Insights into the early-life chemical exposome of Nigerian infants and potential correlations with the developing gut microbiome.

Early-life exposure to natural and synthetic chemicals can impact acute and chronic health conditions. Here, a suspect screening workflow anchored on high-resolution mass spectrometry was applied to elucidate xenobiotics in breast milk and matching stool samples collected from Nigerian mother-infant pairs (n = 11) at three time points. Potential correlations between xenobiotic exposure and the developing gut microbiome, as determined by 16S rRNA gene amplicon sequencing, were subsequently explored. Overall, 12,192 and 16,461 features were acquired in the breast milk and stool samples, respectively. Following quality control and suspect screening, 562 and 864 features remained, respectively, with 149 of these features present in both matrices. Taking advantage of 242 authentic reference standards measured for confirmatory purposes of food bio-actives and toxicants, 34 features in breast milk and 68 features in stool were identified and semi-quantified. Moreover, 51 and 78 features were annotated with spectral library matching, as well as 416 and 652 by in silico fragmentation tools in breast milk and stool, respectively. The analytical workflow proved its versatility to simultaneously determine a diverse panel of chemical classes including mycotoxins, endocrine-disrupting chemicals (EDCs), antibiotics, plasticizers, perfluorinated alkylated substances (PFAS), and pesticides, although it was originally optimized for polyphenols. Spearman rank correlation of the identified features revealed significant correlations between chemicals of the same classification such as polyphenols. One-way ANOVA and differential abundance analysis of the data obtained from stool samples revealed that molecules of plant-based origin elevated as complementary foods were introduced to the infants' diets. Annotated compounds in the stool, such as tricetin, positively correlated with the genus Blautia. Moreover, vulgaxanthin negatively correlated with Escherichia-Shigella. Despite the limited sample size, this exploratory study provides high-quality exposure data of matched biospecimens obtained from mother-infant pairs in sub-Saharan Africa and shows potential correlations between the chemical exposome and the gut microbiome.

Mushroom Tyrosinase: Six Isoenzymes Catalyzing Distinct Reactions.

"Mushroom tyrosinase" from the common button mushroom is the most frequently used source of tyrosinase activity, both for basic and applied research. Here, the complete tyrosinase family from Agaricus bisporus var. bisporus (abPPO1-6) was cloned from mRNA and expressed heterologously using a single protocol. All six isoenzymes accept a wide range of phenolic and catecholic substrates, but display pronounced differences in their specificity and enzymatic reaction rate. AbPPO3 ignores γ-l-glutaminyl-4-hydroxybenzene (GHB), a natural phenol present in mM concentrations in A. bisporus, while AbPPO4 processes 100 µM GHB at 4-times the rate of the catechol l-DOPA. All six AbPPOs are biochemically distinct enzymes fit for different roles in the fungal life cycle, which challenges the traditional concept of isoenzymes as catalyzing the same physiological reaction and varying only in secondary properties. Transferring this approach to other enzymes and organisms will greatly stimulate both the study of the in vivo function(s) of enzymes and the application of these highly efficient catalysts.

Polyphenol exposure of mothers and infants assessed by LC-MS/MS based biomonitoring in breast milk.

Exposure to polyphenols is relevant throughout critical windows of infant development, including the breastfeeding phase. However, the quantitative assessment of polyphenols in human breast milk has received limited attention so far, though polyphenols may positively influence infant health. Therefore, a targeted LC-MS/MS assay was developed to investigate 86 analytes representing different polyphenol classes in human breast milk. The sample preparation consisted of liquid extraction, salting out, freeze-out, and a dilution step. Overall, nearly 70% of the chemically diverse polyphenols fulfilled all strict validation criteria for full quantitative assessment. The remaining analytes did not fulfill all criteria at every concentration level, but can still provide useful semi-quantitative insights into nutritional and biomedical research questions. The limits of detection for all analyzed polyphenols were in the range of 0.0041-87 ng*mL-1, with a median of 0.17 ng*mL-1. Moreover, the mean recovery was determined to be 82% and the mean signal suppression and enhancement effect was 117%. The developed assay was applied in a proof-of-principle study to investigate polyphenols in breast milk samples provided by twelve Nigerian mothers at three distinct time points post-delivery. In total, 50 polyphenol analytes were detected with almost half being phenolic acids. Phase II metabolites, including genistein-7-ß-D-glucuronide, genistein-7-sulfate, and daidzein-7-ß-D-glucuronide, were also detected in several samples. In conclusion, the developed method was demonstrated to be fit-for-purpose to simultaneously (semi-) quantify a wide variety of polyphenols in breast milk. It also demonstrated that various polyphenols including their biotransformation products were present in breast milk and therefore likely transferred to infants where they might impact microbiome development and infant health.

