnology, where enzymes are increasingly deployed in industrial settings at high concentrations and elevated temperatures, and where engineering the chemical environment around a catalyst could prove as important as engineering the catalyst itself. For now, the study stands as a vivid reminder that in the crowded world of the cell, an enzyme’s performance depends not only on its own structure, but on the company it keeps.

Subject of Research: How soft chemical interactions with surrounding sugar molecules modulate the temperature-dependent activity of the glycolytic enzyme phosphofructokinase-1

Article Title: Robust enzymes

Article References: Robust enzymes. (n.d.). Original publication

Image Credits: AI Generated

DOI: Not provided

Keywords: enzymes, glycolysis, phosphofructokinase, molecular crowding, soft interactions, protein stability, metabolism, cellular stress, biophysics, sugar molecules, enzyme kinetics, molecular simulations

The enzyme, named BhEstB, was isolated and characterized by a team of researchers working at the University of Hamburg in Germany in collaboration with partners in Algeria and at Heinrich-Heine-University Düsseldorf and Forschungszentrum Jülich. Its source is Bacillus halotolerans, a salt-tolerant member of a bacterial genus famous for its metabolic versatility and resilience in harsh environments. Writing in the journal Applied Microbiology and Biotechnology, lead author Hadjira Bounabi and colleagues, including corresponding author Wolfgang R. Streit, describe BhEstB as a versatile carboxylesterase whose behavior defies easy categorization within the established families of PET-degrading enzymes. Structurally, it is closely related to an esterase from Bacillus subtilis, yet it diverges markedly from canonical PETases and from most well-characterized MHETases, including the MHETase of Ideonella sakaiensis. Instead, it clusters more closely with carboxylesterases such as TfCa, placing it in a distinct branch of the hydrolase family tree.

That phylogenetic distance turns out to matter enormously for function. When the team measured BhEstB’s activity across a panel of artificial substrates—p-nitrophenyl esters of varying chain lengths—the enzyme displayed broad esterase activity, with optimal performance at a mildly alkaline pH of 8 and at temperatures between 20 and 30 degrees Celsius. Those are gentle, ambient conditions, a striking contrast to the elevated temperatures often required by PET-active enzymes that must approach the polymer’s glass transition temperature to gain access to its chains. The modest thermal optimum initially suggested BhEstB might be a niche player, but the kinetic data told a very different story when the researchers turned to the soluble intermediates of PET breakdown.

Against BHET, BhEstB delivered results that place it among the most efficient BHET-hydrolyzing enzymes characterized to date. The enzyme achieved complete conversion of 50 millimolar BHET within just one hour at 30 degrees Celsius. The underlying kinetics are equally impressive: a turnover number, or kcat, of 15.57 plus or minus 1.25 per second, a Michaelis constant, or Km, of 6.54 plus or minus 1.01 millimolar, and a corresponding catalytic efficiency of 2.38 times ten to the third per molar per second. In practical terms, this means each enzyme molecule processes roughly fifteen BHET molecules every second under saturating conditions, with a substrate affinity that allows meaningful conversion even at moderate concentrations. For a bioprocess engineer, those numbers translate directly into smaller reactors, shorter residence times, and lower costs.

The enzyme’s performance on MHET, the other major soluble intermediate of PET hydrolysis, was nearly as striking. BhEstB converted 39.8 millimolar MHET into terephthalic acid with a yield of 77 percent, plus or minus 0.7 percent, within 24 hours. Terephthalic acid is the prize at the end of this enzymatic relay: it is the monomer from which virgin PET is made, and recovering it in pure form from waste plastic is the central goal of enzymatic recycling. By efficiently finishing the job that depolymerizing enzymes begin, BhEstB functions as a second-stage catalyst that could be paired with PETases or cutinases in a two-step biocatalytic cascade, converting plastic waste all the way back to its building blocks.

Perhaps the most telling comparison came from head-to-head benchmarking. BhEstB significantly outperformed not only the engineered LCC variant known as LCC ICCG—one of the most intensively optimized PET-depolymerizing enzymes in the field—but also PET46, a recently published archaeal feruloyl esterase that had itself drawn attention for its PET-intermediate activity. That a naturally occurring Bacillus enzyme, without the benefit of directed evolution or rational engineering, could surpass these heavily studied catalysts on BHET and MHET underscores how much unexplored enzymatic diversity remains hidden in soil microorganisms. It also validates the authors’ framing of BhEstB as a blueprint: a structural and mechanistic template from which future engineered variants could be derived.

