Microbial ß C-S Lyases: Enzymes with Multifaceted Roles in Flavor Generation.

ß C-S lyases (ß-CSLs; EC 4.4.1.8) are enzymes catalyzing the dissociation of ß carbon-sulfur bonds of cysteine S-conjugates to produce odorant metabolites with a free thiol group. These enzymes are increasingly studied for their role in flavor generation in a variety of food products, whether these processes occur directly in plants, by microbial ß-CSLs during fermentation, or in the mouth under the action of the oral microbiota. Microbial ß-CSLs react with sulfur aroma precursors present in beverages, vegetables, fruits, or aromatic herbs like hop but also potentially with some precursors formed through Maillard reactions in cooked foods such as meat or coffee. ß-CSLs from microorganisms like yeasts and lactic acid bacteria have been studied for their role in the release of polyfunctional thiols in wine and beer during fermentation. In addition, ß-CSLs from microorganisms of the human oral cavity were shown to metabolize similar precursors and to produce aroma in the mouth with an impact on retro-olfaction. This review summarizes the current knowledge on ß-CSLs involved in flavor generation with a focus on enzymes from microbial species present either in the fermentative processes or in the oral cavity. This paper highlights the importance of this enzyme family in the food continuum, from production to consumption, and offers new perspectives concerning the utilization of ß-CSLs as a flavor enhancer.

Characterization of a Human Respiratory Mucosa Model to Study Odorant Metabolism.

Nasal xenobiotic metabolizing enzymes (XMEs) are important for the sense of smell because they influence odorant availability and quality. Since the major part of the human nasal cavity is lined by a respiratory mucosa, we hypothesized that this tissue contributed to nasal odorant metabolism through XME activity. Thus, we built human respiratory tissue models and characterized the XME profiles using single-cell RNA sequencing. We focused on the XMEs dicarbonyl and l-xylulose reductase, aldehyde dehydrogenase (ALDH) 1A1, and ALDH3A1, which play a role in food odorant metabolism. We demonstrated protein abundance and localization in the tissue models and showed the metabolic activity of the corresponding enzyme families by exposing the models to the odorants 3,4-hexandione and benzaldehyde. Using gas chromatography coupled with mass spectrometry, we observed, for example, a significantly higher formation of the corresponding metabolites 4-hydroxy-3-hexanone (39.03 ± 1.5%, p = 0.0022), benzyl alcohol (10.05 ± 0.88%, p = 0.0008), and benzoic acid (8.49 ± 0.57%, p = 0.0004) in odorant-treated tissue models compared to untreated controls (0 ± 0, 0.12 ± 0.12, and 0.18 ± 0.18%, respectively). This is the first study that reveals the XME profile of tissue-engineered human respiratory mucosa models and demonstrates their suitability to study nasal odorant metabolism.

Evaluating the biomolecular interaction between delamanid/formulations and human serum albumin by fluorescence, CD spectroscopy and SPR: Effects on protein conformation, kinetic and thermodynamic parameters.

