Thermal oxidation mechanism of palmitic aicd.

The oxidation and degradation of fats lead to a decrease in the nutritional value of food and pose safety concerns. Saturated fatty acids also hold a significant position in the field of lipid oxidation. In this study, the oxidation products of methyl palmitate were investigated by using gas chromatography mass spectrometry (GC-MS). Seven monohydroperoxides and 72 secondary oxidation products were detected. Combined with density functional theory (DFT) calculations, the formation mechanisms of oxidation products can be summarized into four stages. The initial stage involved the formation of monohydroperoxides and alkanes, followed by the subsequent stage involving methyl x-oxo(hydroxy)hexadecanoates. The third stage involved the formation of methyl ketones, carboxylic acids, and aldehydes, while the final stage involved lactones. Meanwhile, methyl ketones were the most abundant oxidation product, approximately 25 times more abundant than aldehydes; the calculated results agreed well with the experimental results. The establishment of a comprehensive thermal oxidation mechanism for palmitic acid provided a new foundation for future lipid oxidation analyses.

Anti-Biofilm Activity of Oleacein and Oleocanthal from Extra-Virgin Olive Oil toward Pseudomonas aeruginosa.

New antimicrobial molecules effective against Pseudomonas aeruginosa, known as an antibiotic-resistant "high-priority pathogen", are urgently required because of its ability to develop biofilms related to healthcare-acquired infections. In this study, for the first time, the anti-biofilm and anti-virulence activities of a polyphenolic extract of extra-virgin olive oil as well as purified oleocanthal and oleacein, toward P. aeruginosa clinical isolates were investigated. The main result of our study was the anti-virulence activity of the mixture of oleacein and oleocanthal toward multidrug-resistant and intermediately resistant strains of P. aeruginosa isolated from patients with ventilator-associated pneumonia or surgical site infection. Specifically, the mixture of oleacein (2.5 mM)/oleocanthal (2.5 mM) significantly inhibited biofilm formation, alginate and pyocyanin production, and motility in both P. aeruginosa strains (p < 0.05); scanning electron microscopy analysis further evidenced its ability to inhibit bacterial cell adhesion as well as the production of the extracellular matrix. In conclusion, our results suggest the potential application of the oleacein/oleocanthal mixture in the management of healthcare-associated P. aeruginosa infections, particularly in the era of increasing antimicrobial resistance.

Synthesizing Amino Acids Modified with Reactive Carbonyls in Silico to Assess Structural Effects Using Molecular Dynamics Simulations.

Protein carbonylation by reactive aldehydes derived from lipid peroxidation leads to cross-linking, oligomerization, and aggregation of proteins, causing intracellular damage, impaired cell functions, and, ultimately, cell death. It has been described in aging and several age-related chronic conditions. However, the basis of structural changes related to the loss of function in protein targets is still not well understood. Hence, a route to the in silico construction of new parameters for amino acids carbonylated with reactive carbonyl species derived from fatty acid oxidation is described. The Michael adducts for Cys, His, and Lys with 4-hydroxy-2-nonenal (HNE), 4-hydroxy-2-hexenal (HHE), and a furan ring form for 4-Oxo-2-nonenal (ONE), were built, while malondialdehyde (MDA) was directly attached to each residue. The protocol describes details for the construction, geometry optimization, assignment of charges, missing bonds, angles, dihedral angles parameters, and its validation for each modified residue structure. As a result, structural effects induced by the carbonylation with these lipid derivatives have been measured by molecular dynamics simulations on different protein systems such as the thioredoxin enzyme, bovine serum albumin and the membrane Zu-5-ankyrin domain employing root-mean-square deviation (RMSD), root mean square fluctuation (RMSF), structural secondary prediction (DSSP) and the solvent-accessible surface area analysis (SASA), among others.

An unexpected role of EasDaf: catalyzing the conversion of chanoclavine aldehyde to chanoclavine acid.