The blueprint idea carries real weight because BhEstB’s sequence and structure differ from the canonical PETase fold that has dominated the literature since Ideonella sakaiensis was described in 2016. Carboxylesterases of this type appear to have evolved for hydrolyzing ordinary ester bonds in plant and soil chemistry, yet their active-site architecture accommodates the aromatic diesters of PET breakdown with unexpected ease. Understanding which structural features grant BhEstB its extraordinary BHET turnover—features that canonical PETases and MHETases lack—could guide protein engineers in designing hybrid enzymes that combine polymer attack with rapid intermediate processing, eliminating the accumulation of BHET and MHET that can limit the throughput of current enzymatic recycling schemes.

On the bulk polymer itself, the team was careful to keep expectations measured. BhEstB showed modest overall activity on commercial semi-crystalline PET powder, with some activity observed at elevated temperatures, but it is clearly not a polymer-depolymerizing specialist on the order of LCC or the best engineered PETases. The authors frame this honestly: BhEstB is a versatile BHET- and MHET-active carboxylesterase with modest activity toward the bulk polymer, contributing to the expanding diversity of PET-intermediate-degrading enzymes rather than replacing the depolymerization workhorses. In a multi-enzyme recycling process, that division of labor is not a weakness but a design principle—each catalyst doing what it does best under conditions that favor the overall reaction.

The broader significance of the work lies in what it reveals about the ecology and evolution of plastic-degrading chemistry. PET has existed for less than a century, far too short a time for enzymes to have evolved specifically for it, so the catalysts being discovered today are almost certainly repurposed from older metabolic functions such as cutin degradation, ferulate ester hydrolysis, or general ester scavenging. BhEstB, with its carboxylesterase scaffold and ambient-temperature optimum, fits this pattern perfectly and suggests that the search space for PET-intermediate enzymes extends well beyond the known PETase lineages into the vast and poorly sampled diversity of bacterial esterases. Metagenomic and activity-based screening of soil and marine microbiomes, the approach exemplified by the Hamburg group’s broader research program, is likely to yield many more such catalysts.

For a world drowning in PET waste—hundreds of millions of tonnes produced annually, with only a small fraction chemically recycled—enzymes like BhEstB represent a practical step toward genuine circularity. A complete enzymatic recycling process would combine a depolymerase that shreds the polymer into soluble fragments with an intermediate-processing enzyme that finishes the conversion to terephthalic acid and ethylene glycol, both of which can feed back into virgin-quality PET production. By demonstrating complete BHET conversion in an hour and efficient MHET turnover at 30 degrees Celsius, BhEstB shows that the second stage of that process can be fast, mild, and economical. The study, funded in part by Algeria’s Directorate-General for Scientific Research and Technological Development and Germany’s Federal Ministry of Education and Research, and published open access, adds a powerful new name to the growing roster of plastic-eating enzymes—and a reminder that some of biotechnology’s best tools are still waiting quietly in the soil.

Subject of Research: Enzymatic degradation of PET plastic intermediates by a Bacillus carboxylesterase

Article Title: A versatile Bacillus esterase reveals a carboxylesterase blueprint for PET intermediate degradation

Article References: Bounabi, H., Yahiaoui, B., Gasmi, M., Mechouche, M. S., Bäse, N., Gurschke, M., Molitor, R., Pérez-García, P., & Streit, W. R. (2026). A versatile Bacillus esterase reveals a carboxylesterase blueprint for PET intermediate degradation. Applied Microbiology and Biotechnology, 110(1), Article 304. https://doi.org/10.1007/s00253-026-14056-w

Image Credits: AI Generated

DOI: 10.1007/s00253-026-14056-w

Keywords: PET biodegradation, Bacillus halotolerans, carboxylesterase, BHET, MHET, terephthalic acid, enzyme kinetics, plastic recycling, biocatalysis, hydrolases, PETase, microbial enzymes

