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Key points Previous studies demonstrated that the mitochondrial Ca2+ uniporter MCU and the Na+‐Ca2+ exchanger NCLX exist in proximity to the sarcoplasmic reticulum (SR) ryanodine receptor RyR and the Ca2+ pump SERCA, respectively, creating a mitochondria‐SR Ca2+ interaction. However, the physiological relevance of the mitochondria‐SR Ca2+ interaction has remained unsolved. Furthermore, although mitochondrial Ca2+ has been proposed to be an important factor regulating mitochondrial energy metabolism, by activating NADH‐producing dehydrogenases, the contribution of the Ca2+‐dependent regulatory mechanisms to cellular functions under physiological conditions has been controversial. In this study, we constructed a new integrated model of human ventricular myocyte with excitation‐contraction‐energetics coupling and investigated systematically the contribution of mitochondria‐SR Ca2+ interaction, especially focusing on cardiac energetics during dynamic workload transitions in exercise. Simulation analyses revealed that the spatial coupling of mitochondria and SR, particularly via mitochondrial Ca2+ uniport activity‐RyR, was the primary determinant of mitochondrial Ca2+ concentration, and that the Ca2+‐dependent regulatory mechanism facilitated mitochondrial NADH recovery during exercise and contributed to the stability of NADH in the workload transition by about 40%, while oxygen consumption rate and cytoplasmic ATP level were not influenced. We concluded that the mitochondria‐SR Ca2+ interaction, created via the uneven distribution of Ca2+ handling proteins, optimizes the contribution of the mitochondrial Ca2+‐dependent regulatory mechanism to stabilizing NADH during exercise. The mitochondrial Ca2+ uniporter protein MCU and the Na+‐Ca2+ exchanger protein NCLX are reported to exist in proximity to the sarcoplasmic reticulum (SR) ryanodine receptor RyR and the Ca2+ pump SERCA, respectively, creating a mitochondria‐SR Ca2+ interaction in cardiomyocytes. Mitochondrial Ca2+ (Ca2+mit) has been proposed to be an important factor regulating mitochondrial energy metabolism, by activating NADH‐producing dehydrogenases. Here we constructed an integrated model of a human ventricular myocyte with excitation‐contraction‐energetics coupling and investigated the role of the mitochondria‐SR Ca2+ interaction in cardiac energetics during exercise. Simulation analyses revealed that the spatial coupling particularly via mitochondrial Ca2+ uniport activity‐RyR is the primary determinant of Ca2+mit concentration, and that the activation of NADH‐producing dehydrogenases by Ca2+mit contributes to NADH stability during exercise. The mitochondria‐SR Ca2+ interaction optimizes the contribution of Ca2+mit to the activation of NADH‐producing dehydrogenases. [ABSTRACT FROM AUTHOR]

Volatile fatty acid derivatives (VFADs) play a significant role in contributing to flowery–fruity flavor black tea. Innovative black tea is typically crafted from aroma-intensive tea cultivars, such as Jinmudan, using defined production methodologies. In this study, the during-processing tea leaves of innovative black tea were applied as materials, and we selected a total of 45 VFADs, comprising 11 derived aldehydes, nine derived alcohols, and 25 derived esters. Furthermore, the dynamic variations of these VFADs were uncovered. Transcriptome analysis was performed to identify genes involved in the LOX (lipoxygenase) pathway, resulting in the identification of 17 CsLOX genes, one hydrogen peroxide lyase (CsHPL) gene, 11 alcohol dehydrogenases (CsADH) genes, 11 genes as acyl CoA oxidase (CsACOX) genes, and three allene oxide synthase (CsAOS) genes. Additionally, the expression levels of these genes were measured, indicating that the processing treatments of innovative black tea, particularly turn-over and fermentation, had a stimulation effect on most genes. Finally, qRT-PCR verification and correlation analysis were conducted to explain the relationship between VFADs and candidate genes. This study aims to provide a reference for illuminating the formation mechanisms of aroma compounds in innovative black tea, thereby inspiring the optimization of innovative processing techniques and enhancing the overall quality of black tea. [ABSTRACT FROM AUTHOR]

Short-chain dehydrogenase/reductases (SDRs) are the largest NAD(H)-dependent oxidoreductase superfamilies and are involved in diverse metabolisms. This study presents a comprehensive genomic analysis of the SDR superfamily in Cinnamomum camphora, a species that is one of the most significant woody essential oil plants in southern China. We identify a total of 222 CcSDR proteins and classify them into five types based on their cofactor-binding and active sites: 'atypical', 'classic', 'divergent', 'extended', and 'unknown'. Phylogenetic analysis reveals three evolutionary branches within the CcSDR proteins, and further categorization using the SDR-initiative Hidden Markov model resulted in 46 families, with the CcSDR110C, CcSDR108E, and CcSDR460A families being the most populous. Collinearity analysis identified 34 pairs of CcSDR paralogs in C. camphora, 141 pairs of SDR orthologs between C. camphora and Populus trichocarpa, and 59 pairs between C. camphora and Oryza sativa. Expression profile analysis indicates a preference for the expression of 77 CcSDR genes in specific organs such as flowers, bark, twigs, roots, leaves, or fruits. Moreover, 77 genes exhibit differential expression patterns during the four developmental stages of leaves, while 130 genes show variance across the five developmental stages of fruits. Additionally, to explore the biosynthetic mechanism of methyl eugenol, a key component of the leaf essential oil in the methyl eugenol chemotype, this study also identifies eugenol synthase (EGS) within the CcSDR460A family through an integrated strategy. Real-time quantitative PCR analysis demonstrates that the expression of CcEGS in the leaves of the methyl eugenol chemotype is more than fourfold higher compared to other chemotypes. When heterologously expressed in Escherichia coli, it catalyzes the conversion of coniferyl acetate into a mixture predominantly composed of eugenol (71.44%) and isoeugenol (21.35%). These insights pave the way for future research into the functional diversity of CcSDR genes, with a focus on secondary metabolism. [ABSTRACT FROM AUTHOR]

Summary Hydrogen sulfide (H2S) is an endogenous gaseous signaling molecule, which has been shown to play an important role in plant growth and development by coupling with various phytohormones. However, the relationship between H2S and cytokinin (CTK) and the mechanisms by which H2S and CTK affect root growth remain poorly understood. Endogenous CTK was analyzed by UHPLC‐ESI‐MS/MS. Persulfidation of cytokinin oxidase/dehydrogenases (CKXs) was analyzed by mass spectrometry (MS). ckx2/CKX2wild‐type (WT), OE CKX2 and ckx2/CKX2Cys(C)62alanine(A) transgenic lines were isolated with the ckx2 background. H2S is linked to CTK content by CKX2, which regulates root system architecture (RSA). Persulfidation at cysteine (Cys)62 residue of CKX2 enhances CKX2 activity, resulting in reduced CTK content. We utilized 35S‐LCD/oasa1 transgenic lines to investigate the effect of endogenous H2S on RSA, indicating that H2S reduces the gravitropic set‐point angle (GSA), shortens root hairs, and increases the number of lateral roots (LRs). The persulfidation of CKX2Cys62 changes the elongation of cells on the upper and lower flanks of LR elongation zone, confirming that Cys62 of CKX2 is the specificity target of H2S to regulate RSA in vivo. In conclusion, this study demonstrated that H2S negatively regulates CTK content and affects RSA by persulfidation of CKX2Cys62 in Arabidopsis thaliana. [ABSTRACT FROM AUTHOR]

α,ω-Dicarboxylic acids, ω-aminoalkanoic acids, and α,ω-diaminoalkanes are valuable building blocks for the production of biopolyesters and biopolyamides. One of the key steps in producing these chemicals is the oxidation of ω-hydroxycarboxylic acids using alcohol dehydrogenases (e.g., ChnD of Acinetobacter sp. NCIMB 9871). However, the reaction and structural features of these enzymes remain mostly undiscovered. Thereby, we have investigated characteristics of ChnD based on enzyme kinetics, substrate-docking simulations, and mutation studies. Kinetic analysis revealed a distinct preference of ChnD for medium chain ω-hydroxycarboxylic acids, with the highest catalytic efficiency of 18.0 mM−1s−1 for 12-hydroxydodecanoic acid among C6 to C12 ω-hydroxycarboxylic acids. The high catalytic efficiency was attributed to the positive interactions between the carboxyl group of the substrates and the guanidino group of two arginine residues (i.e., Arg62 and Arg266) in the substrate binding site. The ChnD_R62L variant showed the increased efficiency and affinity, particularly for fatty alcohols (i.e., C6–C10) and branched-chain fatty alcohols, such as 3-methyl-2-buten-1-ol. Overall, this study contributes to the deeper understanding of medium-chain primary aliphatic alcohol dehydrogenases and their applications for the production of industrially relevant chemicals such as α,ω-dicarboxylic acids, ω-aminoalkanoic acids, and α,ω-diaminoalkanes from renewable biomass. • ChnD prefers oxidizing longer ω-hydroxycarboxylic acids, particularly 12-hydroxydodecanoic acid. • Carboxyl groups in ω-hydroxycarboxylic acids significantly enhance efficiency by interacting with arginine residue. • The ChnD_R62L variant shows improved efficiency and affinity for fatty and branched-chain alcohols. [ABSTRACT FROM AUTHOR]