Ergot alkaloids (EAs) are a diverse group of indole alkaloids known for their complex structures, significant pharmacological effects, and toxicity to plants. The biosynthesis of these compounds begins with chanoclavine-I aldehyde (CC aldehyde, 2), an important intermediate produced by the enzyme EasDaf or its counterpart FgaDH from chanoclavine-I (CC, 1). However, how CC aldehyde 2 is converted to chanoclavine-I acid (CC acid, 3), first isolated from Ipomoea violacea several decades ago, is still unclear. In this study, we provide in vitro biochemical evidence showing that EasDaf not only converts CC 1 to CC aldehyde 2 but also directly transforms CC 1 into CC acid 3 through two sequential oxidations. Molecular docking and site-directed mutagenesis experiments confirmed the crucial role of two amino acids, Y166 and S153, within the active site, which suggests that Y166 acts as a general base for hydride transfer, while S153 facilitates proton transfer, thereby increasing the acidity of the reaction. KEY POINTS: • EAs possess complicated skeletons and are widely used in several clinical diseases • EasDaf belongs to the short-chain dehydrogenases/reductases (SDRs) and converted CC or CC aldehyde to CC acid • The catalytic mechanism of EasDaf for dehydrogenation was analyzed by molecular docking and site mutations.

Unravelling caramelization and Maillard reactions in glucose and glucose + leucine model cakes: Formation and degradation kinetics of volatile markers extracted during baking.

A large number of volatile compounds are formed during the baking of foods by reactions such as caramelization and Maillard reactions. Elucidating the reaction mechanisms may be useful to predict and control food quality. Ten reaction volatile markers were extracted during baking of solid model cakes implemented with known amounts of precursors (glucose with or without leucine) and then quantified by Thermal desorption-Gas chromatography-Mass spectrometry. The kinetic data showed that the level of air convection in the oven had no significant influence on the reaction rates. In contrast, increasing baking temperatures had a nonlinear accelerating impact on the generation of newly formed volatile compounds with a bell-shaped kinetic curve found for most of the markers at 200 °C. The presence of leucine triggered the activation of the Maillard and Strecker routes with a specific and very rapid formation of 3-Methylbutanal and pyrazines. A dynamic model was developed, combining evaporation flow rate and kinetic formation and consumption of reaction markers. It can be used to describe, for two furanic compounds of different volatilities, the vapor concentrations in the oven from the concentrations measured in the model cakes.

MXene/Hydrogel-based bioelectronic nose for the direct evaluation of food spoilage in both liquid and gas-phase environments.

Various bioelectronic noses have been recently developed for mimicking human olfactory systems. However, achieving direct monitoring of gas-phase molecules remains a challenge for the development of bioelectronic noses due to the instability of receptor and the limitations of its surrounding microenvironment. Here, we report a MXene/hydrogel-based bioelectronic nose for the sensitive detection of liquid and gaseous hexanal, a signature odorant from spoiled food. In this study, a conducting MXene/hydrogel structure was formed on a sensor via physical adsorption. Then, canine olfactory receptor 5269-embedded nanodiscs (cfOR5269NDs) which could selectively recognize hexanal molecules were embedded in the three-dimensional (3D) MXene/hydrogel structures using glutaraldehyde as a linker. Our MXene/hydrogel-based bioelectronic nose exhibited a high selectivity and sensitivity for monitoring hexanal in both liquid and gas phases. The bioelectronic noses could sensitively detect liquid and gaseous hexanal down to 10-18 M and 6.9 ppm, and they had wide detection ranges of 10-18 - 10-6 M and 6.9-32.9 ppm, respectively. Moreover, our bioelectronic nose allowed us to monitor hexanal levels in fish and milk. In this respect, our MXene/hydrogel-based bioelectronic nose could be a practical strategy for versatile applications such as food spoilage assessments in both liquid and gaseous systems.

An aldehyde-crosslinking mitochondrial probe for STED imaging in fixed cells.