Acid phosphatases, classified under the enzyme commission number EC 3.1.3.2, are orthophosphoric monoester phosphohydrolases that hydrolyze phosphate esters and carry out transphosphorylation reactions in acidic environments. In plants, they are indispensable players in phosphate metabolism, signal transduction, nutrient mobilization during germination, and the acquisition of phosphorus when soils are depleted. Beyond plant physiology, these enzymes serve as clinical markers for human diseases, including prostate cancer and infertility, which gives any new insight into their active-site architecture a relevance that stretches far beyond the botanical world. Yet for many plant acid phosphatase isoenzymes, the structural basis of catalysis remains poorly understood, and Erythrina indica, a leguminous tree of medicinal and ecological importance, had never had its seed phosphatases characterized at this level of detail.

The investigative strategy rested on a classic but powerful technique: residue-specific chemical modification. The team treated purified AP-II with three targeted reagents, each designed to attack one class of amino acid. Dicyclohexylcarbodiimide, abbreviated DCHC, was deployed against carboxylate groups; N-bromosuccinimide, or NBS, against tryptophan; and phenylmethylsulfonyl fluoride, PMSF, against serine. The results were unambiguous. Modification with NBS at 5 millimolar concentration produced complete loss of enzymatic activity, PMSF treatment destroyed more than 90 percent of activity, and DCHC treatment caused a roughly 90 percent decline. Because each reagent is selective for a different residue type, the near-total inactivation pointed directly to the participation of tryptophan, serine, and carboxylate in the enzyme’s active site.

Crucially, the researchers then ran substrate protection experiments to confirm that these residues were not merely incidental casualties of the reagents. When 2 millimolar p-nitrophenyl phosphate, the enzyme’s model substrate, was added before the modification reactions, the loss of activity caused by all three modifying agents was significantly prevented. The substrate, in effect, occupied the active site and shielded the vulnerable residues from chemical attack, exactly as would be expected if those residues were genuinely part of the catalytic machinery rather than bystanders elsewhere on the protein. This protective effect provided strong evidence that all three residue classes are directly involved in the catalytic process.

Kinetic analysis of the partially inactivated enzyme added a subtle but important layer of interpretation. When AP-II was partially inactivated with NBS, PMSF, or DCHC, the maximum reaction velocity, Vmax, decreased while the Michaelis constant, Km, remained essentially unchanged. That dissociation is telling: Km reflects substrate binding affinity, while Vmax reflects the rate of catalytic turnover. A drop in Vmax with stable Km means the modified residues contribute primarily to the chemistry of catalysis rather than to substrate recognition. The enzyme could still grab its substrate, but it could no longer efficiently execute the hydrolysis reaction, implicating the three residue classes in the bond-breaking step itself.

The pH dependence of activity supplied independent confirmation of the carboxylate’s role. Measuring kinetic parameters across the pH range from 3 to 6 and plotting log(Vmax/Km) against pH yielded pKa values of 4.6 and 5.25 for AP-II, values squarely consistent with the ionization behavior of a carboxylate group participating in the reaction. Meanwhile, Lineweaver-Burk plots showed that Km for p-nitrophenyl phosphate stayed nearly constant across pH 3.0 to 6.0 while Vmax changed, demonstrating that pH alters the rate of reaction without altering substrate affinity. The stoichiometry of modification told a similar story of economy: although two tryptophan residues were modified by NBS, extrapolating the plot of residual activity against modified residues to zero activity revealed that only a single tryptophan is directly involved in catalysis. Likewise, a log-log plot of inactivation rate against PMSF concentration gave a slope of one, indicating a single serine residue participates, and the same slope-of-one analysis for DCHC indicated that binding of one molecule of reagent suffices to inactivate the enzyme.