Enzyme engineering is a powerful tool for improving or altering the properties of biocatalysts for industrial, research, and therapeutic applications. Fast and accurate screening of variant libraries is often the bottleneck of enzyme engineering and may be overcome by growth-based screening strategies with simple processes to enable high throughput. The currently available growth-based screening strategies have been widely employed for enzymes but not yet for catalytically potent and oxygen-sensitive metalloenzymes. Here, we present a screening system that couples the activity of an oxygen-sensitive formate dehydrogenase to the growth of Escherichia coli. This system relies on the complementation of the E. coli formate hydrogenlyase (FHL) complex by Mo-dependent formate dehydrogenase H (EcFDH-H). Using an EcFDH-Hdeficient strain, we demonstrate that growth inhibition by acidic glucose fermentation products can be alleviated by FHL complementation. This allows the identification of catalytically active EcFDH-H variants at a readily measurable cell density readout, reduced handling efforts, and a low risk of oxygen contamination. Furthermore, a good correlation between cell density and formate oxidation activity was established using EcFDH-H variants with variable catalytic activities. As proof of concept, the growth assay was employed to screen a library of 1,032 EcFDH-H variants and reduced the library size to 96 clones. During the subsequent colorimetric screening of these clones, the variant A12G exhibiting an 82.4% enhanced formate oxidation rate was identified. Since many metal-dependent formate dehydrogenases and hydrogenases form functional complexes resembling E. coli FHL, the demonstrated growth-based screening strategy may be adapted to components of such electron-transferring complexes. [ABSTRACT FROM AUTHOR]

Gluconobacter oxydans succinic semialdehyde reductase (GoxSSAR) and Acetobacter aceti glyoxylate reductase (AacGR) represent a novel class in the β-hydroxyacid dehydrogenases superfamily. Kinetic analyses revealed GoxSSAR's activity with both glyoxylate and succinic semialdehyde, while AacGR is glyoxylate specific. GoxSSAR K167A lost activity with succinic semialdehyde but retained some with glyoxylate, whereas AacGR K175A lost activity. These findings elucidate differences between these homologous enzymes. [ABSTRACT FROM AUTHOR]

This study explored the enantiocomplementary bioreduction of substituted 1-(arylsulfanyl)propan-2-ones in batch mode using four wild-type yeast strains and two different recombinant alcohol dehydrogenases from Lactobacillus kefir and Rhodococcus aetherivorans. The selected yeast strains and recombinant alcohol dehydrogenases as whole-cell biocatalysts resulted in the corresponding 1-(arylsulfanyl)propan-2-ols with moderate to excellent conversions (60–99%) and high selectivities (ee > 95%). The best bioreductions—in terms of conversion (>90%) and enantiomeric excess (>99% ee)—at preparative scale resulted in the expected chiral alcohols with similar conversion and selectivity to the screening reactions. [ABSTRACT FROM AUTHOR]

Stabilized enzymes are crucial for the industrial application of biocatalysis due to their enhanced operational stability, which leads to prolonged enzyme activity, cost-efficiency and consequently scalability of biocatalytic processes. Over the past decade, numerous studies have demonstrated that deep eutectic solvents (DES) are excellent enzyme stabilizers. However, the search for an optimal DES has primarily relied on trial-and-error methods, lacking systematic exploration of DES structure-activity relationships. Therefore, this study aims to rationally design DES to stabilize various dehydrogenases through extensive experimental screening, followed by the development of a straightforward and reliable mathematical model to predict the efficacy of DES in enzyme stabilization. A total of 28 DES were tested for their ability to stabilize three dehydrogenases at 30°C: (S)-alcohol dehydrogenase from Rhodococcus ruber (ADH-A), (R)-alcohol dehydrogenase from Lactobacillus kefir (Lk-ADH) and glucose dehydrogenase from Bacillus megaterium (GDH). The residual activity of these enzymes in the presence of DES was quantified using first-order kinetic models. The screening revealed that DES based on polyols serve as promising stabilizing environments for the three tested dehydrogenases, particularly for the enzymes Lk-ADH and GDH, which are intrinsically unstable in aqueous environments. In glycerol-based DES, increases in enzyme half-life of up to 175-fold for Lk-ADH and 60-fold for GDH were observed compared to reference buffers. Furthermore, to establish the relationship between the enzyme inactivation rate constants and DES descriptors generated by the Conductor-like Screening Model for Real Solvents, artificial neural network models were developed. The models for ADH-A and GDH showed high efficiency and reliability (R² > 0.75) for in silico screening of the enzyme inactivation rate constants based on DES descriptors. In conclusion, these results highlight the significant potential of the integrated experimental and in silico approach for the rational design of DES tailored to stabilize enzymes. [ABSTRACT FROM AUTHOR]

The enzymatic effectiveness in the reaction cascade that reduces CO2 to methanol in water at room‐temperature faces various constraints. One of the major challenges is the short life of costly enzymes: immobilization is used to make them more stable and recyclable. The comparative analysis of the several immobilization techniques reported in the literature is challenging due to the diverse reaction conditions (single enzyme test or pool of enzymes test) and experimental setups, as well as the high variability in the amount of enzymes and cofactor. In the present study, a comparison is presented among three different methods (co‐encapsulation into Ca–alginate beads, co‐absorption onto zirconium(IV) phosphate (ZrP) and covalent binding to dialdehydecellulose [DAC]) of co‐immobilization of the three dehydrogenases Fatedehydrogenase (DH), FaldDH, and alcohol dehydrogenase, used in equal amount and under the same experimental conditions, so to check at what extension the support and the immobilization method can influence the activity of the enzymatic pool. DAC is used for the first time to support the three DHs and results to be the best method of immobilization with respect to those used here, that also allows longer life on enzymes and repeated recycling of the supported enzymes, increasing the overall methanol production with respect to the free enzymes. [ABSTRACT FROM AUTHOR]

In recent decades, there has been an increase in the incidence of autoimmune diseases (AID) among adults and children. The immunopathogenesis of AID is based on an imbalance between autoaggressive and regulatory cells (Tregs), which is regulated by metabolic signaling pathways and the cytokine microenvironment. Understanding the mechanisms of immunometabolism opens up new possibilities for the treatment of patients with AID. The aim was to evaluate the activity of lymphocyte dehydrogenases associated with OXPHOS and glycolysis, depending on the level of proinflammatory and anti-inflammatory cytokines in children with AID.324 children with AID were examined: 80 – Crohn’s disease (CD), 53 – ulcerative colitis (UC), 89 – psoriasis (PS), 66 – multiple sclerosis (MS), 36 – autoimmune hepatitis (AIH). Activity of mitochondrial dehydrogenases (succinate dehydrogenase (SDH) and glycerol-3-phosphate dehydrogenase (GFDH)) were evaluated by immunocytochemical method using flow cytometry. The level of cytokines (CС) in blood sera was determined by multiplex analysis.In each studied group of children, CС with the highest values in exacerbation and remission of the disease were identified. The maximum values of CС were in patients with exacerbation: CD, UC, PS, MS – IL-23; AIH – IL-27. Evaluation of cytokine complexes associated with cells showed significant differences between patients in exacerbation/remission: CD, UC and PS – M1(IL-1+IL-6+TNFα), cTh1(IFNγ+IL-12p70+TNFβ+IL-2), cTh2 (IL-4+IL-5+IL-10+IL-13+IL-17E/IL-25+IL-33), cTh17 (IL-1β+IL-6+IL-17A+IL-17F+IL-21+IL-22+IL-23); MS – M1, cTh1, cTh2; AIH – cTh2. SDH activity in AID remission differed between pathologies in CD4+ cells, Th17 and Tregs. In exacerbation of AID, there were differences in Tregs between patients with UC and PS. The highest GPDH activity in exacerbation was observed in CD4+ lymphocytes, Th17 and Tregs in CD. The ratio of SDH/GPDH in T-lymphocytes in children with CD in exacerbation and remission was lowest and significantly lower than in UC, PS, MS, AIH and apparently healthy children. In the group of children with a low SDH/GPDH ratio, the levels of CCL20/MIP3α, IFNγ, IL-12p70, IL-13, IL-17A, IL-1β, and TNFα were significantly increased. Conclusions. Informative cytokine complexes were identified in children with AID. The relationship between the metabolic activity of lymphocytes and the level of circulating cytokines is shown.