Extraction, Purification, and Characterization of Olive (Olea europaea L., cv. Chemlal) Polyphenol Oxidase.

Among fruits susceptible to enzymatic browning, olive polyphenol oxidase (OePPO) stood out as being unisolated from a natural source until this study, wherein we successfully purified and characterized the enzyme. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) of heated and nonheated OePPO revealed distinct molecular weights of 35 and 54 kDa, respectively, indicative of its oligomeric nature comprising active and C-terminal subunits. OePPO displayed latency, fully activating with 5 mM SDS under optimal conditions of pH 7.5 and 15 °C. The enzyme demonstrated monophenolase activity and showcased the highest efficiency toward hydroxytyrosol. Despite its low optimal temperature, OePPO exhibited high thermal resistance, maintaining stability up to 90 °C. However, beyond this threshold, the oligomeric enzyme disassociated, yielding a denatured main subunit and C-terminal fragments. Six OePPO genes were found in the fruits. Tryptic digestion identified the enzyme as mature OePPO1 (INSDC OY733096), while mass spectrometry detected the active form mass alongside several C-terminal fragments, revealing potential cleavage sites (Gly407, Tyr408).

All-Inorganic Polyoxometalates Act as Superchaotropic Membrane Carriers.

Polyoxometalates (POMs) are known antitumoral, antibacterial, antiviral, and anticancer agents and considered as next-generation metallodrugs. Herein, a new biological functionality in neutral physiological media, where selected mixed-metal POMs are sufficiently stable and able to affect membrane transport of impermeable, hydrophilic, and cationic peptides (heptaarginine, heptalysine, protamine, and polyarginine) is reported. The uptake is observed in both, model membranes as well as cells, and attributed to the superchaotropic properties of the polyoxoanions. In view of the structural diversity of POMs these findings pave the way toward their biomedical application in drug delivery or for cell-biological uptake studies with biological effector molecules or staining agents.

Exposomic Biomonitoring of Polyphenols by Non-Targeted Analysis and Suspect Screening.

Polyphenols, prevalent in plants and fungi, are investigated intensively in nutritional and clinical settings because of their beneficial bioactive properties. Due to their complexity, analysis with untargeted approaches is favorable, which typically use high-resolution mass spectrometry (HRMS) rather than low-resolution mass spectrometry (LRMS). Here, the advantages of HRMS were evaluated by thoroughly testing untargeted techniques and available online resources. By applying data-dependent acquisition on real-life urine samples, 27 features were annotated with spectral libraries, 88 with in silico fragmentation, and 113 by MS1 matching with PhytoHub, an online database containing >2000 polyphenols. Moreover, other exogenous and endogenous molecules were screened to measure chemical exposure and potential metabolic effects using the Exposome-Explorer database, further annotating 144 features. Additional polyphenol-related features were explored using various non-targeted analysis techniques including MassQL for glucuronide and sulfate neutral losses, and MetaboAnalyst for statistical analysis. As HRMS typically suffers a sensitivity loss compared to state-of-the-art LRMS used in targeted workflows, the gap between the two instrumental approaches was quantified in three spiked human matrices (urine, serum, plasma) as well as real-life urine samples. Both instruments showed feasible sensitivity, with median limits of detection in the spiked samples being 10-18 ng/mL for HRMS and 4.8-5.8 ng/mL for LRMS. The results demonstrate that, despite its intrinsic limitations, HRMS can readily be used for comprehensively investigating human polyphenol exposure. In the future, this work is expected to allow for linking human health effects with exposure patterns and toxicological mixture effects with other xenobiotics.