Fluorescence labeling of chemically fixed specimens, especially immunolabeling, plays a vital role in super-resolution imaging as it offers a convenient way to visualize cellular structures like mitochondria or the distribution of biomolecules with high detail. Despite the development of various distinct probes that enable super-resolved stimulated emission depletion (STED) imaging of mitochondria in live cells, most of these membrane-potential-dependent fluorophores cannot be retained well in mitochondria after chemical fixation. This lack of suitable mitochondrial probes has limited STED imaging of mitochondria to live cell samples. In this study, we introduce a mitochondria-specific probe, PK Mito Orange FX (PKMO FX), which features a fixation-driven cross-linking motif and accumulates in the mitochondrial inner membrane. It exhibits high fluorescence retention after chemical fixation and efficient depletion at 775 nm, enabling nanoscopic imaging both before and after aldehyde fixation. We demonstrate the compatibility of this probe with conventional immunolabeling and other strategies commonly used for fluorescence labeling of fixed samples. Moreover, we show that PKMO FX facilitates correlative super-resolution light and electron microscopy, enabling the correlation of multicolor fluorescence images and transmission EM images via the characteristic mitochondrial pattern. Our probe further expands the mitochondrial toolkit for multimodal microscopy at nanometer resolutions.

Regioselective hydroformylation of propene catalysed by rhodium-zeolite.

Hydroformylation is an industrial process for the production of aldehydes from alkenes1,2. Regioselective hydroformylation of propene to high-value n-butanal is particularly important, owing to a wide range of bulk applications of n-butanal in the manufacture of various necessities in human daily life3. Supported rhodium (Rh) hydroformylation catalysts, which often excel in catalyst recyclability, ease of separation and adaptability for continuous-flow processes, have been greatly exploited4. Nonetheless, they usually consist of rotationally flexible and sterically unconstrained Rh hydride dicarbonyl centres, only affording limited regioselectivity to n-butanal5-8. Here we show that proper encapsulation of Rh species comprising Rh(I)-gem-dicarbonyl centres within a MEL zeolite framework allows the breaking of the above model. The optimized catalyst exhibits more than 99% regioselectivity to n-butanal and more than 99% selectivity to aldehydes at a product formation turnover frequency (TOF) of 6,500 h-1, surpassing the performance of all heterogeneous and most homogeneous catalysts developed so far. Our comprehensive studies show that the zeolite framework can act as a scaffold to steer the reaction pathway of the intermediates confined in the space between the zeolite framework and Rh centres towards the exclusive formation of n-butanal.

Synthesis of fluorinated amino acids by low-specificity, promiscuous aldolases coupled to in situ fluorodonor generation.

Fluorine (F) is an important element in the synthesis of molecules broadly used in medicine, agriculture, and materials. F addition to organic structures represents a unique strategy for tuning molecular properties, yet this atom is rarely found in Nature and approaches to produce fluorometabolites (such as fluorinated amino acids, key building blocks for synthesis) are relatively scarce. This chapter discusses the use of L-threonine aldolase enzymes (LTAs), a class of enzymes that catalyze reversible aldol addition to the α-carbon of glycine. The C-C bond formation ability of LTAs, together with their known substrate promiscuity, make them ideal for in vitro F biocatalysis. Here, we describe protocols to harness the activity of the low-specificity LTAs isolated from Escherichia coli and Pseudomonas putida on 2-fluoroacetaldehyde to efficiently synthesize 4-fluoro-L-threonine in vitro. This chapter also provides a comprehensive account of experimental protocols to implement these activities in vivo. These methods are illustrative and can be adapted to produce other fluorometabolites of interest.

Determination of genotoxic impurities of aromatic aldehydes in pharmaceutical preparations by high performance liquid chromatography after derivatization with N-Cyclohexyl-4-hydrazino-1,8-naphthalenediimide.

