Substitution of both histidines in the active site of yeast alcohol dehydrogenase 1 exposes underlying pH dependencies.

Histidine residues 44 and 48 in yeast alcohol dehydrogenase (ADH) bind to the coenzymes NAD(H) and contribute to catalysis. The individual H44R and H48Q substitutions alter the kinetics and pH dependencies, and now the roles of other ionizable groups in the enzyme were studied in the doubly substituted H44R/H48Q ADH. The substitutions make the enzyme more resistant to inactivation by diethyl pyrocarbonate, modestly improve affinity for coenzymes, and substantially decrease catalytic efficiencies for ethanol oxidation and acetaldehyde reduction. The pH dependencies for several kinetic parameters are shifted from pK values for wild-type ADH of 7.3-8.1 to values for H44R/H48Q ADH of 8.0-9.6, and are assigned to the water or alcohol bound to the catalytic zinc. It appears that the rate of binding of NAD+ is electrostatically favored with zinc-hydroxide whereas binding of NADH is faster with neutral zinc-water. The pH dependencies of catalytic efficiencies (V/EtKm) for ethanol oxidation and acetaldehyde reduction are similarly controlled by deprotonation and protonation, respectively. The substitutions make an enzyme that resembles the homologous horse liver H51Q ADH, which has Arg-47 and Gln-51 and exhibits similar pK values. In the wild-type ADHs, it appears that His-48 (or His-51) in the proton relay systems linked to the catalytic zinc ligands modulate catalytic efficiencies.

Preparation, assay, and application of 4-fluorothreonine transaldolase from Streptomyces sp. MA37 for ß-hydroxyl amino acid derivatives.

ß-Hydroxy-α-amino acids (ßHAAs) are an essential class of building blocks of therapeutically important compounds and complex natural products. They contain two chiral centers at Cα and Cß positions, resulting in four possible diastereoisomers. Many innovative asymmetric syntheses have been developed to access structurally diverse ßHAAs. The main challenge, however, is the control of the relative and absolute stereochemistry of the asymmetric carbons in a sustainable way. In this respect, there has been considerable attention focused on the chemoenzymatic synthesis of ßHAAs via a one-step process. Nature has evolved different enzymatic routes to produce these valuable ßHAAs. Among these naturally occurring transformations, L-threonine transaldolases present potential biocatalysts to generate ßHAAs in situ. 4-Fluorothreonine transaldolase from Streptomyces sp. MA37 (FTaseMA) catalyzes the cross-over transaldolation reaction between L-Thr and fluoroacetaldehyde to give 4-fluorothreonine and acetaldehyde (Ad). It has been demonstrated that FTaseMA displays considerable substrate plasticity toward structurally diverse aldehyde acceptors, leading to the production of various ßHAAs. In this chapter, we describe methods for the preparation of FTaseMA, and the chemoenzymatic synthesis of ßHAAs from various aldehydes and L-Thr using FTaseMA.

1H NMR based sulfonation reaction kinetics of wine relevant thiols in comparison with known carbonyls.

Sulfite addition is a common tool for ensuring wines' oxidative stability via the activity of its free and weakly bound molecular fraction. As a nucleophile, bisulfite forms covalent adducts with wine's most relevant electrophiles, such as carbonyls, polyphenols, and thiols. The equilibrium in these reactions is often represented as dissociation rather than formation. Recent studies from our laboratory demonstrate, first, the acetaldehyde sulfonate dissociation, and second, the chemical stability of cysteine and epicatechin sulfonates under wine aging conditions. Thus, the objective of this study was to monitor by 1H NMR the binding specificity of known carbonyl-derived SO2 binders (acetaldehyde and pyruvic acid) in the presence of S-containing compounds (cysteine and glutathione). We report that during simulated wine aging, the sulfur dioxide that is rapidly bound to carbonyl compounds will be released and will bind to cysteine and glutathione, demonstrating the long-term sulfur dioxide binding potential of S-containing compounds. These results are meant to serve as a complement to existing literature reviews focused on molecular markers related to wines' oxidative stability and emphasize once more the importance of S-containing compounds in wine aging chemical mechanisms.

Highly Enhanced Photocatalytic NO Removal and Inhibited Peroxyacetyl Nitrate Formation in Synergistic Acetaldehyde Degradation.