Formate dehydrogenases (FDHs) catalyze the oxidation of formate to CO2 while reducing NAD(P)+ to NAD(P)H and are classified into two main classes: metal‐dependent (Mo‐ or W‐containing) and metal‐independent FDHs. The latter are oxygen‐tolerant and relevant as a cofactor regeneration system for various bioprocesses and gained more and more attention due to their ability to catalyze the reverse CO2 reduction. This review gives an overview of metal‐independent FDHs, the recent advances made in this field, and their relevance for future applications in biocatalysis. This includes the exploitation of novel FDHs which have altered co‐substrate specificity as well as enzyme engineering approaches to improve process stability and general performance. [ABSTRACT FROM AUTHOR]

The goal of the study is to evaluate metabolic and morphological changes of the facultative alkaliphile Bacillus aequororis 5‐DB and the weakly alkali‐resistant B. subtilis ATCC 6633 in a wide pH range and at different NaCl concentrations. The alkaliphile B. aequororis 5‐DB is shown to have a broader general resistance to adverse factors (wide pH range, 50 g/L NaCl) than a weakly alkali‐tolerant strain of the same genus. This alkaliphile is also shown to have a significantly greater resistance not only to high pH but also to low pH in comparison with B. subtilis ATCC 6633. The resistance of B. aequororis 5‐DB to low pH was expressed in higher metabolic activity, maintenance of ΔpH, and no significant cell damage. The selected set of methods (reduction of resazurin to resorufin by cell dehydrogenases, bioluminescent method for determining ATP, AFM, and measurement of intracellular pH) allows us to adequately assess the ability of microbial cells to withstand harsh environmental factors. Nonspecific resistance of B. aequororis 5‐DB was proven using a complex of selected methods. Tolerance to a wide range of pH and high salt concentrations may be useful for biotechnological applications of the strain. [ABSTRACT FROM AUTHOR]

Protection against oxidative stress is a vital defense mechanism for Mycobacterium tuberculosis within the host. However, few transcription factors that control bacterial antioxidant defense are known. Here, we present evidence that SdrR, encoded by the MSMEG_5712 (Ms5712) gene, functions as an oxidative stress response regulator in Mycobacterium smegmatis. SdrR recognizes an 11-bp motif sequence in the operon's upstream regulatory region and negatively regulates the expression of short-chain dehydrogenases/reductases (SDR). Overexpressing sdrR inhibited SDR expression, which rendered the strain oxidative more stress-sensitive. Conversely, sdrR knockout alleviates SDR repression, which increases its oxidative stress tolerance. Thus, SdrR responds to oxidative stress by negatively regulating sdr expression. Therefore, this study elucidated an underlying regulatory mechanism behind mycobacterial oxidative stress adaptation. [ABSTRACT FROM AUTHOR]

Synthetic nicotinamide biomimetics (NCBs) have emerged as alternatives to the use of natural cofactors. The relatively low cost and ease of manufacture of NCBs may enable the scaling of biocatalytic reactions to produce bulk chemicals (e.g., biofuels and plastics). NCBs are also recognized by only a subset of NAD(P)/NAD(P)H-dependent enzymes, which potentially allows access to orthogonal redox cascades that can be run simultaneously within a single reactor. In the work presented here, a series of NCBs was prepared and tested for activity with alcohol dehydrogenases and ene-reductases. While the NCBs did not support enzymatic activity with the alcohol dehydrogenases, the observed rate of the ene-reductases with NCBs was greater than when incubated with the natural cofactor (consistent with previous observations). We obtained the structures of an ene-reductase and an alcohol dehydrogenase with an NCB bound in their active sites. While the NCB bound to the ene-reductases in a productive position and orientation for hydride transfer to the isoalloxazine ring of the flavin cofactor, the NCB failed to adopt a catalytically competent binding mode in the alcohol dehydrogenase. [ABSTRACT FROM AUTHOR]

Androgen receptor signaling is crucial for the development of treatment resistance in prostate cancer. Among steroidogenic enzymes, 3β-hydroxysteroid dehydrogenases (3βHSDs) play critical roles in extragonadal androgen synthesis, especially 3βHSD1. Increased expression of 3βHSDs is observed in castration-resistant prostate cancer tumors compared with primary prostate tumors, indicating their involvement in castration resistance. Recent studies link 3βHSD1 to resistance to androgen receptor signaling inhibitors. The regulation of 3βHSD1 expression involves various factors, including transcription factors, microenvironmental influences, and posttranscriptional modifications. Additionally, the clinical significance of HSD3B1 genotypes, particularly the rs1047303 variant, has been extensively studied. The impact of HSD3B1 genotypes on treatment outcomes varies according to the therapy administered, suggesting the potential of HSD3B1 genotyping for personalized medicine. Targeting 3βHSDs may be a promising strategy for prostate cancer management. Overall, understanding the roles of 3βHSDs and their genetic variations may enable the development and optimization of novel treatments for prostate cancer. [ABSTRACT FROM AUTHOR]

Alcohol dehydrogenases (ADHs) have garnered recognition for their potential in the synthesis of pivotal pharmaceutical compounds. However, their utilization in the context of piperidone synthesis remains an area ripe for exploration. In this study, we examine the performance of an alcohol dehydrogenase derived from Corynebacterium glutamicum (CgADH) using a substrate analogue functional screening (SAFS) method to elucidate its substrate specificity. To improve the catalytic activity of CgADH, a phenylalanine/alanine‐scanning and iterative saturation mutation (PAS‐ISM) method was used. The most active variant, I151F/I195A, exhibited a remarkable 10.6‐fold increase in catalytic activity compared to the wild‐type. Structural analysis revealed that the introduction of residues 151F and 195A led to a remodeling of the substrate‐binding pocket, enabling additional p‐π hydrophobic interactions with the substrate, ultimately promoting a more favorable substrate binding pose. This study introduces the SAFS screening method, which enables the identification of enzymes with no sequence homology to known enzymes. Furthermore, the application of PAS‐ISM presents an efficient approach for the engineering of alcohol dehydrogenases. These findings open up promising avenues to expand the utility of ADHs in the synthesis of piperidone, thereby advancing the field of pharmaceutical chemistry. [ABSTRACT FROM AUTHOR]

Native amine dehydrogenases offer sustainable access to chiral amines, so the search for scaffolds capable of converting more diverse carbonyl compounds is required to reach the full potential of this alternative to conventional synthetic reductive aminations. Here we report a multidisciplinary strategy combining bioinformatics, chemoinformatics and biocatalysis to extensively screen billions of sequences in silico and to efficiently find native amine dehydrogenases features using computational approaches. In this way, we achieve a comprehensive overview of the initial native amine dehydrogenase family, extending it from 2,011 to 17,959 sequences, and identify native amine dehydrogenases with non-reported substrate spectra, including hindered carbonyls and ethyl ketones, and accepting methylamine and cyclopropylamine as amine donor. We also present preliminary model-based structural information to inform the design of potential (R)-selective amine dehydrogenases, as native amine dehydrogenases are mostly (S)-selective. This integrated strategy paves the way for expanding the resource of other enzyme families and in highlighting enzymes with original features. Sustainable chemistry can benefit from biocatalysis, but a high diversity of enzymes is needed. Here, the authors screen billions of protein sequences to provide an overview of the native amine dehydrogenase family for amine synthesis. [ABSTRACT FROM AUTHOR]

Spirobisnaphthalenes (SBNs) are a class of highly oxygenated, fungal bisnaphthalenes containing a unique spiroketal bridge, that displayed diverse bioactivities. Among the reported SBNs, palmarumycins are the major type, which are precursors for the other type of SBNs structurally. However, the biosynthesis of SBNs is unclear. In this study, we elucidated the biosynthesis of palmarumycins, using gene disruption, heterologous expression, and substrate feeding experiments. The biosynthetic gene cluster for palmarumycins was identified to be distant from the polyketide synthase gene cluster, and included two cytochrome P450s (PalA and PalB), and one short chain dehydrogenase/reductase (PalC) encoding genes as key structural genes. PalA is an unusual, multifunctional P450 that catalyzes the oxidative dimerization of 1,8‐dihydroxynaphthalene to generate the spiroketal linkage and 2,3‐epoxy group. Chemical synthesis of key intermediate and in vitro biochemical assays proved that the oxidative dimerization proceeded via a binaphthyl ether. PalB installs the C‐5 hydroxy group, widely found in SBNs. PalC catalyzes 1‐keto reduction, the reverse 1‐dehydrogenation, and 2,3‐epoxide reduction. Moreover, an FAD‐dependent oxidoreductase, encoded by palD, which locates outside the cluster, functions as a 1‐dehydrogenase. These results provided the first genetic and biochemical evidence for the biosynthesis of palmarumycin SBNs. [ABSTRACT FROM AUTHOR]