The detection of aromatic aldehydes, considered potential genotoxic impurities, holds significant importance during drug development and production. Current analytical methods necessitate complex pre-treatment processes and exhibit insufficient specificity and sensitivity. This study presents the utilization of naphthalenediimide as a pre-column derivatisation reagent to detect aromatic aldehyde impurities in pharmaceuticals via high-performance liquid chromatography (HPLC). We screened a series of derivatisation reagents through density functional theory (DFT) and investigated the phenomenon of photoinduced electron transfer (PET) for both the derivatisation reagents and the resulting products. Optimal experimental conditions for derivatisation were achieved at 40 °C for 60 min. This approach has been successfully applied to detect residual aromatic aldehyde genotoxic impurities in various pharmaceutical preparations, including 4-Nitrobenzaldehyde, 2-Nitrobenzaldehyde, 1,4-Benzodioxane-6-aldehyde, and 5-Hydroxymethylfurfural. The pre-column derivatisation method significantly enhanced detection sensitivity and reduced the limit of detection (LOD), which ranged from 0.002 to 0.008 µg/ml for the analytes, with relative standard deviations < 3 %. The correlation coefficient (R2) >0.998 demonstrated high quality. In chloramphenicol eye drops, the concentration of 4-Nitrobenzaldehyde was measured to be 8.6 µg/mL below the specified concentration, with recoveries ranging from 90.0 % to 119.2 %. In comparison to existing methods, our work simplifies the pretreatment process, enhances the sensitivity and specificity of the analysis, and offers comprehensive insights into impurity detection in pharmaceutical preparations.

Investigation of liquid chromatography-mass spectrometry analysis of a peptide aldehyde SJA6017 with identifying its hemiacetal, gem-diol, and enol ether.

The quantitative analysis of SJA6017, a peptide aldehyde inhibitor of calpain (Calpain Inhibitor VI), has encountered challenges in preclinical drug studies. The complex reverse-phase HPLC chromatographic behavior exhibits two peaks, each containing multiple species. An liquid chromatography-mass spectrometry (LC-MS/MS) study proposed an explanation for this phenomenon, caused by the amide aldehyde structure of SJA6017. Four chemical species corresponding to the two HPLC peaks have been identified as SJA6017 and its methyl hemiacetal, methyl enol ether, and gem-diol. In many instances of preclinical studies, methanol is favored as a substitute for DMSO. The hemiacetal is formed when the amide-activated peptide aldehyde reacts with methanol, which can then be further dehydrated in the mass spectrometer ion source under high temperature to form the methyl enol ether. The hemiacetal and gem-diol can also be decomposed to SJA6017 in the ion source. Additionally, the amide-activated peptide aldehyde can easily hydrate to the gem-diol of SJA6017 during sample incubation or sample preparation. The hemiacetal and gem-diol of SJA6017 are stable enough to have different retention times in the liquid chromatography, which explains why SJA6017 appears as two peaks, each containing multiple species. An LC-MS/MS tandem quadrupole mass spectrometer quantitative analysis method is proposed, enabling the analysis of these types of samples. This work serves as both an illustrative example and a cautionary note for mass analysis, sample incubations, and sample preparations involving compounds of peptide aldehyde, including similar aldehyde-containing metabolites, especially when methanol is present. This study provides the information needed to understand peptide aldehyde behavior at various steps of preclinical in vitro studies in the presence of methanol. It has assisted in the development of the SJA6017 bioanalysis method and will also aid in the development of bioanalysis methods for similar peptide aldehydes.

Synthesis and performance of a nanosensing platform for homocysteine detection: A series of iridium(III) complexes containing aldehyde group as probe and MOF as supporting substrate.

