The pathogenic yeast Candida albicans is both a powerful commensal and a pathogen of humans that can infect wide range of organs and body sites. Metabolic flexibility promotes infection and commensal colonization by this opportunistic pathogen. Yeast cell survival depends upon assimilation of fermentable and non-fermentable locally available carbon sources. Physiologically relevant sugars like glucose and fructose are present at low levels in host niches. However, because glucose is the preferred substrate for energy and biosynthesis of structural components, its efficient detection and metabolism are fundamental for the metabolic adaptation of the pathogen. We explored and characterized the C. albicans hexose kinase system composed of one hexokinase ( Ca Hxk2) and two glucokinases ( Ca Glk1 and Ca Glk4). Using a set of mutant strains, we found that hexose phosphorylation is mostly performed by Ca Hxk2, which sustains growth on hexoses. Our data on hexokinase and glucokinase expression point out an absence of cross regulation mechanisms at the transcription level and different regulatory pathways. In the presence of glucose, Ca Hxk2 migrates in the nucleus and contributes to the glucose repression signaling pathway. In addition, Ca Hxk2 participates in oxidative, osmotic and cell wall stress responses, while glucokinases are overexpressed under hypoxia. Hexose phosphorylation is a key step necessary for filamentation that is affected in the hexokinase mutant. Virulence of this mutant is clearly impacted in the Galleria mellonella and macrophage models. Filamentation, glucose phosphorylation and stress response defects of the hexokinase mutant prevent host killing by C. albicans . By contributing to metabolic flexibility, stress response and morphogenesis, hexose kinase enzymes play an essential role in the virulence of C. albicans .

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Ionic liquids (ILs) are important new solvents for electrochemistry and biocatalysis, but dehydrogenases usually do not work in ionic liquids. Adding more than 40 % (v/v) of the water miscible ionic liquid [MMIm][Me2PO4] (MMIm: 1‐methyl‐3‐methyl imidazolium dimethylphosphate) inactivates the formate dehydrogenase (FDH) from Candida boidinii. The grafting of a variety of IL‐inspired hydroxylated cations (hydroxyalkyl imidazolium, hydroxylalkyl pyrrolydinium, and cholinium) on the enzyme through lysine coupling was performed to understand the relationship between grafted cation, enzyme activity, and protein structure. As a general trend, the more a cation was kosmotropic (e.g., presenting a high B coefficient), the larger the resulting modifications were. The ability of these enzymes to bind the substrates was studied by fluorescence quenching in the presence of nicotinamide adenine dinucleotide (NAD+) and azide. The dissociation constant for NAD+ was only slightly affected by the grafting of the cations, however, the quenching efficiency was reduced. Azide binding was more affected by the cations. In the presence of 30 % (v/v) [MMIm][Me2PO4], the catalytic efficiency of the wild‐type enzyme was reduced by 2.8 fold. In comparison, the catalytic efficiency of the modified FDH was preserved in these conditions and even improved after modification by hydroxypropyl imidazolium. The grafting of the chaotropic cations prevented the unfolding of the FDH due to [MMIm][Me2PO4].

The effect of the ionic liquid 1-ethyl-3-methylimidazolium ethylsulfate ([Emim][EtSO(4)]) on the copper-catalyzed luminol chemiluminescence (CL) is reported. A drastic light emission enhancement is observed, related to a strong interaction between Cu(2+) and the imidazolium ring. In these conditions, the CL reaction was able to produce light efficiently at pH as low as 6.5 (amplification factor: Intensity(+IL)/Intensity(-IL) = 2900). Interesting effects of [Emim][EtSO(4)] on the enzyme glucose oxidase activity were also evidenced, and advantages were taken from this enhancement to perform sensitive chemiluminescent glucose detection (LOD = 4 microM) at pH 8.0.