Fluorescence spectroscopy illuminated the physical environment of the catalytic tryptophan. Excited at 280 and 295 nanometers, AP-II emitted with a maximum at 313 nanometers, a blue-shifted signature indicating that the fluorescent tryptophans are buried in the hydrophobic interior of the protein rather than exposed to solvent. Quenching experiments with three agents of different charge, neutral acrylamide, positively charged cesium chloride, and negatively charged potassium iodide, revealed an unusual microenvironment. Acrylamide quenched the fluorescence completely, and remarkably, cesium ions achieved full accessibility as well, while iodide quenched far less effectively. The researchers concluded that the tryptophan microenvironment is strongly negatively charged, which explains why the positively charged cesium ion penetrates so readily while the negatively charged iodide is repelled. Stern-Volmer plots for acrylamide were linear, characteristic of purely dynamic quenching, whereas the upward-curving plots for cesium chloride and potassium iodide indicated a sphere-of-action static component or two populations of tryptophan residues quenched at different rates.

Structural studies rounded out the picture. Thermal denaturation caused activity loss accompanied by a red shift of the fluorescence maximum from 313 to 333 nanometers, the classic signature of protein unfolding exposing buried tryptophans to water. Chemical denaturation with guanidine hydrochloride abolished activity at concentrations as low as 0.2 molar, further supporting the involvement of carboxylate residues in catalysis. Denatured enzyme titrated with NBS revealed five total tryptophans in AP-II, of which two are surface-exposed and three are buried in the core. Circular dichroism spectroscopy showed that AP-II is composed of roughly 45 percent alpha-helix, 23 percent beta-sheet, and 31 percent random coil, and a sixth-degree polynomial fit of the far-UV CD spectrum achieved a coefficient of determination of approximately 0.89, which the authors interpreted as indicating greater structural stability of the major form. In silico structure prediction went further, suggesting a homodimeric enzyme of 102 kilodaltons with about 5.2 percent N-linked glycosylation and conserved histidine, tryptophan, aspartate, and tyrosine residues, an architecture typical of large plant acid phosphatases.

The authors are candid about the limits of their approach. The identification of active-site residues rests primarily on chemical modification rather than high-resolution crystallography, the three-dimensional structure is computationally predicted and awaits experimental validation, and the entire characterization was performed with the artificial substrate p-nitrophenyl phosphate, which may not mirror the enzyme’s physiological substrate preferences. Future work, they suggest, should include site-directed mutagenesis of the identified residues and testing against natural substrates. Even so, the study delivers a coherent and technically rigorous account of how three amino acids, a carboxylate acting in acid-base catalysis, a buried tryptophan stabilizing the substrate, and a serine participating in phosphoryl transfer, cooperate to hydrolyze phosphate esters in the acidic interior of a seed. It is a reminder that even a tree growing on a residential street in Pune can harbor molecular machinery of elegant precision, and that careful classical biochemistry, bolstered by modern statistics and computation, can still resolve that machinery one residue at a time.

Subject of Research: Identification of the catalytic carboxylate, tryptophan, and serine residues in acid phosphatase form II from Erythrina indica seeds

Article Title: Identification of catalytic residues of acid phosphatase form II from Erythrina indica seeds

Article References: Uzgare, A. S., & Shaikh, F. M. R. (2026). Identification of catalytic residues of acid phosphatase form II from Erythrina indica seeds. Discover Chemistry, 3(1), Article 488. https://doi.org/10.1007/s44371-026-00948-3

Image Credits: AI Generated

DOI: 10.1007/s44371-026-00948-3

Keywords: acid phosphatase, Erythrina indica, active site, chemical modification, tryptophan, serine, carboxylate, enzyme kinetics, fluorescence spectroscopy, circular dichroism, plant biochemistry, catalytic mechanism

Biofilms are structured communities of bacteria encased in a self-produced matrix of proteins, extracellular DNA, and polysaccharides. Within these fortresses, pathogens become dramatically more tolerant of antibiotics and immune attack, which is why biofilms complicate the treatment of cystic fibrosis lungs, chronic wounds, medical implants, and catheter-associated infections. Pseudomonas aeruginosa and Staphylococcus aureus are flagship biofilm builders, and both appear on global lists of antibiotic-resistant priority pathogens. An enzyme that can chew through the proteinaceous scaffolding of a biofilm, or disrupt the physiology of the cells inside it, therefore represents a genuinely different therapeutic strategy: rather than killing bacteria outright, it dismantles the architecture that makes them so hard to eradicate.