The low cost and simple detection method for Hcy (homocysteine) is highly desired in analytical and biological fields since Hcy has been regarded as a bio-marker for multiple diseases. In this work, five Ir(C^N)2(N^N)+ compounds having -CHO group in their C^N or N^N ligand were synthesized and tried for Hcy sensing. Electron-donating groups such as -NH2 and -CH3 were incorporated into the C^N or N^N ligand. Their geometric structure, electronic structure, and optical parameters (with or without Hcy) were analyzed and compared carefully to explore their Hcy sensing potential. The sensing mechanism was revealed by NMR titration and theoretical simulation as a cyclization reaction between the -CHO group and Hcy. The optimal compounds, which showed increased emission quantum yield (2.5-fold) and emission blue-shift (by âˆ¼ 100 nm) upon Hcy, were then covalently grafted into a porous host bio-MOF-1. Linear working plots were fitted, with good selectivity, LOD of 0.15 µM, and response time of 33 s. The novelty of this work was the eye-sensitive emission color change of this nanosensing platform from red (without Hcy) to green (with Hcy).

Protein Tyrosine Amination: Detection, Imaging, and Chemoproteomic Profiling with Synthetic Probes.

Protein tyrosine nitration (PTN) by oxidative and nitrative stress is a well-known post-translational modification that plays a role in the initiation and progression of various diseases. Despite being recognized as a stable modification for decades, recent studies have suggested the existence of a reduction in PTN, leading to the formation of 3-aminotyrosine (3AT) and potential denitration processes. However, the vital functions of 3AT-containing proteins are still unclear due to the lack of selective probes that directly target the protein tyrosine amination. Here, we report a novel approach to label and enrich 3AT-containing proteins with synthetic salicylaldehyde (SAL)-based probes: SALc-FL with a fluorophore and SALc-Yn with an alkyne tag. These probes exhibit high selectivity and efficiency in labeling and can be used in cell lysates and live cells. More importantly, SALc-Yn offers versatility when integrated into multiple platforms by enabling proteome-wide quantitative profiling of cell nitration dynamics. Using SALc-Yn, 355 proteins were labeled, enriched, and identified to carry the 3AT modification in oxidatively stressed RAW264.7 cells. These findings provide compelling evidence supporting the involvement of 3AT as a critical intermediate in nitrated protein turnover. Moreover, our probes serve as powerful tools to investigate protein nitration and denitration processes, and the identification of 3AT-containing proteins contributes to our understanding of PTN dynamics and its implications in cellular redox biology.

Stereoselective Synthesis and Catalytical Application of Perillaldehyde-Based 3-Amino-1,2-diol Regioisomers.

A library of regioisomeric monoterpene-based aminodiols was synthesised and applied as chiral catalysts in the addition of diethylzinc to benzaldehyde. The synthesis of the first type of aminodiols was achieved starting from (-)-8,9-dihydroperillaldehyde via reductive amination, followed by Boc protection and dihydroxylation with the OsO4/NMO system. Separation of formed stereoisomers resulted in a library of aminodiol diastereoisomers. The library of regioisomeric analogues was obtained starting from (-)-8,9-dihydroperillic alcohol, which was transformed into a mixture of allylic trichloroacetamides via Overman rearrangement. Changing the protecting group to a Boc function, the protected enamines were subjected to dihydroxylation with the OsO4/NMO system, leading to a 71:16:13 mixture of diastereoisomers, which were separated, affording the three isomers in isolated form. The obtained primary aminodiols were transformed into secondary derivatives. The regioselectivity of the ring closure of the N-benzyl-substituted aminodiols with formaldehyde was also investigated, resulting in 1,3-oxazines in an exclusive manner. To explain the stability difference between diastereoisomeric 1,3-oxazines, a series of comparative theoretical modelling studies was carried out. The obtained potential catalysts were applied in the reaction of aromatic aldehydes and diethylzinc with moderate to good enantioselectivities (up to 94% ee), whereas the opposite chiral selectivity was observed between secondary aminodiols and their ring-closed 1,3-oxazine analogues.

Nanofabrication of citronellal with chitosan biopolymer to boost its efficacy against aflatoxin B1 and Aspergillus flavus mediated biodeterioration of active ingredient of Piper longum.