Ribosomally synthesized and post-translationally modified peptides (RiPPs) are a broad group of compounds mediating microbial competition in nature. Azole/azoline heterocycle formation in the peptide backbone is a key step in the biosynthesis of many RiPPs. Heterocycle formation in RiPP precursors is often carried out by a scaffold protein, an ATP-dependent cyclodehydratase, and an FMN-dependent dehydrogenase. It has generally been assumed that the orchestration of these modifications is carried out by a stable complex including the scaffold, cyclodehydratase, and dehydrogenase. The antimicrobial RiPP micrococcin begins as a precursor peptide (TclE) with a 35-amino acid N-terminal leader and a 14-amino acid C-terminal core containing six Cys residues that are converted to thiazoles. The putative scaffold protein (TclI) presumably presents the TclE substrate to a cyclodehydratase (TclJ) and a dehydrogenase (TclN) to accomplish the two-step installation of the six thiazoles. In this study, we identify a minimal TclE leader region required for thiazole formation, demonstrate complex formation between TclI, TclJ, and TclN, and further define regions of these proteins required for complex formation. Our results point to a mechanism of thiazole installation in which TclI associates with the two enzymes in a mutually exclusive fashion, such that each enzyme competes for access to the peptide substrate in a dynamic equilibrium, thus ensuring complete modification of each Cys residue in the TclE core. [ABSTRACT FROM AUTHOR]

The cytokinin oxidase/dehydrogenase (CKX) enzyme is essential for controlling the fluctuating levels of endogenous cytokinin (CK) and has a significant impact on different aspects of plant growth and development. Nonetheless, there is limited knowledge about CKX genes in tomato (Solanum lycopersicum L.). Here we performed genome-wide identification and analysis of nine SlCKX family members in tomatoes using bioinformatics tools. The results revealed that nine SlCKX genes were unevenly distributed on five chromosomes (Chr.1, Chr.4, Chr.8, Chr.10, and Chr.12). The amino acid length, isoelectric points, and molecular weight of the nine SlCKX proteins ranged from 453 to 553, 5.77 to 8.59, and 51.661 to 62.494 kD, respectively. Subcellular localization analysis indicated that SlCKX2 proteins were located in both the vacuole and cytoplasmic matrix; SlCKX3 and SlCKX5 proteins were located in the vacuole; and SlCKX1, 4, 6, 7, 8, and 9 proteins were located in the cytoplasmic matrix. Furthermore, we observed differences in the gene structures and phylogenetic relationships of SlCKX proteins among different members. SlCKX1-9 were positioned on two out of the three branches of the CKX phylogenetic tree in the multispecies phylogenetic tree construction, revealing their strong conservation within phylogenetic subgroups. Unique patterns of expression of CKX genes were noticed in callus cultures exposed to varying concentrations of exogenous ZT, suggesting their roles in specific developmental and physiological functions in the regeneration system. These results may facilitate subsequent functional analysis of SlCKX genes and provide valuable insights for establishing an efficient regeneration system for tomatoes. [ABSTRACT FROM AUTHOR]

Background: Breast cancer is a serious threat to women's health with high morbidity and mortality. The development of more effective therapies for the treatment of breast cancer is strongly warranted. Growing evidence suggests that targeting glucose metabolism may be a promising cancer treatment strategy. We previously identified a new glyceraldehyde-3-phosphate dehydrogenase (GAPDH) inhibitor, DC-5163, which shows great potential in inhibiting tumor growth. Here, we evaluated the anticancer potential of DC-5163 in breast cancer cells. Methods: The effects of DC-5163 on breast cancer cells were investigated in vitro and in vivo. Seahorse, glucose uptake, lactate production, and cellular ATP content assays were performed to examine the impact of DC-5163 on cellular glycolysis. Cell viability, colony-forming ability, cell cycle, and apoptosis were assessed by CCK8 assay, colony formation assay, flow cytometry, and immunoblotting respectively. The anticancer activity of DC-5163 in vivo was evaluated in a mouse breast cancer xenograft model. Results: DC-5163 suppressed aerobic glycolysis and reduced energy supply of breast cancer cells, thereby inhibiting breast cancer cell growth, inducing cell cycle arrest in the G0/G1 phase, and increasing apoptosis. The therapeutic efficacy was assessed using a breast cancer xenograft mouse model. DC-5163 treatment markedly suppressed tumor growth in vivo without inducing evident systemic toxicity. Micro-PET/CT scans revealed a notable reduction in tumor 18F-FDG and 18F-FLT uptake in the DC-5163 treatment group compared to the DMSO control group. Conclusions: Our results suggest that DC-5163 is a promising GAPDH inhibitor for suppressing breast cancer growth without obvious side effects. 18F-FDG and 18F-FLT PET/CT can noninvasively assess the levels of glycolysis and proliferation in tumors following treatment with DC-5163. [ABSTRACT FROM AUTHOR]

The synthesis of campholenic-based fragrances requires the preservation of specific structural elements to capture the desired sandalwood scent. The most critical step of their preparation is the reduction of α,β-unsaturated carbonyl precursors while preserving the campholenic unsaturation. Classical reductions, especially hydrogenations, often lack complete chemoselectivity, leading to the formation of over-reduced byproducts. In addition, the stereochemistry plays a key role in the olfactory perception of these chiral fragrances. However, none of the current industrial syntheses are stereoselective, resulting in wasteful production of non-contributory isomers. Herein, we explore the untapped potential of biocatalytic reductions using ene-reductases (ERs) and alcohol dehydrogenases (ADHs) to enhance the sustainability of four commercial sandalwood fragrances (Brahmanol®, Firsantol®, Sandalore®, and Ebanol®), focusing on the stereoselective synthesis of their most odorant isomers. A comparison of green metrics, including E-factors and EcoScale, between bio- and chemo-based reductions is presented. [ABSTRACT FROM AUTHOR]

SUMMARY: Alcohol dehydrogenases (ADHs) are a group of zinc‐binding enzymes belonging to the medium‐length dehydrogenase/reductase (MDR) protein superfamily. In plants, these enzymes fulfill important functions involving the reduction of toxic aldehydes to the corresponding alcohols (as well as catalyzing the reverse reaction, i.e., alcohol oxidation; ADH1) and the reduction of nitrosoglutathione (GSNO; ADH2/GSNOR). We investigated and compared the structural and biochemical properties of ADH1 and GSNOR from Arabidopsis thaliana. We expressed and purified ADH1 and GSNOR and determined two new structures, NADH‐ADH1 and apo‐GSNOR, thus completing the structural landscape of Arabidopsis ADHs in both apo‐ and holo‐forms. A structural comparison of these Arabidopsis ADHs revealed a high sequence conservation (59% identity) and a similar fold. In contrast, a striking dissimilarity was observed in the catalytic cavity supporting substrate specificity and accommodation. Consistently, ADH1 and GSNOR showed strict specificity for their substrates (ethanol and GSNO, respectively), although both enzymes had the ability to oxidize long‐chain alcohols, with ADH1 performing better than GSNOR. Both enzymes contain a high number of cysteines (12 and 15 out of 379 residues for ADH1 and GSNOR, respectively) and showed a significant and similar responsivity to thiol‐oxidizing agents, indicating that redox modifications may constitute a mechanism for controlling enzyme activity under both optimal growth and stress conditions. [ABSTRACT FROM AUTHOR]

Gluconobacter strains perform incomplete oxidation of various sugars and alcohols, employing regio- and stereoselective membrane-bound dehydrogenases oriented toward the periplasmic space. This oxidative fermentation process is utilized industrially. The ketogluconate production pathway, characteristic of these strains, begins with the conversion of d -glucose to d -gluconate, which then diverges and splits into 2 pathways producing 5-keto- d -gluconate and 2-keto- d -gluconate and subsequently 2,5-diketo- d -gluconate. These transformations are facilitated by membrane-bound d -glucose dehydrogenase, glycerol dehydrogenase, d -gluconate dehydrogenase, and 2-keto- d -gluconate dehydrogenase. The variance in end products across Gluconobacter strains stems from the diversity of enzymes and their activities. This review synthesizes biochemical and genetic knowledge with biotechnological applications, highlighting recent advances in metabolic engineering and the development of an efficient production process focusing on enzymes relevant to the ketogluconate production pathway in Gluconobacter strains. [ABSTRACT FROM AUTHOR]

The ecotoxicity of Ag particles of different size has been assessed by microbiological, biochemical, and phytotoxic indicators for the upper layer (0–20 cm) of ordinary chernozem (Haplic Chernozem) in a laboratory model experiment. The effect has been studied of nano- (10 and 100 nm) and microparticles (1000 nm) of Ag at concentrations of 1, 10, and 100 mg/kg on the biological parameters of ordinary chernozem 30 days after contamination: the activity of catalase, dehydrogenases, ferrireductase, urease, peroxidase, polyphenol oxidase, invertase, phosphatase, the total number of bacteria, the abundance of bacteria of Azotobacter genus, the number of germinated seeds and the length of radish roots. It was found that the ecotoxicity of Ag particles depended on their size: in most cases, Ag particles 10 nm in size had a stronger ecotoxic effect on the biological parameters than particles 100 and 1000 nm in size. There were no significant differences in the ecotoxicity of 100 and 1000 nm Ag particles. The difference in the effects of Ag particles of different sizes increased with increasing Ag concentration in the soil: the higher the Ag concentration was in the soil (from 1 to 100 mg/kg), the more pronounced the difference was in ecotoxicity between 10 nm Ag particles and 100 and 1000 nm Ag particles. Phytotoxic indicators were more sensitive to contamination by Ag nanoparticles at all concentrations studied (1, 10 and 100 mg/kg); the total number of bacteria, invertase and phosphatase activity at 10 and 100 mg/kg; the abundance of bacteria of Azotobacter genus and the activity of dehydrogenases at 100 mg/kg. It is advisable to use these indicators in biodiagnostics of the ecotoxicity of Ag nanoparticles. [ABSTRACT FROM AUTHOR]