Serratiopeptidase, sometimes marketed as serrapeptase, is an extracellular protease naturally secreted by Serratia marcescens. It has been used for decades, particularly in Japan and India, as an anti-inflammatory and anti-edema agent, and earlier studies have hinted that it can affect Pseudomonas physiology and biofilm integrity. What has been missing is a rigorous, quantitative characterization of a purified preparation, since crude enzyme mixtures make it impossible to attribute activity to a single protein. The new study addressed this gap with a four-step purification pipeline: precipitation, dialysis, ion-exchange chromatography, and gel filtration chromatography, which together yielded a 15.91-fold increase in purity, sufficient for reliable biochemical and kinetic analysis.

With the purified enzyme in hand, the team determined its molecular weight to be approximately 47 kilodaltons, consistent with the serralysin-like metalloproteases produced by Serratia species. The enzyme displayed broad substrate specificity but showed its highest affinity toward casein, a milk protein commonly used as a benchmark substrate in protease assays. Activity peaked at pH 7 and 40 degrees Celsius, close to physiological conditions, and the enzyme proved remarkably durable under these optima, retaining 89.2 percent and 82.4 percent of its activity, respectively, after 60 minutes of incubation. That combination of neutral pH preference and moderate temperature optimum is exactly what one would want in a therapeutic protein intended to function inside the human body.

The kinetic analysis revealed an enzyme of impressive catalytic power. The Michaelis constant, Km, was 0.034 millimolar, equivalent to 842 micrograms per milliliter, indicating tight substrate binding, while the maximum velocity, Vmax, reached 738.2 units per milliliter. The turnover number, kcat, came in at 55.3 per second, meaning each enzyme molecule processes more than 55 substrate molecules every second. Dividing kcat by Km yields a catalytic efficiency of 1.64 × 10⁶ per molar per second, a figure that places this serratiopeptidase firmly in the category of highly efficient catalysts. For a protein being considered as a drug candidate, such numbers matter: an efficient enzyme can deliver biological effects at lower doses, reducing cost and potential side effects.

Equally informative was the enzyme’s response to chemical modulators. Metal ions including zinc, cobalt, barium, and manganese enhanced activity, as did the non-ionic detergent Tween-20 and the reducing agent beta-mercaptoethanol. Conversely, EDTA, a chelator that strips away metal cofactors, and PMSF, a classic serine protease inhibitor, both shut the enzyme down. This dual sensitivity is diagnostic: it confirms that the SP6 enzyme is a serine-metalloprotease, a hybrid classification consistent with the serralysin family, in which a catalytic serine operates within a metal-dependent active site. Knowing this helps predict how the enzyme will behave in complex biological environments and informs strategies for stabilizing it in formulation.

Perhaps the most clinically relevant findings concern stability and resistance to degradation. The enzyme retained 61.31 percent of its activity after 60 days of storage at 4 degrees Celsius, a refrigerated shelf life that simplifies handling and distribution. More strikingly, it resisted digestion by trypsin and by human serum, with in vitro half-lives of 4 and 5 hours, respectively. Therapeutic proteins typically face rapid destruction by circulating proteases and serum components, so a bacterial enzyme that survives hours in serum-like conditions is unusual and valuable. The authors note that extending the half-lives of therapeutic proteins is a major focus of drug development, and intrinsic resistance to proteolysis gives this enzyme a head start.

The anti-biofilm experiments delivered the study’s headline result. Against Pseudomonas aeruginosa MTCC 2453, serratiopeptidase achieved a maximum biofilm inhibition of 41.65 percent at a concentration of 200 micrograms per milliliter, with an IC50 of 56.92 plus or minus 8 micrograms per milliliter. Against Staphylococcus aureus MTCC 1430, the maximum inhibition was 21.87 percent at the same dose, with an IC50 of 124.6 plus or minus 8 micrograms per milliliter. The authors are careful to define these IC50 values relative to the normalized response range, meaning they correspond to 50 percent of the observed inhibitory effect rather than absolute halving of biofilm mass, a methodological honesty that strengthens the credibility of the data. The stronger effect against Pseudomonas aligns with earlier reports that serratiopeptidase alters the physiology of Pseudomonas isolates from cystic fibrosis patients and disrupts Pseudomonas biofilms and functional amyloids.