The study reports the efficacy of nanofabricated citronellal inside the chitosan biopolymer (NeCn) against Aspergillus flavus growth, aflatoxin B1 (AFB1) production, and active ingredient biodeterioration (Piperine) in Piper longum L. The prepared NeCn was characterized by Scanning Electron Microscopy (SEM), Dynamic Light Scattering (DLS), and Fourier Transform Infrared Spectroscopy (FTIR). The results revealed that the NeCn exhibited distantly improved antifungal (1.25 µL/mL) and AFB1 inhibition (1.0 µL/mL) compared to free Cn. The perturbances in membrane function, mitochondrial membrane potential, antioxidant defense system, and regulatory genes (Ver-1 and Nor-1) of AFB1 biosynthesis were reported as probable modes of action of NeCn. The NeCn (1.25 µL/mL) effectively protects the P. longum from A. flavus (78.8%), AFB1 contamination (100%), and deterioration of Piperine (62.39%), thus demonstrating its potential as a promising novel antifungal agent for food preservation.

Insights into the relative contribution of four precursors to 3-sulfanylhexan-1-ol and 3-sulfanylhexylacetate biogenesis during fermentation.

The desirable wine aroma compounds 3-sulfanylhexan-1-ol (3SH) and 3-sulfanylhexyl acetate (3SHA) are released during fermentation from non-volatile precursors present in the grapes. This work explores the relative contribution of four precursors (E-2-hexenal, 3-S-glutathionylhexan-1-ol, 3-S-glutathionylhexanal, and 3-S-cysteinylhexan-1-ol) to 3SH and 3SHA. Through the use of isotopically labelled analogues of these precursors in defined fermentation media, new insights into the role of each precursor have been identified. E-2-Hexenal was shown to contribute negligible amounts of thiols, while 3-S-glutathionylhexan-1-ol was the main precursor of both 3SH and 3SHA. The glutathionylated precursors were both converted to 3SHA more efficiently than 3-S-cysteinylhexan-1-ol. Interestingly, 3-S-glutathionylhexanal generated 3SHA without detectable concentrations of 3SH, suggesting possible differences in the way this precursor is metabolised compared to 3-S-glutathionylhexan-1-ol and 3-S-cysteinylhexan-1-ol. We also provide the first evidence for chemical conversion of 3-S-glutathionylhexan-1-ol to 3-S-(γ-glutamylcysteinyl)-hexan-1-ol in an oenological system.

14C-Isotope Use to Quantify Covalent Reactions between Flavor Compounds and ß-Lactoglobulin.

A 14C-based method was developed to study the rate and extent of covalent bond formation between ß-lactoglobulin and three model flavor compounds: a ketone (2-undecanone UDO), an aldehyde (decanal DAL), an isothiocyanate (2-phenylethyl isothiocyanate PEITC), and an unreactive "methods blank" (decane DEC). Aqueous protein solutions with one of the 14C-labeled model flavor compounds were placed in water baths at 25, 45, and 65 °C for 4 weeks measuring the amount of flavor: protein reaction at 1, 3, 7, 14, 21, and 28 days. UDO showed lowest reactivity (max of 0.9% of added compound reacted), DAL (max of 16.4% reacted), and PEITC (max of 71.8% reacted). All compounds showed a rapid initial reaction rate which slowed after ca. 7 days. It appears that only PEITC (at 65 °C) saturated all potential protein-reactive sites over the storage period.

All-polysaccharide, self-healing, pH-sensitive, in situ-forming hydrogel of carboxymethyl chitosan and aldehyde-functionalized hydroxyethyl cellulose.

