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Mesoporous titania nanoparticles (NPs) can be used for encapsulation polyphenols, with applications in the food industry, cosmetics, or biomedicine. TiO2 NPs were synthesized using the sol-gel method combined with solvothermal treatment. TiO2 NPs were characterized through X-ray diffraction, FTIR spectroscopy, the N2 adsorption method, scanning and transmission electron microscopy, and thermal analysis. The sample prepared using Pluronic F127 presented a higher surface area and less agglomerated NPs than the samples synthesized with Pluronic P123. Grape marc (GM), a by-product from wine production, can be exploited for preparing extracts with good antioxidant properties. In this regard, we prepared hydroethanolic and ethanolic GM extracts from two cultivars, Feteasca Neagra (FN) and Pinot Noir. The extract components were determined by spectrometric analyses and HPLC. The extract with the highest radical scavenging activity, the hydroethanolic FN extract, was encapsulated in titania (FN@TiO2) and compared with SBA-15 silica support. Both resulting materials showed biocompatibility on the NCTC fibroblast cell line in a 50–300 µg/mL concentration range after 48 h of incubation and even better radical scavenging potential than the free extract. Although titania has a lower capacity to host polyphenols than SBA-15, the FN@TiO2 sample shows better cytocompatibility (up to 700 µmg/mL), and therefore, it could be used for skin-care products. [ABSTRACT FROM AUTHOR]

While it is well established that thermally-activated quantum mechanical tunnelling of light particles (electrons and light atoms, typically hydrogen) plays a role in many enzyme-catalysed reactions, there are few definitive experimental signatures of atomic tunnelling and no clear methods of directly estimating the relative tunnelling contribution from typical experimental data. As most enzyme reactions involve the binding/capture of freely diffusing substrate(s), reactions are typically initiated by mixing and experimental conditions must then be compatible with liquid water (the solvent). This precludes the classic test of tunnelling: the observation of temperature-independent rate constants at cryogenic temperatures. Instead, H-tunnelling is usually inferred from kinetic isotope effects that are larger than the semiclassical limit. Often, the temperature dependence of the reaction is also measured over the experimentally accessible range (∼278–313 K for mesophilic enzymes), with resulting data analysed and interpreted using variations of Arrhenius, Eyring or Marcus theory. The apparent Arrhenius and Eyring activation parameters allow some quantitative comparison of different reactions, but do not directly provide any information about tunnelling, while the validity of parameters derived from non-adiabatic models such as Marcus theory are questionable due to the partially adiabatic nature of these reactions. Here, we use the correlation found between apparent activation enthalpy and entropy across several series of enzyme variants and tunnelling contributions determined using computational chemistry in an attempt to question and define new signatures of hydrogen tunnelling, which can be used to interpret typical experimental kinetic data measured for enzyme-catalysed reactions. [ABSTRACT FROM AUTHOR]

Most ene‐reductases belong to the Old Yellow Enzyme (OYE) family of flavin‐dependent oxidoreductases. OYEs use nicotinamide coenzymes as hydride donors to catalyze the reduction of alkenes that contain an electron‐withdrawing group. There have been many investigations of the structures and catalytic mechanisms of OYEs. However, the origin of coenzyme specificity in the OYE family is unknown. Structural NMR and X‐ray crystallographic data were used to rationally design variants of two OYEs, pentaerythritol tetranitrate reductase (PETNR) and morphinone reductase (MR), to discover the basis of coenzyme selectivity. PETNR has dual‐specificity and reacts with NADH and NADPH; MR accepts only NADH as hydride donor. Variants of a β‐hairpin motif in an active site loop of both these enzymes were studied using stopped‐flow spectroscopy. Specific attention was placed on the potential role of arginine residues within the β‐hairpin motif. Mutagenesis demonstrated that Arg130 governs the preference of PETNR for NADPH, and that Arg142 interacts with the coenzyme pyrophosphate group. These observations were used to switch coenzyme specificity in MR by replacing either Glu134 or Leu146 with arginine residues. These variants had increased (~15‐fold) affinity for NADH. Mutagenesis enabled MR to accept NADPH as a hydride donor, with E134R MR showing a significant (55‐fold) increase in efficiency in the reductive half‐reaction, when compared to the essentially unreactive wild‐type enzyme. Insight into the question of coenzyme selectivity in OYEs has therefore been addressed through rational redesign. This should enable coenzyme selectivity to be improved and switched in other OYEs. [ABSTRACT FROM AUTHOR]

Pentaerythritol tetranitrate reductase (PETNR) is a flavoenzyme possessing a broad substrate specificity and is a member of the Old Yellow Enzyme family of oxidoreductases. As well as having high potential as an industrial biocatalyst, PETNR is an excellent model system for studying hydrogen transfer reactions. Mechanistic studies performed with PETNR using stopped-flow methods have shown that tunneling contributes towards hydride transfer from the NAD(P)H coenzyme to the flavin mononucleotide (FMN) cofactor and fast protein dynamics have been inferred to facilitate this catalytic step. Herein, we report the near-complete 1H, 15N and 13C backbone resonance assignments of PETNR in a stoichiometric complex with the FMN cofactor in its native oxidized form, which were obtained using heteronuclear multidimensional NMR spectroscopy. A total of 97% of all backbone resonances were assigned, with 333 out of a possible 344 residues assigned in the 1H-15N TROSY spectrum. This is the first report of an NMR structural study of a flavoenzyme from the Old Yellow Enzyme family and it lays the foundation for future investigations of functional dynamics in hydride transfer catalytic mechanism. [ABSTRACT FROM AUTHOR]

Highlights: [•] TiO2 doped with Nb or Mg synthesized by hydrolysis of metal alkoxides. [•] Mg–TiO2 is basic, pure TiO2 is amphoteric, Nb–TiO2 is acidic. [•] Pd–Cu/(Mg or Nb)–TiO2 active catalysts for NO3 − hydrogenation. [•] Activity moderately influenced by support acid–base properties. [•] NH4 + selectivity unaffected by support acid–base properties. [ABSTRACT FROM AUTHOR]

The incorporation of stable isotopes into proteins is beneficial or essential for a range of experiments, including NMR, neutron scattering and reflectometry, proteomic mass spectrometry, vibrational spectroscopy and "heavy" enzyme kinetic isotope effect (KIE) measurements. Here, we present detailed protocols for the stable isotopic labeling of pentaerythritol tetranitrate reductase (PETNR) via recombinant expression in E. coli. PETNR is an ene-reductase belonging to the Old Yellow Enzyme (OYE) family of flavoenzymes, and is regarded as a model system for studying hydride transfer reactions. Included is a discussion of how efficient back-exchange of amide protons in the protein core can be achieved and how the intrinsic flavin mononucleotide (FMN) cofactor can be exchanged, allowing the production of isotopologues with differentially labeled protein and cofactor. In addition to a thorough description of labeling strategies, we briefly exemplify how data analysis and interpretation of "heavy" enzyme KIEs can be performed., (© 2019 Elsevier Inc. All rights reserved.)