What makes these numbers exciting is not that the enzyme obliterates biofilms on its own, but that partial disruption is often enough to restore the vulnerability of embedded bacteria. Anti-biofilm agents are increasingly viewed as adjuncts: by loosening or thinning the matrix, they allow antibiotics and immune cells to reach cells that would otherwise remain sheltered. A 41.65 percent reduction in Pseudomonas biofilm formation, achieved by a single enzyme at a modest concentration, could meaningfully amplify the efficacy of co-administered antimicrobials. The enzyme’s activity at neutral pH and 40 degrees Celsius, its serum stability, and its resistance to trypsin all support the plausibility of such combination approaches.

Significant hurdles remain before serratiopeptidase reaches the clinic as an anti-biofilm drug. The current work used a partially purified preparation, and further purification, scale-up of production, formulation studies, toxicity testing, and ultimately animal and clinical trials will be required. The enzyme’s origin from Serratia marcescens, an opportunistic pathogen itself, raises production and safety questions that recombinant expression systems, which have already been used to produce serratiopeptidase in cell-free platforms, could help resolve. Nevertheless, by systematically documenting the enzyme’s molecular weight, kinetics, inhibitor profile, storage stability, serum resistance, and anti-biofilm potency, Kumar and Bhattacharya have transformed a supplement-shelf enzyme into a well-characterized candidate for rational development. As antibiotic resistance continues to erode the effectiveness of conventional drugs, enzymes that dismantle bacterial fortresses rather than merely poisoning the bacteria inside them may prove to be one of the most important weapons in the next generation of anti-infective therapy.

Subject of Research: Biochemical characterization and anti-biofilm activity of serratiopeptidase from Serratia marcescens SP6

Article Title: Elucidation of the biochemical properties of a partially purified Serratia marcescens SP6 serratiopeptidase with anti-biofilm efficacy

Article References: Kumar, S., & Bhattacharya, S. (2026). Elucidation of the biochemical properties of a partially purified Serratia marcescens SP6 serratiopeptidase with anti-biofilm efficacy. 3 Biotech, 16(10), Article 433. https://doi.org/10.1007/s13205-026-05063-9

Image Credits: AI Generated

DOI: 10.1007/s13205-026-05063-9

Keywords: serratiopeptidase, Serratia marcescens, biofilm, Pseudomonas aeruginosa, Staphylococcus aureus, protease, enzyme kinetics, antibiotic resistance, metalloprotease, anti-biofilm therapy, protein purification, therapeutic enzymes

Cinnamyl acetate, an ester that carries warm, balsamic, cinnamon-like notes, is a defining contributor to the aroma bouquet of P. mume flowers. While previous work had traced parts of the scent pathway, including the production of cinnamyl alcohol by the enzyme PmCAD1 and the role of benzyl alcohol acetyltransferase genes in other scent compounds, the gene responsible for the final acetylation step that converts cinnamyl alcohol into cinnamyl acetate had never been pinpointed. The new study set out to find it by systematically mining the plant’s genome.

The researchers began with a genome-wide survey of the variety P. mume var. tortuosa, identifying 116 members of the acyltransferase gene family, a large group of enzymes known to attach acyl groups to alcohols and other acceptor molecules. Because floral scent genes typically show activity patterns that mirror the accumulation of their volatile products, the team then cross-referenced transcriptome data from different cultivars with reverse-transcription quantitative PCR measurements taken from petals at four distinct flowering developmental stages. This screening strategy narrowed the field to three candidate cinnamyl alcohol acyltransferase genes, which the researchers named PmCAAT1, PmCAAT2, and PmCAAT3.

Sequence analysis of the three cloned genes revealed that their protein products carry the hallmarks of the BAHD acyltransferase superfamily, a widespread class of plant enzymes named after its first characterized members. In particular, all three proteins contain the conserved HXXXD catalytic motif, which positions a key histidine residue for the transfer reaction, and the DFGWG motif that forms part of the substrate-binding pocket. These motifs are the structural fingerprints that biochemists use to recognize enzymes that shuttle activated acyl groups, typically from acetyl-coenzyme A, onto alcohol substrates to form esters.