Delamanid is an anti-tuberculosis drug used for the treatment of drug-resistant tuberculosis. Since delamanid has a high protein bound potential, even patients with low albumin levels should experience high and rapid delamanid clearance. However, the interaction between delamanid and albumin should be better controlled to optimize drug efficacy. This study was designed to evaluate the binding characteristics of delamanid to human serum albumin (HSA) using various methods: fluorescence spectroscopy, circular dichroism (CD), surface plasmon resonance (SPR), and molecular docking simulation. The fluorescence emission band without any shift indicated the interaction was not affected by the polarity of the fluorophore microenvironment. The reduction of fluorescence intensity at 344 nm was proportional to the increment of delamanid concentration as a fluorescence quencher. UV-absorbance measurement at the maximum wavelength (λmax, 280 nm) was evaluated using inner filter effect correction. The HSA conformation change was explained by the intermolecular energy transfer between delamanid and HSA during complex formation. The study, which was conducted at temperatures of 298 K, 303 K, and 310 K, revealed a static quenching mechanism that correlated with a decreased of bimolecular quenching rate constant (kq) and binding constant (Ka) at increased temperatures. The Ka was 1.75-3.16 × 104 M-1 with a specific binding site with stoichiometry 1:1. The negative enthalpy change, negative entropy change, and negative Gibbs free energy change demonstrated an exothermic-spontaneous reaction while van der Waals forces and hydrogen bonds played a vital role in the binding. The molecular displacement approach and molecular docking confirmed that the binding occurred mainly in subdomain IIA, which is a hydrophobic pocket of HSA, with a theoretical binding free energy of -9.33 kcal/mol. SPR exhibited a real time negative sensorgram that resulted from deviation of the reflex angle due to ligand delamanid-HSA complex forming. The binding occurred spontaneously after delamanid was presented to the HSA surface. The SPR mathematical fitting model revealed that the association rate constant (kon) was 2.62 × 108 s-1M-1 and the dissociation rate constant (koff) was 5.65 × 10-3 s-1. The complexes were performed with an association constant (KA) of 4.64 × 1010 M-1 and the dissociation constant (KD) of 2.15 × 10-11 M. The binding constant indicated high binding affinity and high stability of the complex in an equilibrium. Modified CD spectra revealed that conformation of the HSA structure was altered by the presence of delamanid during preparation of the proliposomes that led to the reduction of secondary structure stabilization. This was indicated by the percentage decrease of α-helix. These findings are beneficial to understanding delamanid-HSA binding characteristics as well as the drug administration regimen.

Rattus norvegicus Glutathione Transferase Omega 1 Localization in Oral Tissues and Interactions with Food Phytochemicals.

Glutathione transferases are xenobiotic-metabolizing enzymes with both glutathione-conjugation and ligandin roles. GSTs are present in chemosensory tissues and fluids of the nasal/oral cavities where they protect tissues from exogenous compounds, including food molecules. In the present study, we explored the presence of the omega-class glutathione transferase (GSTO1) in the rat oral cavity. Using immunohistochemistry, GSTO1 expression was found in taste bud cells of the tongue epithelium and buccal cells of the oral epithelium. Buccal and lingual extracts exhibited thiol-transferase activity (4.9 ± 0.1 and 1.8 ± 0.1 µM/s/mg, respectively). A slight reduction from 4.9 ± 0.1 to 4.2 ± 0.1 µM/s/mg (p < 0.05; Student's t test) was observed in the buccal extract with 100 µM GSTO1-IN-1, a specific inhibitor of GSTO1. RnGSTO1 exhibited the usual activities of omega GSTs, i.e., thiol-transferase (catalytic efficiency of 8.9 × 104 M-1·s-1), and phenacyl-glutathione reductase (catalytic efficiency of 8.9 × 105 M-1·s-1) activities, similar to human GSTO1. RnGSTO1 interacts with food phytochemicals, including bitter compounds such as luteolin (Ki = 3.3 ± 1.9 µM). Crystal structure analysis suggests that luteolin most probably binds to RnGSTO1 ligandin site. Our results suggest that GSTO1 could interact with food phytochemicals in the oral cavity.

Correction: The odorant metabolizing enzyme UGT2A1: Immunolocalization and impact of the modulation of its activity on the olfactory response.

[This corrects the article DOI: 10.1371/journal.pone.0249029.].

Development of New Models of Oral Mucosa to Investigate the Impact of the Structure of Transmembrane Mucin-1 on the Mucosal Pellicle Formation and Its Physicochemical Properties.

The mucosal pellicle (MP) is a biological film protecting the oral mucosa. It is composed of bounded salivary proteins and transmembrane mucin MUC1 expressed by oral epithelial cells. Previous research indicates that MUC1 expression enhances the binding of the main salivary protein forming the MP, MUC5B. This study investigated the influence of MUC1 structure on MP formation. A TR146 cell line, which does not express MUC1 natively, was stably transfected with genes coding for three MUC1 isoforms differing in the structure of the two main extracellular domains: the VNTR domain, exhibiting a variable number of tandem repeats, and the SEA domain, maintaining the two bound subunits of MUC1. Semi-quantification of MUC1 using dot blot chemiluminescence showed comparable expression levels in all transfected cell lines. Semi-quantification of MUC5B by immunostaining after incubation with saliva revealed that MUC1 expression significantly increased MUC5B adsorption. Neither the VNTR domain nor the SEA domain was influenced MUC5B anchoring, suggesting the key role of the MUC1 N-terminal domain. AFM-IR nanospectroscopy revealed discernible shifts indicative of changes in the chemical properties at the cell surface due to the expression of the MUC1 isoform. Furthermore, the observed chemical shifts suggest the involvement of hydrophobic effects in the interaction between MUC1 and salivary proteins.