Astrocytes play a pivotal role in maintaining brain homeostasis. Recent research has highlighted the significance of palmitic acid (PA) in triggering pro-inflammatory pathways contributing to neurotoxicity. Furthermore, Genomic-scale metabolic models and control theory have revealed that metabolic switches (MSs) are metabolic pathway regulators by potentially exacerbating neurotoxicity, thereby offering promising therapeutic targets. Herein, we characterized these enzymatic MSs in silico as potential therapeutic targets, employing protein–protein and drug–protein interaction networks alongside structural characterization techniques. Our findings indicate that five MSs (P00558, P04406, Q08426, P09110, and O76062) were functionally linked to nervous system drug targets and may be indirectly regulated by specific neurological drugs, some of which exhibit polypharmacological potential (e.g., Trifluperidol, Trifluoperazine, Disulfiram, and Haloperidol). Furthermore, four MSs (P00558, P04406, Q08426, and P09110) feature ligand-binding or allosteric cavities with druggable potential. Our results advocate for a focused exploration of P00558 (phosphoglycerate kinase 1), P04406 (glyceraldehyde-3-phosphate dehydrogenase), Q08426 (peroxisomal bifunctional enzyme, enoyl-CoA hydratase, and 3-hydroxyacyl CoA dehydrogenase), P09110 (peroxisomal 3-ketoacyl-CoA thiolase), and O76062 (Delta(14)-sterol reductase) as promising targets for the development or repurposing of pharmacological compounds, which could have the potential to modulate lipotoxic-altered metabolic pathways, offering new avenues for the treatment of related human diseases such as neurological diseases. [ABSTRACT FROM AUTHOR]

Simple Summary: This study aimed to elucidate the factors contributing to the limited presence of Cheilotoma musciformis in Poland, with a specific focus on soil characteristics affecting both vegetation and insect populations. It examined how soil enzyme activity influences the occurrence of Ch. musciformis in the xerothermic grasslands of Southern Poland. Sites inhabited by the beetle were typically subject to extensive grazing by farm animals or recent bush clearance, contrasting with control plots situated on unused or overgrazed xerothermic grasslands. Soils in beetle-inhabited sites exhibited significantly higher levels of enzyme activity, total organic carbon, and total nitrogen, along with lower pHKCl compared to control sites. These findings suggest the beetle's reliance on extensively grazed xerothermic grasslands. Given that grazing practices influence the behavior of preferred host plant species, effective protection planning for Ch. musciformis-inhabited grasslands should carefully consider changes in soil biochemical properties and vegetation structure. This work attempts to find the reasons for the rather limited range of occurrence of Cheilotoma musciformis in Poland, based on soil properties, which affects both the plant cover and the entomofauna. The aim of the study was to assess the influence of soil enzyme activity on the occurrence of Ch. musciformis in xerothermic grasslands in Southern Poland. The sites inhabited by the beetle were most often extensively grazed by farm animals or had recently been cleared of bushes. The control plots were in wasteland. The soils of most sites with Ch. musciformis were characterized by significantly higher activity of the tested enzymes and higher content of total organic carbon and total nitrogen, as well as lower pHKCl compared to the control sites. The higher enzymatic activity of soils in sites with the beetle than in the control sites may indicate the dependence of the occurrence of this beetle on the presence of patches of extensively grazed xerothermic grasslands. Grazing influences the behavior of preferred host plant species. Therefore, when planning active protection of xerothermic grasslands inhabited by Ch. musciformis, changes in the biochemical properties of the soil and vegetation structure should be taken into account. [ABSTRACT FROM AUTHOR]

Molybdenum‐ or tungsten‐dependent formate dehydrogenases have emerged as significant catalysts for the chemical reduction of CO2 to formate, with biotechnological applications envisaged in climate‐change mitigation. The role of Met405 in the active site of Desulfovibrio vulgaris formate dehydrogenase AB (DvFdhAB) has remained elusive. However, its proximity to the metal site and the conformational change that it undergoes between the resting and active forms suggests a functional role. In this work, the M405S variant was engineered, which allowed the active‐site geometry in the absence of methionine Sδ interactions with the metal site to be revealed and the role of Met405 in catalysis to be probed. This variant displayed reduced activity in both formate oxidation and CO2 reduction, together with an increased sensitivity to oxygen inactivation. [ABSTRACT FROM AUTHOR]

Pentachlorophenol (PCP) is a widely used pesticide. However, whether PCP and its metabolite chloranil have endocrine-disrupting effects by inhibiting placental 3β-hydroxysteroid dehydrogenase 1 (3β-HSD1) remains unclear. The study used in vitro assays with human and rat placental microsomes to measure 3β-HSD activity as well as human JAr cells to evaluate progesterone production. The results showed that PCP exhibited moderate inhibition of human 3β-HSD1, with an IC 50 value of 29.83 μM and displayed mixed inhibition in terms of mode of action. Conversely, chloranil proved to be a potent inhibitor, demonstrating an IC 50 value of 147 nM, and displaying a mixed mode of action. PCP significantly decreased progesterone production by JAr cells at 50 μM, while chloranil markedly reduced progesterone production at ≥1 μM. Interestingly, PCP and chloranil moderately inhibited rat placental homolog 3β-HSD4, with IC 50 values of 27.94 and 23.42 μM, respectively. Dithiothreitol (DTT) alone significantly increased human 3β-HSD1 activity. Chloranil not PCP mediated inhibition of human 3β-HSD1 activity was completely reversed by DTT and that of rat 3β-HSD4 was partially reversed by DTT. Docking analysis revealed that both PCP and chloranil can bind to the catalytic domain of 3β-HSDs. The difference in the amino acid residue Cys83 in human 3β-HSD1 may explain why chloranil is a potent inhibitor through its interaction with the cysteine residue of human 3β-HSD1. In conclusion, PCP is metabolically activated to chloranil as a potent inhibitor of human 3β-HSD1. [Display omitted] • Pentachlorophenol is a moderate inhibitor of human and rat placental 3β-HSDs. • Pentachlorophenol is converted to chloranil that is a potent inhibitor of human 3β-HSD1. • Chloranil inhibits progesterone production by human JAr cells. • DTT can completely or partially antagonize chloranil-mediated inhibition of 3β-HSDs. [ABSTRACT FROM AUTHOR]

Asymmetric reduction of the conjugated CC bonds by the old yellow enzymes (OYEs) presents a promising field in the synthesis of chiral chemicals. Nevertheless, few natural OYEs have been applied in large-scale applications due to the requirement of costly NADPH and low operational stability. Herein, a stable and efficient fusion of YqjM from Bacillus subtilis and glucose dehydrogenase (GDH) from Bacillus megaterium was constructed to stereoselectively reduce the conjugated CC bonds in a self-sufficient continuous process. The effects of the enzyme order and different linkers on the fusions were investigated by structural analysis and all-atom molecular dynamics simulation. The best fusion YqjM_G_GDH gave 98% conversion of 100 ​mmol/L 2-methylcyclopentenone with an excellent ee value (>99%) in 3 ​h, while the mixture of individual enzymes only obtained 68% conversion after more than 8 ​h. The improved substrate conversion of YqjM_G_GDH fusion was probably attributed to the increased flexibility of each fused enzyme and the shortening of the diffusion distance of NADPH regenerated. A one-pot process was designed to purify and immobilize the fusion on the Ni2+-nitrilotriacetic acid functionalized magnetic mesoporous silica nanoflowers. The resulting immobilized biocatalyst not only catalyzed the asymmetric reduction of various α,β-unsaturated ketones (20 ​mmol/L) continuously with only 50 ​μmol/L NADP+ to initiate the whole process, but also retained more than 82% of the initial activity after seven cycles, serving as a good candidate for the industrial applications. [ABSTRACT FROM AUTHOR]

The enzymatic effectiveness in the reaction cascade that reduces CO2 to methanol in water at room‐temperature faces various constraints. One of the major challenges is the short life of costly enzymes: immobilization is used to make them more stable and recyclable. The comparative analysis of the several immobilization techniques reported in the literature is challenging due to the diverse reaction conditions (single enzyme test or pool of enzymes test) and experimental setups, as well as the high variability in the amount of enzymes and cofactor. In the present study, a comparison is presented among three different methods (co‐encapsulation into Ca–alginate beads, co‐absorption onto zirconium(IV) phosphate (ZrP) and covalent binding to dialdehydecellulose [DAC]) of co‐immobilization of the three dehydrogenases Fatedehydrogenase (DH), FaldDH, and alcohol dehydrogenase, used in equal amount and under the same experimental conditions, so to check at what extension the support and the immobilization method can influence the activity of the enzymatic pool. DAC is used for the first time to support the three DHs and results to be the best method of immobilization with respect to those used here, that also allows longer life on enzymes and repeated recycling of the supported enzymes, increasing the overall methanol production with respect to the free enzymes.