The coexistence of NO and CH3CHO in the air is considered to produce secondary peroxyacetyl nitrate (PAN) under sunlight irradiation, threatening the ecological environment and public health. Herein, we provide a simple strategy for the photocatalytic removal of NO and acetaldehyde (CH3CHO) on Sr2Sb2O7. In comparison with the single removal, the nearly complete removal of NO is reached by deep oxidation to NO3- with the assistance of CH3CHO. The underlying mechanism is revealed by GC-MS, in situ DRIFTS, and density functional theory calculations. The intermediates •CH3 from CH3CHO and NO2- from NO tend to bond and further oxidize to CH3ONO2, thus promoting NO removal. CH3NO2 and CH3ONO2 are the key products instead of PAN on Sr2Sb2O7 from the synergistic degradation of NO and CH3CHO. This work brings new insights into reaction pathway regulation for promoting performance and suppressing byproducts during synergistic air pollutant removal.

Optimized enantioselective (S)-2-hydroxypropiophenone synthesis by free- and encapsulated-resting cells of Pseudomonas putida.

BACKGROUND: Aromatic α-hydroxy ketones, such as S-2-hydroxypropiophenone (2-HPP), are highly valuable chiral building blocks useful for the synthesis of various pharmaceuticals and natural products. In the present study, enantioselective synthesis of 2-HPP was investigated by free and immobilized whole cells of Pseudomonas putida ATCC 12633 starting from readily-available aldehyde substrates. Whole resting cells of P. putida, previously grown in a culture medium containing ammonium mandelate, are a source of native benzoylformate decarboxylase (BFD) activity. BFD produced by induced P. putida resting cells is a highly active biocatalyst without any further treatment in comparison with partially purified enzyme preparations. These cells can convert benzaldehyde and acetaldehyde into the acyloin compound 2-HPP by BFD-catalyzed enantioselective cross-coupling reaction. RESULTS: The reaction was carried out in the presence of exogenous benzaldehyde (20 mM) and acetaldehyde (600 mM) as substrates in 6 mL of 200 mM phosphate buffer (pH 7) for 3 h. The optimal biomass concentration was assessed to be 0.006 g dry cell weight (DCW) mL- 1. 2-HPP titer, yield and productivity using the free cells were 1.2 g L- 1, 0.56 g 2-HPP/g benzaldehyde (0.4 mol 2-HPP/mol benzaldehyde), 0.067 g 2-HPP g- 1 DCW h- 1, respectively, under optimized biotransformation conditions (30 °C, 200 rpm). Calcium alginate (CA)-polyvinyl alcohol (PVA)-boric acid (BA)-beads were used for cell entrapment. Encapsulated whole-cells were successfully employed in four consecutive cycles for 2-HPP production under aerobic conditions without any noticeable beads degradation. Moreover, there was no production of benzyl alcohol as an unwanted by-product. CONCLUSIONS: Bioconversion by whole P. putida resting cells is an efficient strategy for the production of 2-HPP and other α-hydroxyketones.

Malondialdehyde trapping by food phenolics.

The reactions between malondialdehyde and 2,5-dimethylresorcinol, orcinol, olivetol, and alkylresocinols were studied in an attempt to investigate both if this lipid oxidation product is trapped by phenolics analogously to other reactive carbonyls and to elucidate the chemical structures of the produced adducts. After being formed, malondialdehyde is both partially fractionated to acetaldehyde and oligomerized into dimers and trimers. All these compounds react with phenolics producing three main kinds of derivatives: 5(or 7)-alkyl-7(or 5)-hydroxy-4-methyl-4H-chromene-3-carbaldehydes, 7-alkyl-9-hydroxy-6H-2,6-methanobenzo[d][1,3]dioxocine-5-carbaldehydes, and 4-(3-formylphenyl)-7-hydroxy-4H-chromene-3-carbaldehydes. A total of twenty-four adducts were isolated by semipreparative high-performance liquid chromatography (HPLC) and characterized by mono- and bi-dimensional nuclear magnetic resonance (NMR) spectroscopy and mass spectrometry (MS). Reaction pathways to explain the formation of all these compounds are proposed. Obtained results show that phenolics can trap malondialdehyde producing stable derivatives. The function(s) that such derivatives can play in foods remain(s) to be elucidated.