Unlocking Flavor Potential Using Microbial ß-Glucosidases in Food Processing.

Aroma is among of the most important criteria that indicate the quality of food and beverage products. Aroma compounds can be found as free molecules or glycosides. Notably, a significant portion of aroma precursors accumulates in numerous food products as nonvolatile and flavorless glycoconjugates, termed glycosidic aroma precursors. When subjected to enzymatic hydrolysis, these seemingly inert, nonvolatile glycosides undergo transformation into fragrant volatiles or volatiles that can generate odor-active compounds during food processing. In this context, microbial ß-glucosidases play a pivotal role in enhancing or compromising the development of flavors during food and beverage processing. ß-glucosidases derived from bacteria and yeast can be utilized to modulate the concentration of particular aroma and taste compounds, such as bitterness, which can be decreased through hydrolysis by glycosidases. Furthermore, oral microbiota can influence flavor perception by releasing volatile compounds that can enhance or alter the perception of food products. In this review, considering the glycosidic flavor precursors present in diverse food and beverage products, we underscore the significance of glycosidases with various origins. Subsequently, we delve into emerging insights regarding the release of aroma within the human oral cavity due to the activity of oral microbial glycosidases.

Structure-activity analysis suggests an olfactory function for the unique antennal delta glutathione transferase of Apis mellifera.

Glutathione transferases (GST) are detoxification enzymes that conjugate glutathione to a wide array of molecules. In the honey bee Apis mellifera, AmGSTD1 is the sole member of the delta class of GSTs, with expression in antennae. Here, we structurally and biochemically characterized AmGSTD1 to elucidate its function. We showed that AmGSTD1 can efficiently catalyse the glutathione conjugation of classical GST substrates. Additionally, AmGSTD1 exhibits binding properties with a range of odorant compounds. AmGSTD1 has a peculiar interface with a structural motif we propose to call 'sulfur sandwich'. This motif consists of a cysteine disulfide bridge sandwiched between the sulfur atoms of two methionine residues and is stabilized by CH…S hydrogen bonds and S…S sigma-hole interactions. Thermal stability studies confirmed that this motif is important for AmGSTD1 stability and, thus, could facilitate its functions in olfaction.

Significant influence of four highly conserved amino-acids in lipochaperon-active sHsps on the structure and functions of the Lo18 protein.

To cope with environmental stresses, bacteria have developed different strategies, including the production of small heat shock proteins (sHSP). All sHSPs are described for their role as molecular chaperones. Some of them, like the Lo18 protein synthesized by Oenococcus oeni, also have the particularity of acting as a lipochaperon to maintain membrane fluidity in its optimal state following cellular stresses. Lipochaperon activity is poorly characterized and very little information is available on the domains or amino-acids key to this activity. The aim in this paper is to investigate the importance at the protein structure and function level of four highly conserved residues in sHSP exhibiting lipochaperon activity. Thus, by combining in silico, in vitro and in vivo approaches the importance of three amino-acids present in the core of the protein was shown to maintain both the structure of Lo18 and its functions.

Perspectives on Nasal Odorant Metabolism Research.

Olfaction is a multi-step process. At a peripheral level, nasal odorant metabolism contributes to olfaction via signal termination, variation, and regulation. We summarize current techniques used to investigate nasal odorant metabolism and give an outlook on future approaches, such as nasal tissue models and their potential contributions in future research directions.

Characterization of human oxidoreductases involved in aldehyde odorant metabolism.