Stabilized enzymes are crucial for the industrial application of biocatalysis due to their enhanced operational stability, which leads to prolonged enzyme activity, cost-efficiency and consequently scalability of biocatalytic processes. Over the past decade, numerous studies have demonstrated that deep eutectic solvents (DES) are excellent enzyme stabilizers. However, the search for an optimal DES has primarily relied on trial-and-error methods, lacking systematic exploration of DES structure-activity relationships. Therefore, this study aims to rationally design DES to stabilize various dehydrogenases through extensive experimental screening, followed by the development of a straightforward and reliable mathematical model to predict the efficacy of DES in enzyme stabilization. A total of 28 DES were tested for their ability to stabilize three dehydrogenases at 30°C: (S)-alcohol dehydrogenase from Rhodococcus ruber (ADH-A), (R)-alcohol dehydrogenase from Lactobacillus kefir (Lk-ADH) and glucose dehydrogenase from Bacillus megaterium (GDH). The residual activity of these enzymes in the presence of DES was quantified using first-order kinetic models. The screening revealed that DES based on polyols serve as promising stabilizing environments for the three tested dehydrogenases, particularly for the enzymes Lk-ADH and GDH, which are intrinsically unstable in aqueous environments. In glycerol-based DES, increases in enzyme half-life of up to 175-fold for Lk-ADH and 60-fold for GDH were observed compared to reference buffers. Furthermore, to establish the relationship between the enzyme inactivation rate constants and DES descriptors generated by the Conductor-like Screening Model for Real Solvents, artificial neural network models were developed. The models for ADH-A and GDH showed high efficiency and reliability (R2 > 0.75) for in silico screening of the enzyme inactivation rate constants based on DES descriptors. In conclusion, these results highlight the significant potential of the integrated experimental and in silico approach for the rational design of DES tailored to stabilize enzymes.

Background: Oral squamous cell carcinoma (OSCC) remains a rampant oral cavity neoplasm with high degree of aggressiveness. Aldo–keto reductase 1B10 (AKR1B10) that is an oxidoreductase dependent on nicotinamide adenine dinucleotide phosphate (NADPH) has been introduced to possess prognostic potential in OSCC. The present work was focused on specifying the involvement of AKR1B10 in the process of OSCC and its latent functional mechanism. Methods: AKR1B10 expression in OSCC tissues and cells were detected by RT-qPCR and Western blot analysis. CCK-8 method, EdU staining, wound healing and transwell assays respectively assayed cell viability, proliferation, migration and invasion. Immunofluorescence staining and Western blot evaluated epithelial mesenchymal transition (EMT). Adenosine triphosphate (ATP) contents, glucose consumption and extracellular acidification rate (ECAR) were measured by relevant commercially available kits and Seahorse XF96 Glycolysis Analyzer, severally. The expressions of proteins associated with metastasis and glycolysis were examined with Western blot. Co-IP assay confirmed the binding between AKR1B10 and hexokinase 2 (HK2). Results: It was observed that AKR1B10 expression was increased in OSCC tissues and cells. After AKR1B10 was knocked down, the proliferation, migration, invasion and EMT of OSCC cells were all hampered. Additionally, AKR1B10 silencing suppressed glycolysis and bound to HK2 in OSCC cells. Up-regulation of HK2 partially abolished the hampered glycolysis, proliferation, migration, invasion and EMT of AKR1B10-silenced OSCC cells. Conclusion: To sum up, AKR1B10 could bind to HK2 to accelerate glycolysis, thereby facilitating the proliferation, migration, invasion and EMT of OSCC cells. [ABSTRACT FROM AUTHOR]

There are two paralogs of glutamate dehydrogenase (GDH) in humans encoded by the GLUD1 and GLUD2 genes as a result of a recent retroposition during the evolution of primates. The two human GDHs possess significantly different regulation by allosteric ligands, which is not fully characterized at the structural level. Recent advances in identification of the GDH ligand binding sites provide a deeper perspective on the significance of the accumulated substitutions within the two GDH paralogs. In this review, we describe the evolution of GLUD1 and GLUD2 after the duplication event in primates using the accumulated sequencing and structural data. A new gibbon GLUD2 sequence questions the indispensability of ancestral R496S and G509A mutations for GLUD2 irresponsiveness to GTP, providing an alternative with potentially similar regulatory features. The data of both GLUD1 and GLUD2 evolution not only confirm substitutions enhancing GLUD2 mitochondrial targeting, but also reveal a conserved mutation in ape GLUD1 mitochondrial targeting sequence that likely reduces its transport to mitochondria. Moreover, the information of GDH interactors, posttranslational modification and subcellular localization are provided for better understanding of the GDH mutations. Medically significant point mutations causing deregulation of GDH are considered from the structural and regulatory point of view. [ABSTRACT FROM AUTHOR]

The hair follicle (HF) is a self-renewing adult miniorgan that undergoes drastic metabolic and morphological changes during precisely timed cyclic organogenesis. The HF cycle is known to be regulated by steroid hormones, growth factors and circadian clock genes. Recent data also suggest a role for a vitamin A derivative, all-trans-retinoic acid (ATRA), the activating ligand of transcription factors, retinoic acid receptors, in the regulation of the HF cycle. Here we demonstrate that ATRA signaling cycles during HF regeneration and this pattern is disrupted by genetic deletion of epidermal retinol dehydrogenases 2 (RDHE2, SDR16C5) and RDHE2-similar (RDHE2S, SDR16C6) that catalyze the rate-limiting step in ATRA biosynthesis. Deletion of RDHEs results in accelerated anagen to catagen and telogen to anagen transitions, altered HF composition, reduced levels of HF stem cell markers, and dysregulated circadian clock gene expression, suggesting a broad role of RDHEs in coordinating multiple signaling pathways. Epidermal retinol dehydrogenases 2 (SDR16C5) and RDHE2-similar (SDR16C6) are the major retinol dehydrogenases in skin, which regulate hair cycling, hair composition, hair follicle stem cell markers, and circadian clock genes. [ABSTRACT FROM AUTHOR]

Introduction: Breast cancer (BC) is the most frequent cancer in women and is a serious worldwide health issue. Phosphoglycerate dehydrogenase (PHGDH) is an enzyme that catalyses the first steps in the serine biosynthetic pathways downstream of glycolysis. Phosphoglycerate and glycine are produced by a series of enzymatic processes from the glycolysis intermediate 3-phosphoglycerate. The aim of the study was to indicate the levels of PHGDH and glycine in patients with breast cancer. Material and methods: The study was performed from December 2022 to March 2023. The total number of samples was 135 samples -- 65 samples were collected from women with breast cancer, and 70 samples were from healthy women as a control group. Blood samples (5 ml) were obtained from all study group members. The complete blood count (CBC) neutrophils/lymphocytes, and haemoglobin ratio analysis was done on a CBC haematology analyser (Sysmex, Japan). Serum PHGDH and glycine were measure by enzyme-linked immunosorbent assay. Results: The study findings revealed a significant increase in the neutrophil/lymphocytes ratio and a decrease in PHGDH level in patients with BC compare to controls (p < 0.01), while the serum glycine level showed a significant increase in patients with BC compare to the control group (p < 0.01). Conclusions: Reduced PHGDH level and high glycine concentration in patients with BC could act as a prognostic factor in cancer development. [ABSTRACT FROM AUTHOR]

Malonyl-Coenzyme A Reductase (MCR) in Chloroflexus aurantiacus , a characteristic enzyme of the 3-hydroxypropionate (3-HP) cycle, catalyses the reduction of malonyl-CoA to 3-HP. MCR is a bi-functional enzyme; in the first step, malonyl-CoA is reduced to the free intermediate malonate semialdehyde by the C-terminal region of MCR, and this is further reduced to 3-HP by the N-terminal region of MCR. Here we present the crystal structures of both N-terminal and C-terminal regions of the MCR from C. aurantiacus. A catalytic mechanism is suggested by ligand and substrate bound structures, and structural and kinetic studies of MCR variants. Both MCR structures reveal one catalytic, and one non-catalytic SDR (short chain dehydrogenase/reductase) domain. C-terminal MCR has a lid domain which undergoes a conformational change and controls the reaction. In the proposed mechanism of the C-terminal MCR, the conversion of malonyl-CoA to malonate semialdehyde is based on the reduction of malonyl-CoA by NADPH, followed by the decomposition of the hemithioacetal to produce malonate semialdehyde and coenzyme A. Conserved arginines, Arg734 and Arg773 are proposed to play key roles in the mechanism and conserved Ser719, and Tyr737 are other essential residues forming an oxyanion hole for the substrate intermediates. • Crystal structures of split Ca MCR (N-terminal and C-terminal) are presented. • MCR-C lid domain undergoes a conformational change and controls the reaction. • Arg734 and Arg773 are proposed to be active residues in the MCR-C mechanism. • In MCR-N, Thr158, Tyr165, Arg169 and Tyr171 form the substrate binding site. • MCR-C is "slower but tightly binding", and MCR-N is "fast, but loosely binding". [ABSTRACT FROM AUTHOR]