Acetaldehyde makes a distinct mutation signature in single-stranded DNA.

Acetaldehyde (AA), a by-product of ethanol metabolism, is acutely toxic due to its ability to react with various biological molecules including DNA and proteins, which can greatly impede key processes such as replication and transcription and lead to DNA damage. As such AA is classified as a group 1 carcinogen by the International Agency for Research on Cancer (IARC). Previous in vitro studies have shown that AA generates bulky adducts on DNA, with signature guanine-centered (GG→TT) mutations. However, due to its weak mutagenicity, short chemical half-life, and the absence of powerful genetic assays, there is considerable variability in reporting the mutagenic effects of AA in vivo. Here, we used an established yeast genetic reporter system and demonstrate that AA treatment is highly mutagenic to cells and leads to strand-biased mutations on guanines (G→T) at a high frequency on single stranded DNA (ssDNA). We further demonstrate that AA-derived mutations occur through lesion bypass on ssDNA by the translesion polymerase Polζ. Finally, we describe a unique mutation signature for AA, which we then identify in several whole-genome and -exome sequenced cancers, particularly those associated with alcohol consumption. Our study proposes a key mechanism underlying carcinogenesis by acetaldehyde-mutagenesis of single-stranded DNA.

Malondialdehyde Acetaldehyde-Adduction Changes Surfactant Protein D Structure and Function.

Alcohol consumption with concurrent cigarette smoking produces malondialdehyde acetaldehyde (MAA)-adducted lung proteins. Lung surfactant protein D (SPD) supports innate immunity via bacterial aggregation and lysis, as well as by enhancing macrophage-binding and phagocytosis. MAA-adducted SPD (SPD-MAA) has negative effects on lung cilia beating, macrophage function, and epithelial cell injury repair. Because changes in SPD multimer structure are known to impact SPD function, we hypothesized that MAA-adduction changes both SPD structure and function. Purified human SPD and SPD-MAA (1 mg/mL) were resolved by gel filtration using Sephadex G-200 and protein concentration of each fraction determined by Bradford assay. Fractions were immobilized onto nitrocellulose by slot blot and assayed by Western blot using antibodies to SPD and to MAA. Binding of SPD and SPD-MAA was determined fluorometrically using GFP-labeled Streptococcus pneumoniae (GFP-SP). Anti-bacterial aggregation of GFP-SP and macrophage bacterial phagocytosis were assayed by microscopy and permeability determined by bacterial phosphatase release. Viral injury was measured as LDH release in RSV-treated airway epithelial cells. Three sizes of SPD were resolved by gel chromatography as monomeric, trimeric, and multimeric forms. SPD multimer was the most prevalent, while the majority of SPD-MAA eluted as trimer and monomer. SPD dose-dependently bound to GFP-SP, but SPD-MAA binding to bacteria was significantly reduced. SPD enhanced, but MAA adduction of SPD prevented, both aggregation and macrophage phagocytosis of GFP-SP. Likewise, SPD increased bacterial permeability while SPD-MAA did not. In the presence of RSV, BEAS-2B cell viability was enhanced by SPD, but not protected by SPD-MAA. Our results demonstrate that MAA adduction changes the quaternary structure of SPD from multimer to trimer and monomer leading to a decrease in the native anti-microbial function of SPD. These findings suggest one mechanism for increased pneumonia observed in alcohol use disorders.

The reaction of acetaldehyde, glyoxal, and ammonia to yield 2-methylimidazole: thermodynamic and kinetic analyses of the mechanism.