Immobilization of Agaricus bisporus Polyphenol Oxidase 4 on mesoporous silica: Towards mimicking key enzymatic processes in peat soils.

HYPOTHESIS: The use of immobilized enzyme-type biocatalysts to mimic specific processes in soil can be considered one of the most promising alternatives to overcome the difficulties behind the structural elucidation of riverine humic-derived iron-complexes. Herein, we propose that the immobilization of the functional mushroom tyrosinase, Agaricus bisporus Polyphenol Oxidase 4 (AbPPO4) on mesoporous SBA-15-type silica could contribute to the study of small aquatic humic ligands such as phenols. EXPERIMENTS: The silica support was functionalized with amino-groups in order to investigate the impact of surface charge on the tyrosinase loading efficiency as well as on the catalytic performance of adsorbed AbPPO4. The oxidation of various phenols was catalyzed by the AbPPO4-loaded bioconjugates, yielding high levels of conversion and confirming the retention of enzyme activity after immobilization. The structures of the oxidized products were elucidated by integrating chromatographic and spectroscopic techniques. We also evaluated the stability of the immobilized enzyme over a wide range of pH values, temperatures, storage-times and sequential catalytic cycles. FINDINGS: This is the first report where the latent AbPPO4 is confined within silica mesopores. The improved catalytic performance of the adsorbed AbPPO4 shows the potential use of these silica-based mesoporous biocatalysts for the preparation of a column-type bioreactor for in situ identification of soil samples.

Biochemical characterization of Dimocarpus longan polyphenol oxidase provides insights into its catalytic efficiency.

The "dragon-eye" fruits produced by the tropical longan tree are rich in nutrients and antioxidants. They suffer from post-harvest enzymatic browning, a process for which mainly the polyphenol oxidase (PPO) family of enzymes is responsible. In this study, two cDNAs encoding the PPO have been cloned from leaves of Dimocarpus longan (Dl), heterologously expressed in Escherichia coli and purified by affinity chromatography. The prepro-DlPPO1 contains two signal peptides at its N-terminal end that facilitate transportation of the protein into the chloroplast stroma and to the thylakoid lumen. Removal of the two signal peptides from prepro-DlPPO1 yields pro-DlPPO1. The prepro-DlPPO1 exhibited higher thermal tolerance than pro-DlPPO1 (unfolding at 65 °C vs. 40 °C), suggesting that the signal peptide may stabilize the fold of DlPPO1. DlPPO1 can be classified as a tyrosinase because it accepts both monophenolic and diphenolic substrates. The pro-DlPPO1 exhibited the highest specificity towards the natural diphenol (-)-epicatechin (kcat/KM of 800 ± 120 s-1 mM-1), which is higher than for 4-methylcatechol (590 ± 99 s-1 mM-1), pyrogallol (70 ± 9.7 s-1 mM-1) and caffeic acid (4.3 ± 0.72 s-1 mM-1). The kinetic efficiencies of prepro-DlPPO1 are 23, 36, 1.7 and 4.7-fold lower, respectively, than those observed with pro-DlPPO1 for the four aforementioned diphenolic substrates. Additionally, docking studies showed that (-)-epicatechin has a lower binding energy than any other investigated substrate. Both kinetic and in-silico studies strongly suggest that (-)-epicatechin is a good substrate of DlPPO1 and ascertain the affinity of PPOs towards specific flavonoid compounds.

Inhibition of SERCA and PMCA Ca2+-ATPase activities by polyoxotungstates.