Oxidoreductases are major enzymes of xenobiotic metabolism. Consequently, they are essential in the chemoprotection of the human body. Many xenobiotic metabolism enzymes have been shown to be involved in chemosensory tissue protection. Among them, some were additionally shown to be involved in chemosensory perception, acting in signal termination as well as in the generation of metabolites that change the activation pattern of chemosensory receptors. Oxidoreductases, especially aldehyde dehydrogenases and aldo-keto reductases, are the first barrier against aldehyde compounds, which include numerous odorants. Using a mass spectrometry approach, we characterized the most highly expressed members of these families in the human nasal mucus sampled in the olfactory vicinity. Their expression was also demonstrated using immunohistochemistry in human epitheliums sampled in the olfactory vicinity. Recombinant enzymes corresponding to three highly expressed human oxidoreductases (ALDH1A1, ALDH3A1, AKR1B10) were used to demonstrate the high enzymatic activity of these enzymes toward aldehyde odorants. The structure‒function relationship set based on the enzymatic parameters characterization of a series of aldehyde odorant compounds was supported by the X-ray structure resolution of human ALDH3A1 in complex with octanal.

Role of Insect and Mammal Glutathione Transferases in Chemoperception.

Glutathione transferases (GSTs) are ubiquitous key enzymes with different activities as transferases or isomerases. As key detoxifying enzymes, GSTs are expressed in the chemosensory organs. They fulfill an essential protective role because the chemosensory organs are located in the main entry paths of exogenous compounds within the body. In addition to this protective function, they modulate the perception process by metabolizing exogenous molecules, including tastants and odorants. Chemosensory detection involves the interaction of chemosensory molecules with receptors. GST contributes to signal termination by metabolizing these molecules. By reducing the concentration of chemosensory molecules before receptor binding, GST modulates receptor activation and, therefore, the perception of these molecules. The balance of chemoperception by GSTs has been shown in insects as well as in mammals, although their chemosensory systems are not evolutionarily connected. This review will provide knowledge supporting the involvement of GSTs in chemoperception, describing their localization in these systems as well as their enzymatic capacity toward odorants, sapid molecules, and pheromones in insects and mammals. Their different roles in chemosensory organs will be discussed in light of the evolutionary advantage of the coupling of the detoxification system and chemosensory system through GSTs.

Structural insights into the interactions of glutathione transferases with a nitric oxide carrier and sodium nitroprusside.

Glutathione transferases are detoxification enzymes with multifaceted roles, including a role in the metabolism and scavenging of nitric oxide (NO) compounds in cells. Here, we explored the ability of Trametes versicolor glutathione transferases (GSTs) from the Omega class (TvGSTOs) to bind metal-nitrosyl compounds. TvGSTOs have been studied previously for their ligandin role and are interesting models to study protein‒ligand interactions. First, we determined the X-ray structure of the TvGSTO3S isoform bound to the dinitrosyl glutathionyl iron complex (DNGIC), a physiological compound involved in the storage of nitric oxide. Our results suggested a different binding mode compared to the one previously described in human GST Pi 1 (GSTP1). Then, we investigated the manner in which TvGSTO3S binds three nonphysiological metal-nitrosyl compounds with different metal cores (iron, ruthenium and osmium). We assayed sodium nitroprusside, a well-studied vasodilator used in cases of hypertensive crises or heart failure. Our results showed that the tested GST can bind metal-nitrosyls at two distinct binding sites. Thermal shift analysis with six isoforms of TvGSTOs identified TvGSTO6S as the best interactant. Using the Griess method, TvGSTO6S was found to improve the release of nitric oxide from sodium nitroprusside in vitro, whereas the effects of human GST alpha 1 (GSTA1) and GSTP1 were moderate. Our results open new structural perspectives for understanding the interactions of glutathione transferases with metal-nitrosyl compounds associated with the biochemical mechanisms of NO uptake/release in biological systems.

Metabolism of Cysteine Conjugates and Production of Flavor Sulfur Compounds by a Carbon-Sulfur Lyase from the Oral Anaerobe Fusobacterium nucleatum.