The most thermodynamically and kinetically favorable pathways for the formation of 2-methylimidazole (2MI) in the reaction of glyoxal and acetaldehyde with ammonia in aqueous solution have been determined. The formation of 2MI proceeds through a number of successive intermediates of acyclic and cyclic structures, and the most favorable route (thermodynamically and kinetically) for the formation of the imidazole ring is the condensation of amine intermediates, in contrast to the existing concepts of imine structures. The limiting stage is the stage of cyclization involving the intramolecular attack by the amino group of the precyclic intermediate on the carbon atom bound to the hydroxyl group with the simultaneous release of a water molecule according to the SN2 mechanism. Further stages of stepwise dehydration lead to the formation of a cyclic diazine, the intramolecular migration of the proton of the tertiary carbon atom to the nitrogen atom of which completes the formation of 2MI. Experimental studies on the effect of the order of mixing of initial reagents on the 2MI yield confirmed the quantum-chemically substantiated favorable pathway for the formation of 2MI during the interaction of amine intermediates, and also revealed that the selectivity of the 2MI formation is achieved by successive mixing of acetaldehyde with ammonia until the formation of hydroxyamine products and their further interaction with glyoxal.

Photocatalytic exoskeleton: Chitin nanofiber for retrievable and sustainable TiO2 carriers for the decomposition of various pollutants.

Loading a photocatalytic TiO2 to organic carriers has been desired for volumetric TiO2 incorporation, facile retrieval, and sustainable utilization. Traditionally, suspended TiO2 nanoparticles or its thin film on two-dimensional substrate are popularly fabricated for pollutants decomposition without carriers; due to poor thermomechanical properties of the organic carriers. Herein, a combination of the chitin nanofiber carrier and atomic layer deposition proves relevance for formation of anatase TiO2 thin layer so that photocatalytic decomposition in three-dimensional surface. Moreover, chitin nanofiber is capable of holding the TiO2 nanoparticles for multiple cycles of photocatalysis. Those types of TiO2 show characteristic degradation performance for gaseous (acetaldehyde) and aqueous pollutants (4-chlorophenol and rhodamine B). After catalytic reaction, chitin/TiO2 is retrievable owing to carrier's robustness even in water without TiO2 aggregation and loss. This work suggests that chitin-based photocatalyst is applicable to numerous pollutants through chitin's relatively high chemical resistance and stably wedged TiO2 during photocatalytic reaction.

Chemoselective and Highly Sensitive Quantification of Gut Microbiome and Human Metabolites.

The microbiome has a fundamental impact on the human host's physiology through the production of highly reactive compounds that can lead to disease development. One class of such compounds are carbonyl-containing metabolites, which are involved in diverse biochemical processes. Mass spectrometry is the method of choice for analysis of metabolites but carbonyls are analytically challenging. Herein, we have developed a new chemical biology tool using chemoselective modification to overcome analytical limitations. Two isotopic probes allow for the simultaneous and semi-quantitative analysis at the femtomole level as well as qualitative analysis at attomole quantities that allows for detection of more than 200 metabolites in human fecal, urine and plasma samples. This comprehensive mass spectrometric analysis enhances the scope of metabolomics-driven biomarker discovery. We anticipate that our chemical biology tool will be of general use in metabolomics analysis to obtain a better understanding of microbial interactions with the human host and disease development.

Modification of a spectrophotometric method for assessment of monoamine oxidase activity with 2,4-dinitrophenylhydrazine as a derivatizing reagent.

The aim of the study was to modify a simple and widely used spectrophotometric assay for MAO activity evaluation with 2,4-dinitrophenylhydrazine. A modified procedure includes molar absorption coefficients of 2,4-DNP-hydrazone benzaldehyde and 2,4-DNP-hydrazone 5-hydroxyindolylacetaldehyde as 2.3 × 104mol-1l cm-1 and 1.0 × 104 mol-1l cm-1, respectively. Such an approach allows to express specific enzyme activity as nmol product formed/min/mg protein.

The Management of Dissolved Oxygen by a Polypropylene Hollow Fiber Membrane Contactor Affects Wine Aging.

BACKGROUND: Numerous oenological practices can cause an excess of dissolved oxygen in wine, thus determining sensory and chromatic defects in the short- to long-term. Hence, it is necessary to manage the excess of oxygen before bottling. METHODS: In this study, the management of the dissolved oxygen content by a polypropylene hollow fiber membrane contactor apparatus was performed in two wines from different grape varieties (Aglianico and Falanghina). The wines were analyzed after an 11-month aging. Anthocyanins and acetaldehyde content were evaluated by HPLC. In addition, other phenolic compounds and chromatic characteristics were analyzed by spectrophotometric methods. NMR and HR ESIMS analyses were conducted to evaluate the amount of pyranoanthocyanins and polymeric pigments. RESULTS: After 11 months of aging, in both wines a decrease of free and total SO2 with respect to initial values was detected. In the wines with the highest dissolved oxygen levels, a more remarkable loss was observed. No significant differences in terms of color parameters were detected. In red wine with the highest oxygen content, a massive formation of polymeric pigments and BSA reactive tannins was observed, as opposed to wines with lower oxygen levels. CONCLUSION: The study demonstrated that the membrane contactor can prove a successful tool to manage dissolved oxygen in wines as to prevent their oxidative spoilage.