Plasma membrane calcium ATPases (PMCA) and sarco(endo) reticulum calcium ATPases (SERCA) are key proteins in the maintenance of calcium homeostasis. Herein, we compare for the first time the inhibition of SERCA and PMCA calcium pumps by several polyoxotungstates (POTs), namely by Wells-Dawson phosphotungstate anions [P2W18O62]6- (intact, {P2W18}), [P2W17O61]10- (monolacunary, {P2W17}), [P2W15O56]12- (trilacunary, {P2W15}), [H2P2W12O48]12- (hexalacunary, {P2W12}), [H3P2W15V3O62]6- (trivanadium-substituted, {P2W15V3}) and by Preyssler-type anion [NaP5W30O110]14- ({P5W30}). The speciation in the solutions of tested POTs was investigated by 31P and 51V NMR spectroscopy. The tested POTs inhibited SERCA Ca2+-ATPase activity, whereby the Preyssler POT showed the strongest effect, with an IC50 value of 0.37 µM. For {P2W17} and {P2W15V3} higher IC50 values were determined: 0.72 and 0.95 µM, respectively. The studied POTs showed to be more potent inhibitors of PMCA Ca2+-ATPase activity, with lower IC50 values for {P2W17}, {P5W30} and {P2W15V3}.

Quantifying up to 90 polyphenols simultaneously in human bio-fluids by LC-MS/MS.

Establishing a method for human biomonitoring (HBM) of polyphenols enables the assessment of internal concentrations of these food bio-actives and the correlation with potential health effects such as antioxidant or anti-inflammatory properties. Thus, a targeted LC-MS/MS method for quantifying up to 90 analytes, representing the main polyphenol classes including flavanones, isoflavones, stilbenes, and phenolic acids, was developed for human urine, serum, and plasma. The method was established for low sample volumes and with a cost and time efficient sample preparation protocol for high-throughput, which is critical for its application in large cohort and exposome-wide association studies. On average, the sample preparation yielded extraction efficiencies of 98% for urine, 98% for serum, and 87% for plasma. Limits of detection were between 0.11 ng mL-1 and 300 ng mL-1 for urine, 0.12 ng mL-1 and 190 ng mL-1 for serum, and 0.12 ng mL-1 and 340 ng mL-1 for plasma, excluding one analyte. In-house validation revealed that 66, 49, and 64 analytes for urine, serum, and plasma, respectively, fulfilled all stringent requirements, that are usually utilized for tailored single analyte methods, at all evaluated concentration levels. After validation, this method was applied in a proof-of-principle study that detected 39 polyphenols in urine. Changes in the concentrations of the analytes after the ingestion of a high polyphenol smoothie was examined over 24 h. The study further confirmed that the majority of polyphenols detected were phenolic acids, and phase II conjugated metabolites were more abundant than their respective non-conjugated forms.

Polyphenol oxidase and enzymatic browning in apricot (Prunus armeniaca L.): Effect on phenolic composition and deduction of main substrates.

In this study, we investigate the effect of enzymatic browning on the phenolic composition of apricot in vivo and in vitro. The in vitro browning was caused by the recombinant latent apricot polyphenol oxidase (L-PaPPO). Successful heterologous expression of PaPPO in Escherichia coli yielded substantial amounts of enzyme containing both copper ions in the catalytic active site. The expressed L-PaPPO was characterized with regard to its molecular mass (56531.3 Da), pH optimum (7.0), activation by SDS, and enzyme kinetics. LC-MS/MS was used to compare the phenolic profiles of brown and non-brown apricots. The browning reactions did significantly decrease total phenolics and antioxidant capacity (measured with DPPH and CUPRAC assays). Catechin, epicatechin, and B-type procyanidins were the individual phenolics most affected by browning, followed by chlorogenic and neochlorogenic acid. These phenolics are most likely the main endogenous substrates of L-PaPPO, as they were oxidized much faster than the other identified phenolics.

Wells-Dawson phosphotungstates as mushroom tyrosinase inhibitors: a speciation study.