Flavor perception is a key factor in the acceptance or rejection of food. Aroma precursors such as cysteine conjugates are present in various plant-based foods and are metabolized into odorant thiols in the oral cavity. To date, the involved enzymes are unknown, despite previous studies pointing out the likely involvement of carbon-sulfur lyases (C-S lyases) from the oral microbiota. In this study, we show that saliva metabolizes allyl-cysteine into odorant thiol metabolites, with evidence suggesting that microbial pyridoxal phosphate-dependent C-S lyases are involved in the enzymatic process. A phylogenetic analysis of PatB C-S lyase sequences in four oral subspecies of Fusobacterium nucleatum was carried out and led to the identification of several putative targets. FnaPatB1 from F. nucleatum subspecies animalis, a putative C-S lyase, was characterized and showed high activity with a range of cysteine conjugates. Enzymatic and X-ray crystallographic data showed that FnaPatB1 metabolizes cysteine derivatives within a unique active site environment that enables the formation of flavor sulfur compounds. Using an enzymatic screen with a library of pure compounds, we identified several inhibitors able to reduce the C-S lyase activity of FnaPatB1 in vitro, which paves the way for controlling the release of odorant sulfur compounds from their cysteine precursors in the oral cavity.

Functional Characterization of the Venus Flytrap Domain of the Human TAS1R2 Sweet Taste Receptor.

The human sweet taste receptor is a heterodimeric receptor composed of two distinct G-protein-coupled receptors (GPCRs), TAS1R2 and TAS1R3. The TAS1R2 and TAS1R3 subunits are members of a small family of class C GPCRs whose members share the same architecture, comprising a Venus Flytrap (VFT) module linked to the seven transmembrane domains (TMDs) by a short cysteine-rich region (CRR). The VFT module of TAS1R2 contains the primary binding site for most of the sweet-tasting compounds, including natural sugars and artificial and natural sweeteners. However, cellular assays, molecular docking and site-directed mutagenesis studies have revealed that the VFT, CRR and TMD of TAS1R3 interact with some sweeteners, including the sweet-tasting protein brazzein. The aim of this study was to better understand the contribution of TAS1R2-VFT in the binding of sweet stimuli. To achieve this, we heterologously expressed human TAS1R2-VFT (hTAS1R2-VFT) in Escherichia coli. Circular dichroism spectroscopic studies revealed that hTAS1R2-VFT was properly folded with evidence of secondary structures. Using size-exclusion chromatography coupled with light scattering, we found that hTAS1R2-VFT behaves as a monomer. Ligand binding quantified by intrinsic tryptophan fluorescence showed that hTAS1R2-VFT is capable of binding sweet stimuli with Kd values, in agreement with physiological detection. Furthermore, we investigated whether the impact of point mutations, already shown to have deleterious effects on cellular assays, could impact the ability of hTAS1R2-VFT to bind sweet ligands. As expected, the ligand affinities of hTAS1R2-VFT were drastically reduced through the introduction of single amino acid substitutions (D278A and E382A) known to abolish the response of the full-length TAS1R2/TAS1R3 receptor. This study demonstrates the feasibility of producing milligram quantities of hTAS1R2-VFT to further characterize the mechanism of binding interaction and perform structural studies.

Nasal Odorant Competitive Metabolism Is Involved in the Human Olfactory Process.

Within the peripheral olfactory process, odorant metabolizing enzymes are involved in the active biotransformation of odorants, thus influencing the intensity and quality of the signal, but little evidence exists in humans. Here, we characterized the fast nasal metabolism of the food aroma pentane-2,3-dione in vivo and identified two resulting metabolites in the nasal-exhaled air, supporting the metabolizing role of the dicarbonyl/l-xylulose reductase. We showed in vitro, using the recombinant enzyme, that pentane-2,3-dione metabolism was inhibited by a second odorant (e.g., butanoic acid) according to an odorant-odorant competitive metabolic mechanism. Hypothesizing that such mechanism exists in vivo, pentane-2,3-dione, presented with a competitive odorant, both at subthreshold concentrations, was actually significantly perceived, suggesting an increase in its nasal availability. Our results, suggesting that odorant metabolizing enzymes can balance the relative detection of odorants in a mixture, in turn influencing the intensity of the signal, should be considered to better manage flavor perception in food.

Expression Patterns of Drosophila Melanogaster Glutathione Transferases.