Ensembling machine learning models to boost molecular affinity prediction.

This study unites six popular machine learning approaches to enhance the prediction of a molecular binding affinity between receptors (large protein molecules) and ligands (small organic molecules). Here we examine a scheme where affinity of ligands is predicted against a single receptor - human thrombin, thus, the models consider ligand features only. However, the suggested approach can be repurposed for other receptors. The methods include Support Vector Machine, Random Forest, CatBoost, feed-forward neural network, graph neural network, and Bidirectional Encoder Representations from Transformers. The first five methods use input features based on physico-chemical properties of molecules, while the last one is based on textual molecular representations. All approaches do not rely on atomic spatial coordinates, avoiding a potential bias from known structures, and are capable of generalizing for compounds with unknown conformations. Within each of the methods, we have trained two models that solve classification and regression tasks. Then, all models are grouped into a pipeline of two subsequent ensembles. The first ensemble aggregates six classification models which vote whether a ligand binds to a receptor or not. If a ligand is classified as active (i.e., binds), the second ensemble predicts its binding affinity in terms of the inhibition constant Ki.

Molecular recognition of a single-chain Fv antibody specific for GA-pyridine, an advanced glycation end-product (AGE), elucidated using biophysical techniques and synthetic antigen analogues.

Advanced glycation end-products (AGEs) are a heterogeneous group of compounds formed by non-enzymatic reaction between reducing-sugar and Arg/Lys in proteins and are involved in various diabetic complications. GA-pyridine is derived from glycolaldehyde and is one of the most cytotoxic AGEs. Here, we established a single-chain Fv (scFv) antibody against GA-pyridine, 73MuL9-scFv, and examined the details of its specificity and antigen recognition by using various techniques involving biophysics, chemical biology and structural biology. We also synthesized several compounds that differ slightly in regard to the position and number of GA-pyridine substituent groups, and revealed that GA-pyridine was specifically bound to 73MuL9-scFv. Thermodynamic analysis revealed that the association of GA-pyridine to 73MuL9-scFv was an exothermic and enthalpy driven reaction, and thus that the antigen recognition involved multiple specific interactions. Crystallographic analysis of the Fv fragment of 73MuL9-scFv revealed that several CH-π and hydrogen bond interactions took place between the Fv-fragment and GA-pyridine, which was consistent with the results of thermodynamic analysis. Further studies using 73MuL9-scFv as a tool to clarify the relevance of GA-pyridine to diabetic complications are warranted.

Insights on the role of acetaldehyde and other aldehydes in the odour and tactile nasal perception of red wine.

Wine models with or without a dearomatised and lyophilized red wine extract containing a young red aroma base (control) plus one vector with one or several aroma compounds (unsaturated-aldehydes, saturated-aldehydes, benzaldehyde, isoamyl-alcohol, methoxypyrazines and (Z)-1,5-octadien-3-one) were prepared. Models were spiked with increasing amounts of acetaldehyde whose headspace concentrations were controlled. Odour and nasal chemesthesic properties were assessed by a trained sensory panel. Results confirm the contribution of the different players, notably isoamyl-alcohol, (Z)-1,5-octadien-3-one, benzaldehyde and methoxypyrazines, to wine aroma and tactile nasal characteristics and demonstrate that acetaldehyde levels play an outstanding role in their modulation. At low levels, it can play positive roles in some specific aromatic contexts, while at higher levels, enhance the negative effects associated to the generic presence of other aldehydes (saturated, unsaturated and Strecker aldehydes) by enhancing "green vegetable" notes and "itching" character and the "burning" effects linked to high levels of isoamyl alcohol.

Interaction pattern of histidine, carnosine and histamine with methylglyoxal and other carbonyl compounds.