In order to elucidate the active polyoxotungstate (POT) species that inhibit fungal polyphenol oxidase (AbPPO4) in sodium citrate buffer at pH 6.8, four Wells-Dawson phosphotungstates [α/ß-PV2WVI18O62]6- (intact form), [α2-PV2WVI17O61]10- (monolacunary), [PV2WVI15O56]12- (trilacunary) and [H2PV2WVI12O48]12- (hexalacunary) were investigated. The speciation of the POT solutions under the dopachrome assay (50 mM Na-citrate buffer, pH 6.8; L-3,4-dihydroxyphenylalanine as a substrate) conditions were determined by 183W-NMR, 31P-NMR spectroscopy and mass spectrometry. The intact Wells-Dawson POT [α/ß-PV2WVI18O62]6- shows partial (~ 69%) disintegration into the monolacunary [α2-PV2WVI17O61]10- anion with moderate activity (Ki = 9.7 mM). The monolacunary [α2-PV2WVI17O61]10- retains its structural integrity and exhibits the strongest inhibition of AbPPO4 (Ki = 6.5 mM). The trilacunary POT [PV2WVI15O56]12- rearranges to the more stable monolacunary [α2-PV2WVI17O61]10- (~ 62%) accompanied by release of free phosphates and shows the weakest inhibition (Ki = 13.6 mM). The hexalacunary anion [H2PV2WVI12O48]12- undergoes time-dependent hydrolysis resulting in a mixture of [H2PV2WVI12O48]12-, [PV8WVI48O184]40-, [PV2WVI19O69(H2O)]14- and [α2-PV2WVI17O61]10- which together leads to comparable inhibitory activity (Ki = 7.5 mM) after 48 h. For the solutions of [α/ß-PV2WVI18O62]6-, [α2-PV2WVI17O61]10- and [PV2WVI15O56]12- the inhibitory activity is correlated to the degree of their rearrangement to [α2-PV2WVI17O61]10-. The rearrangement of hexalacunary [H2PV2WVI12O48]12- into at least four POTs with a negligible amount of monolacunary anion interferes with the correlation of activity to the degree of their rearrangement to [α2-PV2WVI17O61]10-. The good inhibitory effect of the Wells-Dawson [α2-PV2WVI17O61]10- anion is explained by the low charge density of its protonated forms Hx[α2-PV2WVI17O61](10-x)- (x = 3 or 4) at pH 6.8.

Aluminum-Substituted Keggin Germanotungstate [HAl(H2O)GeW11O39]4-: Synthesis, Characterization, and Antibacterial Activity.

We report on the new monosubstituted aluminum Keggin-type germanotungstate (C4H12N)4[HAlGeW11O39(H2O)]·11H2O ([Al(H2O)GeW11]4-), which has been synthesized at room temperature via rearrangement of the dilacunary [γ-GeW10O36]8- polyoxometalate precursor. [Al(H2O)GeW11]4- has been characterized thoroughly both in the solid state by single-crystal and powder X-ray diffraction, IR spectroscopy, thermogravimetric analysis, and elemental analysis as well as in solution by cyclic voltammetry (CV) 183W, 27Al NMR and UV-vis spectroscopy. A study on the antibacterial properties of [Al(H2O)GeW11]4- and the known aluminum(III)-centered Keggin polyoxotungstates (Al-POTs) α-Na5[AlW12O40] (α-[AlW12O40]5-) and Na6[Al(AlOH2)W11O39] ([Al(AlOH2)W11O39]6-) revealed enhanced activity for all three Al-POTs against the Gram-negative bacterium Moraxella catarrhalis (minimum inhibitory concentration (MIC) up to 4 µg mL-1) and the Gram-positive Enterococcus faecalis (MIC up to 128 µg mL-1) compared to the inactive Al(NO3)3 salt (MIC > 256 µg mL-1). CV indicates the redox activity of the Al-POTs as a dominating factor for the observed antibacterial activity with increased tendency to reduction, resulting in increased antibacterial activity of the POT.

Polyphenol Exposure, Metabolism, and Analysis: A Global Exposomics Perspective.

Polyphenols are generally known for their health benefits and estimating actual exposure levels in health-related studies can be improved by human biomonitoring. Here, the application of newly available exposomic and metabolomic technology, notably high-resolution mass spectrometry, in the context of polyphenols and their biotransformation products, is reviewed. Comprehensive workflows for investigating these important bioactives in biological fluids or microbiome-related experiments are scarce. Consequently, this new era of nontargeted analysis and omic-scale exposure assessment offers a unique chance for better assessing exposure to, as well as metabolism of, polyphenols. In clinical and nutritional trials, polyphenols can be investigated simultaneously with the plethora of other chemicals to which we are exposed, i.e., the exposome, which may interact abundantly and modulate bioactivity. This research direction aims at ultimately eluting into atrue systems biology/toxicology evaluation of health effects associated with polyphenol exposure, especially during early life, to unravel their potential for preventing chronic diseases.