Evolutionary comparisons added an important layer of insight. Phylogenetic analysis showed that PmCAAT1 sits closest to the clade of coniferyl alcohol acetyltransferases, enzymes previously implicated in phenylpropanoid volatile production, whereas PmCAAT2 and PmCAAT3 clustered together on a separate evolutionary branch. This split hinted that the three paralogs, despite their shared ancestry, may have diverged in function, a hypothesis the team put to the test using both cellular and biochemical assays.

Subcellular localization experiments showed that all three proteins reside in the nucleus and the cytoplasm. This dual distribution is notable because acyltransferases have increasingly been recognized as enzymes that can moonlight in the nucleus, and the localization pattern suggests the enzymes may encounter their substrates in multiple cellular compartments. To assess their roles in living tissue, the researchers used transient expression in P. mume petals, demonstrating that all three genes could regulate the biosynthesis of cinnamyl acetate when expressed in the flower tissue itself.

The decisive evidence came from the biochemistry. Using prokaryotic expression systems to produce the enzymes and in vitro activity assays to test them, the team confirmed that PmCAAT1 and PmCAAT2 can both catalyze the conversion of cinnamyl alcohol to cinnamyl acetate, while PmCAAT3 did not show this activity. Enzyme kinetics then revealed a crucial difference between the two active enzymes: although both display broad substrate selectivity, PmCAAT1 exhibits a strong preference for cinnamyl alcohol, making it the most likely primary driver of cinnamyl acetate production in the flowers. PmCAAT2, by contrast, accepts a wider range of substrates, including the monoterpene linalool, suggesting it may contribute to multiple scent compounds.

The findings place P. mume within a growing body of work showing how plants fine-tune their volatile profiles through the evolution of specialized acyltransferases. Comparable enzymes have been shown to shape the ester aromas of strawberry, banana, melon, rose, grape, and peach, where differences in enzyme activity and substrate preference translate directly into differences in flavor and fragrance. In peach, for example, variation in the activity of a single alcohol acyltransferase explains why high-aroma varieties produce far more of the lactone volatiles prized by consumers. The P. mume study extends this paradigm to ornamental floral scent and to the phenylpropanoid pathway in particular.

Beyond its fundamental interest, the work carries practical weight for horticulture. Because the fragrance of P. mume is a major part of its ornamental and cultural value, knowing which gene supplies the key ester gives breeders a molecular handle for selecting or engineering plants with enhanced aroma. The authors suggest that the results provide a theoretical foundation for molecular breeding of floral fragrance in the species, potentially allowing marker-assisted selection for high PmCAAT1 activity or the use of genome editing to boost scent output. Understanding the enzyme’s substrate preference may also guide metabolic engineering efforts aimed at producing cinnamyl acetate biotechnologically.

The study, led by corresponding author Tengxun Zhang with co-first authors Jinhong Li and Jialu Guan, was supported by the National Natural Science Foundation of China and benefited from gas chromatography time-of-flight mass spectrometry support for volatile detection. As genome resources for ornamental plants continue to expand, enzyme-by-enzyme dissections of scent pathways like this one are transforming fragrance from a subjective trait into a set of manipulable biochemical reactions, promising gardens and orchards where the aromas of spring can be designed as deliberately as their colors.

Subject of Research: Biosynthesis of the floral volatile cinnamyl acetate by cinnamyl alcohol acyltransferases in Prunus mume

Article Title: Cinnamyl alcohol acyltransferase PmCAAT1 specifically catalyzes the formation of the characteristic volatile cinnamyl acetate of Prunus mume

Article References: Li, J., Guan, J., Zhang, S., Lei, M., Li, J., Zhang, Y., & Zhang, T. (2026). Cinnamyl alcohol acyltransferase PmCAAT1 specifically catalyzes the formation of the characteristic volatile cinnamyl acetate of Prunus mume. Plant Cell Reports, 45(10), Article 306. https://doi.org/10.1007/s00299-026-03992-6

Image Credits: AI Generated

DOI: 10.1007/s00299-026-03992-6

Keywords: Prunus mume, cinnamyl acetate, PmCAAT1, BAHD acyltransferase, floral scent, volatile organic compounds, phenylpropanoid pathway, enzyme kinetics, plant biochemistry, molecular breeding, ornamental horticulture, Plant Cell Reports