The ability of histidine to scavenge sugar-derived 1,2-dicarbonyl compounds was investigated using aqueous methanolic model systems containing histidine or histamine in the presence of glucose, methylglyoxal, or glyoxal. The samples were prepared either at room temperature (RT) or at 150 °C and analyzed using ESI-qTOF-MS/MS and isotope labeling technique. Replacing glucose with [U-13C6]glucose allowed the identification of glucose carbon atoms incorporated in the products. Various sugar-generated carbonyl compounds ranging in size from C1 to C6 were captured by histidine or histamine. The majority of the fragments incorporated were either C3 or C2 units originating from glyoxal (C2) or methylglyoxal (C3). The ESI-qTOF-MS/MS analysis indicated that histamine could react with either of the two carbonyl carbons of methylglyoxal utilizing the α-amino group and/or the imidazolium moiety. Furthermore, when histidine was added to 2-amino-1-methyl-6-phenylimidazo(4,5-b)pyridine (PhIP) generating model system, it completely suppressed the formation of PhIP due to scavenging of phenylacetaldehyde.

Identification of New Markers of Alcohol-Derived DNA Damage in Humans.

Alcohol consumption is a risk factor for the development of several cancers, including those of the head and neck and the esophagus. The underlying mechanisms of alcohol-induced carcinogenesis remain unclear; however, at these sites, alcohol-derived acetaldehyde seems to play a major role. By reacting with DNA, acetaldehyde generates covalent modifications (adducts) that can lead to mutations. Previous studies have shown a dose dependence between levels of a major acetaldehyde-derived DNA adduct and alcohol exposure in oral-cell DNA. The goal of this study was to optimize a mass spectrometry (MS)-based DNA adductomic approach to screen for all acetaldehyde-derived DNA adducts to more comprehensively characterize the genotoxic effects of acetaldehyde in humans. A high-resolution/-accurate-mass data-dependent constant-neutral-loss-MS3 methodology was developed to profile acetaldehyde-DNA adducts in purified DNA. This resulted in the identification of 22 DNA adducts. In addition to the expected N2-ethyldeoxyguanosine (after NaBH3CN reduction), two previously unreported adducts showed prominent signals in the mass spectra. MSn fragmentation spectra and accurate mass were used to hypothesize the structure of the two new adducts, which were then identified as N6-ethyldeoxyadenosine and N4-ethyldeoxycytidine by comparison with synthesized standards. These adducts were quantified in DNA isolated from oral cells collected from volunteers exposed to alcohol, revealing a significant increase after the exposure. In addition, 17 of the adducts identified in vitro were detected in these samples confirming our ability to more comprehensively characterize the DNA damage deriving from alcohol exposures.

Efficient biosynthesis of (2S, 3R)-4-methylsulfonylphenylserine by artificial self-assembly of enzyme complex combined with an intensified acetaldehyde elimination system.

(2S, 3R)-4-methylsulfonylphenylserine [(2S, 3R)-MPS], a key chiral precursor for antibiotics florfenicol and thiamphenicol, could be asymmetrically synthesized by l-threonine transaldolase (LTTA) coupled with an acetaldehyde elimination system. The low efficiency of acetaldehyde elimination system blocked further accumulation of (2S, 3R)-MPS. To address this issue, strengthening acetaldehyde elimination system and enzyme self-assembly strategy were combined to accelerate biosynthesis of (2S, 3R)-MPS. The new multi-enzyme cascade with intensified acetaldehyde elimination system BL21 (PsLTTAD2/ScADH/BtGDH) could produce (2S, 3R)-MPS with a titer of 157.6 mM, 1.7-folds than that produced by the original system BL21 (PsLTTAD2/ApADH/CbFDH). Moreover, self-assembly of PsLTTAD2 and ScADH by respective fusion of SpyTag and SpyCatcher were carried out to develop a self-assembled multi-enzyme cascade BL21 (ST-PsLTTAD2/SC-ScADH/BtGDH). As a result, the yield of (2S, 3R)-MPS was up to 248.1 mM with 95% de. As far as we knew, that represented the highest yield of (2S, 3R)-MPS by enzymatic synthesis, and therefore was a promising and green route for industrial production of this valuable compound.