Similar but Still Different: Which Amino Acid Residues Are Responsible for Varying Activities in Type-III Copper Enzymes?

Type-III copper enzymes like polyphenol oxidases (PPOs) are ubiquitous among organisms and play a significant role in the formation of pigments. PPOs comprise different enzyme groups, including tyrosinases (TYRs) and catechol oxidases (COs). TYRs catalyze the o-hydroxylation of monophenols and the oxidation of o-diphenols to the corresponding o-quinones (EC 1.14.18.1). In contrast, COs only catalyze the oxidation of o-diphenols to the corresponding o-quinones (EC 1.10.3.1). To date (August 2020), 102 PDB entries encompassing 18 different proteins from 16 organisms and several mutants have been reported, identifying key residues for tyrosinase activity. The structural similarity between TYRs and COs, especially within and around the active center, complicates the elucidation of their modes of action on a structural basis. However, mutagenesis studies illuminate residues that influence the two activities and show that crystallography on its own cannot elucidate the enzymatic activity mode. Several amino acid residues around the dicopper active center have been proposed to play an essential role in the two different activities. Herein, we critically review the role of all residues identified so far that putatively affect the two activities of PPOs.

Identification of Amino Acid Residues Responsible for C-H Activation in Type-III Copper Enzymes by Generating Tyrosinase Activity in a Catechol Oxidase.

Tyrosinases (TYRs) catalyze the hydroxylation of phenols and the oxidation of the resulting o-diphenols to o-quinones, while catechol oxidases (COs) exhibit only the latter activity. Aurone synthase (AUS) is not able to react with classical tyrosinase substrates, such as tyramine and l-tyrosine, while it can hydroxylate its natural substrate isoliquiritigenin. The structural difference of TYRs, COs, and AUS at the heart of their divergent catalytic activities is still a puzzle. Therefore, a library of 39 mutants of AUS from Coreopsis grandiflora (CgAUS) was generated and the activity studies showed that the reactivity of the three conserved histidines (HisA2 , HisB1 , and HisB2 ) is tuned by their adjacent residues (HisB1 +1, HisB2 +1, and waterkeeper residue) either to react as stronger bases or / and to stabilize a position permissive for substrate proton shuffling. This provides the understanding for C-H activation based on the type-III copper center to be used in future biotechnological processes.

Identification of the amino acid position controlling the different enzymatic activities in walnut tyrosinase isoenzymes (jrPPO1 and jrPPO2).

Polyphenol oxidases (PPOs) are ubiquitously distributed among plants, bacteria, fungi and animals. They catalyze the hydroxylation of monophenols (monophenolase activity) and the oxidation of o-diphenols (diphenolase activity) to o-quinones. PPOs are commonly present as an isoenzyme family. In walnut (Juglans regia), two different genes (jrPPO1 and jrPPO2) encoding PPOs have been identified. In this study, jrPPO2 was, for the first time, heterologously expressed in E. coli and characterized as a tyrosinase (TYR) by substrate scope assays and kinetic investigations, as it accepted tyramine and L-tyrosine as substrates. Moreover, the substrate acceptance and kinetic parameters (kcat and Km values) towards 16 substrates naturally present in walnut were assessed for jrPPO2 (TYR) and its isoenzyme jrPPO1 (TYR). The two isoenzymes prefer different substrates, as jrPPO1 shows a higher activity towards monophenols, whereas jrPPO2 is more active towards o-diphenols. Molecular docking studies performed herein revealed that the amino acid residue in the position of the 1st activity controller (HisB1 + 1; in jrPPO1 Asn240 and jrPPO2 Gly240) is responsible for the different enzymatic activities. Additionally, interchanging the 1st activity controller residue of the two enzymes in two mutants (jrPPO1-Asn240Gly and jrPPO2-Gly240Asn) proved that the amino acid residue located in this position allows plants to selectively target or dismiss substrates naturally present in walnut.

The Aquaporin-3-Inhibiting Potential of Polyoxotungstates.