Background: 5-Oxo-6,8,11,14-eicosatetraenoic acid (5-Oxo-ETE) is a metabolite of arachidonic acid shown to promote biological activities in different cell types. Summary: 5-Oxo-ETE is synthesized from the 5-lipoxygenase product 5S-HETE (5S-hydroxy-6,8,11,14-eicosatetraenoic acid) in the presence of the nicotinamide adenine dinucleotide phosphate (NADP)+-dependent enzyme 5-hydroxyeicosanoid dehydrogenase (5-HEDH). Under some conditions that promote oxidation of NADPH to NADP+, such as the respiratory burst in phagocytic cells, eosinophils, and neutrophils, oxidative stress in monocytes and dendritic cells, and cell death, 5-Oxo-ETE synthesis can be dramatically increased. In addition, 5-Oxo-ETE can also be formed in the absence of 5-lipoxygenase in cells through transcellular biosynthesis by inflammatory cell-derived 5S-HETE. This compound performs its biological activities by the highly selective Gi/o-coupled OXE receptor, which is highly expressed on eosinophils, neutrophils, basophils, and monocytes. As such, 5-Oxo-ETE is a potent chemoattractant for these inflammatory cells, especially for eosinophils. Key Messages: Although the pathophysiological role of 5-Oxo-ETE is not clearly understood, 5-Oxo-ETE may be a significant mediator in allergic diseases, such as allergic asthma, allergic rhinitis, and atopic dermatitis. And targeting the OXE receptor may be a novel therapy for this kind of inflammatory condition. Nowadays, selective OXE receptor antagonists are currently under investigation and could become potential therapeutic agents in allergy. [ABSTRACT FROM AUTHOR]

Bacillus subtilis is a natural producer of 2,3-butanediol (2,3-BDO) and has acquired "Generally Regarded as Safe" status. It is reported to produce 2,3-BDO from synthetic sugars as well as complex and economic sugar sources such as molasses. However, the rate-limiting step in the formation of 2,3-BDO is its conversion from acetoin to 2,3-BDO by the enzyme butanediol dehydrogenase (2,3-BDH). Such 2,3-BDHs were screened based on higher affinity (lower Km) towards acetoin as substrate. The in silico docking studies were conducted for further validation, and they showed a high interaction profile for the PpBDH protein towards acetoin. Heterologous expression of these genes was studied in engineered Bacillus subtilis (BS1A1). In this study, it was seen that 2,3-BDH from Paenibacillus polymyxa ZJ-9 was reported to have higher enzyme activity levels, and in the fermentation studies, it was seen that the ratio of 2,3-BDO to acetoin was increased by 80.25%. The insights encourage further bioprocess optimization for increasing the fermentative production of 2,3-BDO. Our results provide a potential strategy to avoid the back conversion of 2,3-BDO to acetoin in an engineered Bacillus system. [ABSTRACT FROM AUTHOR]

The study objective was to assess microbiological activity of sediments during the application of an EM preparation on the in Lake Skiertąg. Samples were taken before and after microbial bioremediation in the period 2020–2021 from five sediment layers. The abundance, biomass, and efficiency of the respiratory chain of sediment bacteria were determined by the direct methods in a fluorescence microscope. The rate of organic carbon oxidation was measured by the respiratory method. The abundance of benthic bacteria showed significant variability in the profile of each core, and decreased with an increase in sediment depth. The significant decrease in the number and biomass of bacteria in the sediment after the application of the EM technology, was observed during the second year of application of the preparation, and not immediately after its application. In the tested sediments, an significant increase in the percentage of cell with active dehydrogenases ETS+ in the total number of bacteria after application of the preparation was found, but at the same time significantly higher values of this parameter were demonstrated in 2021 compared to the first year of using microbial bioremediation. A significant positive correlation between the potential rate of organic carbon oxidation in the surface layer of sediments and the temperature in the water above the sediment indicates that the temperature conditions stimulated the respiration activity of the sediment microflora. [ABSTRACT FROM AUTHOR]

Berteroin is a bioactive substance classified as an isothiocyanate found in cruciferous vegetables such as cabbage, arugula, and salad leaves. In this study, we aimed to determine whether berteroin exerts anti-inflammatory effects on human periodontal ligament cells (HPDLCs), a resident cells of periodontal tissue. Berteroin suppressed interleukin (IL)-1β or tumor necrosis factor (TNF)-α-induced chemokines (C–C motif chemokine ligand (CCL)2, CCL20, C-X-C motif chemokine ligand (CXCL)10, IL-8, and IL-6) production and intercellular adhesion molecule (ICAM)-1 expression in HPDLCs. In addition, berteroin inhibited phosphorylation of IκB kinase (IKK)- α/ β, nuclear factor (NF)- κB p65, and IκB- α and degradation of IκB- α in the NF-κB pathway induced by IL-1 β or TNF- α stimulation. Moreover, berteroin could inhibit signal transducer and activator of transcription (STAT)3 phosphorylation in TNF- α -stimulated HPDLC. Furthermore, berteroin increased the expression of the antioxidant enzymes, heme oxygenase (HO)-1 and NAD(P)H quinone dehydrogenase (NQO)1, in IL-1 β or TNF- α -stimulated HPDLCs. These results suggest that berteroin may decrease the production of inflammatory mediators in HPDLCs by suppressing the NF-κB pathway, and may also decrease the local reactive oxygen species (ROS) production in periodontal lesions by increasing the production of antioxidant enzymes. [ABSTRACT FROM AUTHOR]

Background: Oxidative enzymes targeting lignocellulosic substrates are presently classified into various auxiliary activity (AA) families within the carbohydrate-active enzyme (CAZy) database. Among these, the fungal AA3 glucose–methanol–choline (GMC) oxidoreductases with varying auxiliary activities are attractive sustainable biocatalysts and important for biological function. CAZy AA3 enzymes are further subdivided into four subfamilies, with the large AA3_2 subfamily displaying diverse substrate specificities. However, limited numbers of enzymes in the AA3_2 subfamily are currently biochemically characterized, which limits the homology-based mining of new AA3_2 oxidoreductases. Importantly, novel enzyme activities may be discovered from the uncharacterized parts of this large subfamily. Results: In this study, phylogenetic analyses employing a sequence similarity network (SSN) and maximum likelihood trees were used to cluster AA3_2 sequences. A total of 27 AA3_2 proteins representing different clusters were selected for recombinant production. Among them, seven new AA3_2 oxidoreductases were successfully produced, purified, and characterized. These enzymes included two glucose dehydrogenases (TaGdhA and McGdhA), one glucose oxidase (ApGoxA), one aryl alcohol oxidase (PsAaoA), two aryl alcohol dehydrogenases (AsAadhA and AsAadhB), and one novel oligosaccharide (gentiobiose) dehydrogenase (KiOdhA). Notably, two dehydrogenases (TaGdhA and KiOdhA) were found with the ability to utilize phenoxy radicals as an electron acceptor. Interestingly, phenoxy radicals were found to compete with molecular oxygen in aerobic environments when serving as an electron acceptor for two oxidases (ApGoxA and PsAaoA), which sheds light on their versatility. Furthermore, the molecular determinants governing their diverse enzymatic functions were discussed based on the homology model generated by AlphaFold. Conclusions: The phylogenetic analyses and biochemical characterization of AA3_2s provide valuable guidance for future investigation of AA3_2 sequences and proteins. A clear correlation between enzymatic function and SSN clustering was observed. The discovery and biochemical characterization of these new AA3_2 oxidoreductases brings exciting prospects for biotechnological applications and broadens our understanding of their biological functions. [ABSTRACT FROM AUTHOR]

Terrestrial geothermal ecosystems are hostile habitats, characterized by large emissions of environmentally relevant gases such as CO2, CH4, H2S and H2. These conditions provide a niche for chemolithoautotrophic microorganisms. Methanotrophs of the phylum Verrucomicrobia, which inhabit these ecosystems, can utilize these gases and grow at pH levels below 1 and temperatures up to 65°C. In contrast, methanotrophs of the phylum Proteobacteria are primarily found in various moderate environments. Previously, novel verrucomicrobial methanotrophs were detected and isolated from the geothermal soil of the Favara Grande on the island of Pantelleria, Italy. The detection of pmoA genes, specific for verrucomicrobial and proteobacterial methanotrophs in this environment, and the partially overlapping pH and temperature growth ranges of these isolates suggest that these distinct phylogenetic groups could coexist in the environment. In this report, we present the isolation and characterization of a thermophilic and acid‐tolerant gammaproteobacterial methanotroph (family Methylococcaceae) from the Favara Grande. This isolate grows at pH values ranging from 3.5 to 7.0 and temperatures from 35°C to 55°C, and diazotrophic growth was demonstrated. Its genome contains genes encoding particulate and soluble methane monooxygenases, XoxF‐ and MxaFI‐type methanol dehydrogenases, and all enzymes of the Calvin cycle. For this novel genus and species, we propose the name 'Candidatus Methylocalor cossyra' CH1. [ABSTRACT FROM AUTHOR]