Glutathione transferases (GSTs) are ubiquitous enzymes that catalyze the conjugation of glutathione to various molecules. Among the 42 GSTs identified in Drosophila melanogaster, Delta and Epsilon are the largest classes, with 25 members. The Delta and Epsilon classes are involved in different functions, such as insecticide resistance and ecdysone biosynthesis. The insect GST number variability is due mainly to these classes. Thus, they are generally considered supports during the evolution for the adaptability of the insect species. To explore the link between Delta and Epsilon GST and their evolution, we analyzed the sequences using bioinformatic tools. Subgroups appear within the Delta and Epsilon GSTs with different levels of diversification. The diversification also appears in the sequences showing differences in the active site. Additionally, amino acids essential for structural stability or dimerization appear conserved in all GSTs. Quantitative real-time polymerase chain reaction (qRT-PCR) analysis revealed that the transcripts corresponding to these two classes are heterogeneously expressed within D. melanogaster. Some GSTs, such as GSTD1, are highly expressed in all tissues, suggesting their general function in detoxification. Conversely, some others, such as GSTD11 or GSTE4, are specifically expressed at a high level specifically in antennae, suggesting a potential role in olfaction.

Role of human salivary enzymes in bitter taste perception.

The molecules that elicit taste sensation are perceived by interacting with the taste receptors located in the taste buds. Enzymes involved in the detoxification processes are found in saliva as well as in type II cells, where taste receptors, including bitter taste receptors, are located. These enzymes are known to interact with a large panel of molecules. To explore a possible link between these enzymes and bitter taste perception, we demonstrate that salivary glutathione transferases (GSTA1 and GSTP1) can metabolize bitter molecules. To support these abilities, we solve three X-ray structures of these enzymes in complexes with isothiocyanates. Salivary GSTA1 and GSTP1 are expressed in a large panel of subjects. Additionally, GSTA1 levels in the saliva of people suffering from taste disorders are significantly lower than those in the saliva of the control group.

Leptin-Induced HLA-G Inhibits Myometrial Contraction and Differentiation.

Maternal obesity is associated with a wide spectrum of labour disorders, including preterm birth. Leptin, a pro-inflammatory adipokine and a key factor of obesity, is suspected to play a major role in these disorders. OB-R, its receptor, is expressed on macrophages and myocytes, two cell types critical for labour onset. Macrophages secrete reactive oxygen species/pro-inflammatory cytokines, responsible for myometrial differentiation while myocytes control uterine contractions. In this study, we assessed the effect of leptin on myometrial contraction and differentiation using our validated co-culture model of human primary macrophages and myocytes. We demonstrated that leptin had a different effect on myocytes and macrophages depending on the dose. A low leptin concentration induced a tocolytic effect by preventing myocytes' contraction, differentiation, and macrophage-induced ROS production. Additionally, leptin led to an increase in HLA-G expression, suggesting that the tocolytic effect of leptin may be driven by HLA-G, a tolerogenic molecule. Finally, we observed that recombinant HLA-G also prevented LPS-induced ROS production by macrophages. Altogether, these data provide a putative molecular mechanism by which leptin may induce immune tolerance and therefore interfere with labour-associated mechanisms. Therefore, HLA-G represents a potential innovative therapeutic target in the pharmacological management of preterm labour.

Effects of oenological tannins on aroma release and perception of oxidized and non-oxidized red wine: A dynamic real-time in-vivo study coupling sensory evaluation and analytical chemistry.

Addition of oenological tannins claims to have a positive impact on wine stability, protection from oxidation and likely sensory persistence. However, their role on red wine aroma during oxidation is controversial. The present study aims at investigating the effect of addition of oenological tannins on wine flavour (mainly aroma) before and after air exposure. Temporal Dominance of Sensations, a dynamic sensory evaluation, was coupled with a dynamic chemical measurement (nosespace analysis) using a Proton-Transfer-Reaction Mass-Spectrometer connected to the nasal cavity of 17 assessors. Results showed that the oxidation of a non-oaked Pinot Noir red wine decreases the fruity aroma dominance and increases the maderised and prune one. A contextual decrease of the fruity ethyl decanoate and increase of oxidative Strecker aldehydes are observed. Ellagitannins but not proanthocyanidins preserved perception of fruitiness and prevented increase of maderised notes. Moreover, ellagitannins increase the aroma persistence mainly in the non-oxidized wine.