Polyoxometalates (POMs) are of increasing interest due to their proven anticancer activities. Aquaporins (AQPs) were found to be overexpressed in tumors bringing particular attention to their inhibitors as anticancer drugs. Herein, we report for the first time the ability of polyoxotungstates (POTs), such as of Wells-Dawson P2W18, P2W12, and P2W15, and Preyssler P5W30 structures, to affect aquaporin-3 (AQP3) activity and impair melanoma cell migration. The tested POTs were revealed to inhibit AQP3 function with different effects, with P2W18, P2W12, and P5W30 being the most potent (50% inhibitory concentration (IC50) = 0.8, 2.8, and 3.2 µM), and P2W15 being the weakest (IC50 > 100 µM). The selectivity of P2W18 toward AQP3 was confirmed in yeast cells transformed with human aquaglyceroporins. The effect of P2W12 and P2W18 on melanoma cells that highly express AQP3 revealed an impairment of cell migration between 55% and 65% after 24 h, indicating that the anticancer properties of these compounds may in part be due to the blockage of AQP3-mediated permeability. Altogether, our data revealed that P2W18 strongly affects AQP3 activity and cancer cell growth, unveiling its potential as an anticancer drug against tumors where AQP3 is highly expressed.

Binding of a Fatty Acid-Functionalized Anderson-Type Polyoxometalate to Human Serum Albumin.

The Anderson-type hexamolybdoaluminate functionalized with lauric acid (LA), (TBA)3[Al(OH)3Mo6O18{(OCH2)3CNHCOC11H23}]·9H2O (TBA-AlMo6-LA, where TBA = tetrabutylammonium), was prepared via two synthetic routes and characterized by thermogravimetric and elemental analyses, mass spectrometry, IR and 1H NMR spectroscopy, and powder and single-crystal X-ray diffraction. The interaction of TBA-AlMo6-LA with human serum albumin (HSA) was investigated via fluorescence and circular dichroism spectroscopy. The results revealed that TBA-AlMo6-LA binds strongly to HSA (63% quenching at an HSA/TBA-AlMo6-LA ratio of 1:1), exhibiting static quenching. In contrast to TBA-AlMo6-LA, the nonfunctionalized polyoxometalate, Na3(H2O)6[Al(OH)6Mo6O18]·2H2O (AlMo6), showed weak binding toward HSA (22% quenching at a HSA/AlMo6 ratio of 1:25). HSA binding was confirmed by X-ray structure analysis of the HSA-Myr-AlMo6-LA complex (Myr = myristate). These results provide a promising lead for the design of novel polyoxometalate-based hybrids that are able to exploit HSA as a delivery vehicle to improve their pharmacokinetics and bioactivity.

Conversion of walnut tyrosinase into a catechol oxidase by site directed mutagenesis.

Polyphenol oxidases (PPOs) comprise tyrosinases (TYRs) and catechol oxidases (COs), which catalyse the initial reactions in the biosynthesis of melanin. TYRs hydroxylate monophenolic (monophenolase activity) and oxidize diphenolic (diphenolase activity) substrates, whereas COs react only with diphenols. In order to elucidate the biochemical basis for the different reactions in PPOs, cDNA from walnut leaves was synthesized, the target gene encoding the latent walnut tyrosinase (jrPPO1) was cloned, and the enzyme was heterologously expressed in Escherichia coli. Mutations targeting the two activity controller residues (Asn240 and Leu244) as well as the gatekeeper residue (Phe260) were designed to impair monophenolase activity of jrPPO1. For the first time, monophenolase activity of jrPPO1 towards L-tyrosine was blocked in two double mutants (Asn240Lys/Leu244Arg and Asn240Thr/Leu244Arg) while its diphenolase activity was partially preserved, thereby converting jrPPO1 into a CO. Kinetic data show that recombinant jrPPO1 resembles the natural enzyme, and spectrophotometric investigations proved that the copper content remains unaffected by the mutations. The results presented herein provide experimental evidence that a precisely tuned interplay between the amino acids located around the active center controls the substrate specificity and therewith the mono- versus diphenolase activity in the type-III copper enzyme jrPPO1.