The rapid electrochemical screening of enzyme activities in bioelectronics is still a challenging issue. In order to solve this problem, we propose to use a 96-well electrochemical assay. This system is composed of 96 screen-printed electrodes on a printed circuit board adapted from a commercial system (carbon is used as the working electrode and silver chloride as the counter/reference electrode). The associated device allows for the measurements on the 96 electrodes to be performed within a few seconds. In this work, we demonstrate the validity of the screening method with the commercial laccase from the fungus Trametes versicolor. The signal-to-noise ratio (S/N) is found to be the best way to analyze the electrochemical signals. The S/N follows a saturation-like mechanism with a dynamic linear range of two decades ranging from 0.5 to 75 ng of laccase (corresponding to enzymatic activities from 62 × 10(-6) to 9.37 × 10(-3) μmol min(-1)) and a sensitivity of 3027 μg(-1) at +100 mV versus Ag/AgCl. Laccase inhibitors (azide and fluoride anions), pH optima, and interfering molecules could also be identified within a few minutes.

The study of protein conformation in ionic liquids (ILs) is crucial to understand enzymatic activity. Steady-state fluorescence is a proven, rapid and easy method to evaluate the protein structure in aqueous solutions, but it is discussed when used in ILs. In this work, the structure of the formate dehydrogenase from Candida boidinii (FDH, EC: 1.2.1.2) in three imidazolium-based ILs (dimethylimidazolium dimethylphosphate [MMIm][Me(2)PO(4)], 1-butyl-3-methylimidazolium acetate [BMIm][CH(3)COO], and dimethylimidazolium methylphosphonate [MMIm][CH(3)HPO(2)(OCH(3))]) is studied by fluorescence spectroscopy. The UV-vis spectroscopic analysis shows that the decrease of the FDH fluorescence is not only due to the high light absorption of these ILs. The Stern-Volmer analysis clearly shows that these ILs are quenchers of the indole fluorescence, while this quenching property is not found when imidazole is used. Fluorescence spectra of the FDH in the presence of the ILs show that a maximal ionic liquid concentration (MILc), which could be used for steady-state fluorescence study, should be defined. Therefore, FDH conformation could not be directly related to the decrease of its fluorescence in ILs. Nevertheless, the structure of the FDH could be evaluated with dynamic and static quenchers like iodide or acrylamide, used below the MILc, demonstrating the relevance of this parameter. The Stern-Volmer constants (K(SV)(Q)), calculated in the presence of the different ILs, demonstrate that these ILs are strong denaturing agents, each one acting with a different mechanism. This report provides a suitable and easy-to-apply method to study any enzyme structures in ILs by steady-state fluorescence.

A series of alkyl thioglycosides and alkyl thiodiglycosides bearing glucose and N-acetylglucosamine residues were prepared by thiol–ene coupling in moderate to good yields (40–85%). Their binding ability towards wheat germ agglutinin was measured by competitive enzyme-linked lectin assays. One of the synthetic compounds presenting two GlcNAc units showed the highest inhibitory effect of this study with an IC50 of 11 µM corresponding to a 3182-fold improvement compared to GlcNAc. These synthetic molecules were used to produce giant vesicles, alone or in mixture with phospholipids, mimicking bacterial outer membrane vesicles (OMV) with potential antiadhesive properties.

Transketolases (TKs) are ubiquitous thiamine pyrophosphate (TPP)-dependent enzymes of the nonoxidative branch of the pentose phosphate pathway. They are considered as interesting therapeutic targets in numerous diseases and infections (e.g., cancer, tuberculosis, malaria), for which it is important to find specific and efficient inhibitors. Current TK assays require important amounts of enzyme, are time-consuming, and are not specific. Here, we report a new high throughput electrochemical assay based on the oxidative trapping of the TK-TPP intermediate. After electrode characterization, the enzyme loading, electrochemical protocol, and substrate concentration were optimized. Finally, 96 electrochemical assays could be performed in parallel in only 7 min, which allows a rapid screening of TK inhibitors. Then, 1360 molecules of an in-house chemical library were screened and one early lead compound was identified to inhibit TK from E. coli with an IC of 63 μM and an inhibition constant ( K) of 3.4 μM. The electrochemical assay was also used to propose an inhibition mechanism.