Isocitrate dehydrogenase is an enzyme converting isocitrate to α-ketoglutarate in the canonical tricarboxylic acid (TCA) cycle. There are three different types of isocitrate dehydrogenase documented in eukaryotes. Our study points out the complex evolutionary history of isocitrate dehydrogenases across kinetoplastids, where the common ancestor of Trypanosomatidae and Bodonidae was equipped with two isoforms of the isocitrate dehydrogenase enzyme: the NADP+-dependent isocitrate dehydrogenase 1 with possibly dual localization in the cytosol and mitochondrion and NADP+-dependent mitochondrial isocitrate dehydrogenase 2. In the extant trypanosomatids, isocitrate dehydrogenase 1 is present only in a few species suggesting that it was lost upon separation of Trypanosoma spp. and replaced by the mainly NADP+-dependent cytosolic isocitrate dehydrogenase 3 of bacterial origin in all the derived lineages. In this study, we experimentally demonstrate that the omnipresent isocitrate dehydrogenase 2 has a dual localization in both mitochondrion and cytosol in at least four species that possess only this isoform. The apparent lack of the NAD+-dependent isocitrate dehydrogenase activity in trypanosomatid mitochondrion provides further support to the existence of the noncanonical TCA cycle across trypanosomatids and the bidirectional activity of isocitrate dehydrogenase 3 when operating with NADP+ cofactor instead of NAD+. This observation can be extended to all 17 species analyzed in this study, except for Leishmania mexicana , which showed only low isocitrate dehydrogenase activity in the cytosol. The variability in isocitrate oxidation capacity among species may reflect the distinct metabolic strategies and needs for reduced cofactors in particular environments. [ABSTRACT FROM AUTHOR]

The rapidly increasing threat of multi-drug-resistant Acinetobacter baumannii infections globally, encompassing a range of clinical manifestations from skin and soft tissue infections to life-threatening conditions like meningitis and pneumonia, underscores an urgent need for novel therapeutic strategies. These infections, prevalent in both hospital and community settings, present a formidable challenge to the healthcare system due to the bacterium's widespread nature and dwindling effective treatment options. Against this backdrop, the exploration of bacterial short-chain dehydrogenase reductases (SDRs) emerges as a promising avenue. These enzymes play pivotal roles in various critical bacterial processes, including fatty acid synthesis, homeostasis, metabolism, and contributing to drug resistance mechanisms. In this study, we present the first examination of the X-ray crystallographic structure of an uncharacterized SDR enzyme from A. baumannii. The tertiary structure of this SDR is distinguished by a central parallel β-sheet, consisting of seven strands, which is flanked by eight α-helices. This configuration exhibits structural parallels with other enzymes in the SDR family, underscoring a conserved architectural theme within this enzyme class. Despite the current ambiguity regarding the enzyme's natural substrate, the importance of many SDR enzymes as targets in anti-bacterial agent design is well-established. Therefore, the detailed structural insights provided in this study open new pathways for the in-silico design of therapeutic agents. By offering a structural blueprint, our findings may provide a platform for future research aimed at developing targeted treatments against this and other multi-drug-resistant infections. [ABSTRACT FROM AUTHOR]

Anthropogenic climate change has been caused by over‐exploitation of fossil fuels and CO2 emissions. To counteract this, the chemical industry has shifted its focus to sustainable chemical production and the valorization of renewable resources. However, the biggest challenges in biomanufacturing are technical efficiency and profitability. In our minimal cell‐free enzyme cascade generating pyruvate as the central intermediate, the NAD+‐dependent, selective oxidation of D‐glyceraldehyde was identified as a key reaction step to improve the overall cascade flux. Successive genome mining identified one candidate enzyme with 24‐fold enhanced activity and another whose stability is unaffected in 10 % (v/v) ethanol, the final product of our model cascade. Semi‐rational engineering improved the substrate selectivity of the enzyme up to 21‐fold, thus minimizing side reactions in the one‐pot enzyme cascade. The final biotransformation of D‐glucose showed a continuous linear production of ethanol (via pyruvate) to a final titer of 4.9 % (v/v) with a molar product yield of 98.7 %. Due to the central role of pyruvate in diverse biotransformations, the optimized production module has great potential for broad biomanufacturing applications. [ABSTRACT FROM AUTHOR]

Background: Oxaliplatin resistance usually leads to therapeutic failure and poor prognosis in colorectal cancer (CRC), while the underlying mechanisms are not yet fully understood. Metabolic reprogramming is strongly linked to drug resistance, however, the role and mechanism of metabolic reprogramming in oxaliplatin resistance remain unclear. Here, we aim to explore the functions and mechanisms of purine metabolism on the oxaliplatin-induced apoptosis of CRC. Methods: An oxaliplatin-resistant CRC cell line was generated, and untargeted metabolomics analysis was conducted. The inosine 5ʹ-monophosphate dehydrogenase type II (IMPDH2) expression in CRC cell lines was determined by quantitative real-time polymerase chain reaction (qPCR) and western blotting analysis. The effects of IMPDH2 overexpression, knockdown and pharmacological inhibition on oxaliplatin resistance in CRC were assessed by flow cytometry analysis of cell apoptosis in vivo and in vitro. Results: Metabolic analysis revealed that the levels of purine metabolites, especially guanosine monophosphate (GMP), were markedly elevated in oxaliplatin-resistant CRC cells. The accumulation of purine metabolites mainly arose from the upregulation of IMPDH2 expression. Gene set enrichment analysis (GSEA) indicated high IMPDH2 expression in CRC correlates with PURINE_METABOLISM and MULTIPLE-DRUG-RESISTANCE pathways. CRC cells with higher IMPDH2 expression were more resistant to oxaliplatin-induced apoptosis. Overexpression of IMPDH2 in CRC cells resulted in reduced cell death upon treatment with oxaliplatin, whereas knockdown of IMPDH2 led to increased sensitivity to oxaliplatin through influencing the activation of the Caspase 7/8/9 and PARP1 proteins on cell apoptosis. Targeted inhibition of IMPDH2 by mycophenolic acid (MPA) or mycophenolate mofetil (MMF) enhanced cell apoptosis in vitro and decreased in vivo tumour burden when combined with oxaliplatin treatment. Mechanistically, the Wnt/β-catenin signalling was hyperactivated in oxaliplatin-resistant CRC cells, and a reciprocal positive regulatory mechanism existed between Wnt/β-catenin and IMPDH2. Blocking the Wnt/β-catenin pathway could resensitize resistant cells to oxaliplatin, which could be restored by the addition of GMP. Conclusions: IMPDH2 is a predictive biomarker and therapeutic target for oxaliplatin resistance in CRC. [ABSTRACT FROM AUTHOR]

The medium-chain dehydrogenase/reductase (MDR) superfamily contains many members that are widely present in organisms and play important roles in growth, metabolism, and stress resistance but have not been studied in Trichosporon asahii. In this study, bioinformatics and RNA sequencing methods were used to analyze the MDR superfamily of T. asahii and its regulatory effect on fluconazole resistance. A phylogenetic tree was constructed using Saccharomyces cerevisiae, Candida albicans, Cryptococcus neoformans, and T. asahii, and 73 MDRs were identified, all of which contained NADPH-binding motifs. T. asahii contained 20 MDRs that were unevenly distributed across six chromosomes. T. asahii MDRs (TaMDRs) had similar 3D structures but varied greatly in their genetic evolution at different phylum levels. RNA-seq and gene expression analyses revealed that the fluconazole-resistant T. asahii strain upregulates xylitol dehydrogenase, and downregulated alcohol dehydrogenase and sorbitol dehydrogenase concluded that the fluconazole-resistant T. asahii strain was less selective toward carbon sources and had higher adaptability to the environment. Overall, our study contributes to our understanding of TaMDRs, providing a basis for further analysis of the genes associated with drug resistance in T. asahii. [ABSTRACT FROM AUTHOR]

The use of immobilized alcohol dehydrogenases (ADHs) offers numerous advantages, especially in the reaction conditions required by industrial applications. Looking for more efficient and cost-effective methods of ADH immobilization, in this study we explored silica-based supports as an alternative to the use of functionalized polymeric resins. Three commercially available ADHs were immobilized by adsorption and covalent bond formation. The obtained supported biocatalysts were applied for the bioreduction of acetophenone and some derivatives with good yields and excellent enantioselectivity. The important intermediate (S)-1-[3,5-bis(trifluoromethyl)phenyl]ethanol was obtained with a high enantiomeric excess (>99%) by using the highest performing immobilized ADH sample. The reusability of this biocatalyst was investigated in a flow system for five consecutive runs; the experiments showed that the biocatalyst could be recycled without a loss of activity and enantioselectivity. Finally, cross-linking with the glutaraldehyde of the supported biocatalyst was also carried out to prevent the leaching of the enzyme during the catalytic reactions. [ABSTRACT FROM AUTHOR]