In situ forming hydrogels are promising for biomedical applications, especially in drug delivery. The precursor solution can be injected at the target site, where it undergoes a sol-gel transition to afford a hydrogel. In this sense, the most significant characteristic of these hydrogels is fast gelation behavior after injection. This study describes an all-polysaccharide, rapidly in situ-forming hydrogel composed of carboxymethyl chitosan (CMCHT) and hydroxyethyl cellulose functionalized with aldehyde groups (HEC-Ald). The HEC-Ald was synthesized through acetal functionalization, followed by acid deprotection. This innovative approach avoids cleavage of pyran rings, as is inherent in the periodate oxidation approach, which is the most common method currently employed for adding aldehyde groups to polysaccharides. The resulting hydrogel exhibited fast stress relaxation, self-healing properties, and pH sensitivity, which allowed it to control the release of an encapsulated model drug in response to the medium pH. Based on the collected data, the HEC-Ald/CMCHT hydrogels show promise as pH-sensitive drug carriers.

Molecular dynamics of structural effects of reactive carbonyl species derivate of lipid peroxidation on bovine serum albumin.

BACKGROUND: Serum albumin is the most abundant protein in the Mammalia blood plasma at where plays a decisive role in the transport wide variety of hydrophobic ligands. BSA undergoes oxidative modifications like the carbonylation by the reactive carbonyl species (RCSs) 4-hydroxy-2-nonenal (HNE), 4 hydroxy-2-hexenal (HHE), malondialdehyde (MDA) and 4-oxo-2-nonenal (ONE), among others. The structural and functional changes induced by protein carbonylation have been associated with the advancement of neurodegenerative, cardiovascular, metabolic and cancer diseases. METHODS: To elucidate structural effects of protein carbonylation with RCSs on BSA, parameters for six new non-standard amino acids were designated and molecular dynamics simulations of its mono­carbonylated-BSA systems were conducted in the AMBER force field. Trajectories were evaluated by RMSD, RMSF, PCA, RoG and SASA analysis. RESULTS: An increase in the conformational instability for all proteins modified with local changes were observed, without significant changes on the BSA global three-dimensional folding. A more relaxed compaction level and major solvent accessible surface area for modified systems was found. Four regions of high molecular fluctuation were identified in all modified systems, being the subdomains IA and IIIB those with the most remarkable local conformational changes. Regarding essential modes of domain movements, it was evidenced that the most representatives were those related to IA subdomain, while IIIB subdomain presented discrete changes. CONCLUSIONS: RCSs induces local structural changes on mono­carbonylated BSA. Also, this study extends our knowledge on how carbonylation by RCSs induce structural effects on proteins.

Theoretical Analysis of Selectivity Differences in Ketoreductases toward Aldehyde and Ketone Carbonyl Groups.

Lactobacillus kefir alcohol dehydrogenase (LkADH) and ketoreductase from Chryseobacterium sp. CA49 (ChKRED12) exhibit different chemoselectivity and stereoselectivity toward a substrate with both keto and aldehyde carbonyl groups. LkADH selectively reduces the keto carbonyl group while retaining the aldehyde carbonyl group, producing optically pure R-alcohols. In contrast, ChKRED12 selectively reduces the aldehyde group and exhibits low reactivity toward ketone carbonyls. This study investigated the structural basis for these differences and the role of specific residues in the active site. Molecular dynamics (MD) simulations and quantum chemical calculations were used to investigate the interactions between the substrate and the enzymes and the essential cause of this phenomenon. The present study has revealed that LkADH and ChKRED12 exhibit significant differences in the structure of their respective active pockets, which is a crucial determinant of their distinct chemoselectivity toward the same substrate. Moreover, residues N89, N113, and E144 within LkADH as well as Q151 and D190 within ChKRED12 have been identified as key contributors to substrate stabilization within the active pocket through electrostatic interactions and van der Waals forces, followed by hydride transfer utilizing the coenzyme NADPH. Furthermore, the enantioselectivity mechanism of LkADH has been elucidated using quantum chemical methods. Overall, these findings not only provide fundamental insights into the underlying reasons for the observed differences in selectivity but also offer a detailed mechanistic understanding of the catalytic reaction.