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The effect of salt stress on the activity of decarboxylating malate dehydrogenases in maize leaves during the adaptive response of cellular metabolism to salt stress was studied. It was shown that excess NaCl concentrations cause an increase in the enzymatic activity of both NAD+-ME and NADP+-ME. The greatest contribution to the increase in the activity of NAD+-ME is made by the intensification of the expression of the nad-me2 gene. At the same time, the expression of nad-me1 under salinity increases to a lesser extent. Methyl-specific PCR showed an inverse relationship between the expression of nad-me2 and the degree of methylation of the promoter of this gene, while nad-me1 and nadf-me, apparently, are regulated by different mechanisms. A different functional role of each isoform of the studied malic enzymes in the adaptive response of cellular metabolism is assumed. [ABSTRACT FROM AUTHOR]

Polysaccharides, being biocompatible and biodegradable polymers, are highly attractive as materials for protein delivery systems. However, protein–polysaccharide interactions may lead to protein structural transformation. In the current study, we analyze the structural adjustment of a homotetrameric protein, glyceraldehyde-3-phosphate dehydrogenase (GAPDH), upon its interactions with both flexible coil chain and the rigid helix of κ-carrageenan. FTIR spectroscopy was used to probe the secondary structures of both protein and polysaccharide. Electrostatically driven protein–polysaccharide interactions in dilute solutions resulted in an insoluble complex formation with a constant κ-carrageenan/GAPDH ratio of 0.2, which amounts to 75 disaccharide units per mole of protein tetramer. Upon interactions with both coiled and helical polysaccharides, a weakening of the intersubunit interactions was revealed and attributed to a partial GAPDH tetramer dissociation. In turn, protein distorted the helical conformation of κ-carrageenan when co-gelled. Molecular modeling showed the energy favorable interactions between κ-carrageenan and GAPDH at different levels of oligomerization. κ-Carrageenan binds in the region of the NAD-binding groove and the S-loop in OR contact, which may stabilize the OP dimers. The obtained results highlight the mutual conformational adjustment of oligomeric GAPDH and κ-carrageenan upon interaction and the stabilization of GAPDH's dissociated forms upon immobilization in polysaccharide gels. [ABSTRACT FROM AUTHOR]

The physical and chemical properties of the soil are important factors influencing the yield of crops. One of the agrotechnical factors influencing the biochemical properties of soil is sowing density. It affects the yield components, light, moisture and thermal conditions in the canopy and the pressure of pests. Secondary metabolites, many of which are known to act as a defense mechanism against insects, are of importance in the interaction between the crop and abiotic and biotic factors of the habitat. To the best of our knowledge, the studies conducted so far do not sufficiently reveal the impacts of the wheat species and the sowing density, together with the biochemical properties of the soil, on the accumulation of bioactive ingredients in the crop plants, and the subsequent impacts on the occurrence of phytophagic entomofauna in various management systems. Explaining these processes creates an opportunity for more sustainable development of agriculture. The study aimed to determine the effect of wheat species and sowing density on the biochemical properties of the soil, concentrations of biologically active compounds in the plant and the occurrence of insect pests in organic (OPS) and conventional (CPS) production systems. The research was conducted on spring wheat species (Indian dwarf wheat—Triticum sphaerococcum Percival and Persian wheat—Triticum persicum Vavilov) grown in OPS and CPS at sowing densities 400, 500, 600 (seeds m−2 ). The following analyzes were performed: (i) soil analysis: the activity of catalases (CAT), dehydrogenases (DEH), peroxidases (PER); (ii) plant analysis: total phenolic compounds (TP), chlorogenic acid (CA), antioxidant capacity (FRAP); (iii) entomological analysis of the number of insects—Oulema spp. adults and larvae. Performing analyzes in such a wide (interdisciplinary) scope will allow for a comprehensive understanding of the soil-plant-insect biological transformation evaluation. Our results showed that an increase in soil enzyme activity caused a decrease in TP contents in the wheat grown the OPS. Despite this, both the content of TP and the anti-oxidative activity of the ferric reducing ability of plasma (FRAP) were higher in these wheats. Bioactive compound contents and FRAP were most favoured by the lowest sowing density. Regardless of the production system, the occurrence of the Oulema spp. adults on T. sphaerococcum was the lowest at a sowing density of 500 seeds m−2 . The occurrence of this pest’s larvae was lowest at a sowing density of 400 seeds m−2 . Research on bioactive compounds in plants, biochemical properties of soil and the occurrence of pests make it possible to comprehensively assess the impact of the sowing density of ancient wheat in the ecological and conventional production system, which is necessary for the development of environmentally sustainable agriculture. [ABSTRACT FROM AUTHOR]

The physical and chemical properties of the soil are important factors influencing the yield of crops. One of the agrotechnical factors influencing the biochemical properties of soil is sowing density. It affects the yield components, light, moisture and thermal conditions in the canopy and the pressure of pests. Secondary metabolites, many of which are known to act as a defense mechanism against insects, are of importance in the interaction between the crop and abiotic and biotic factors of the habitat. To the best of our knowledge, the studies conducted so far do not sufficiently reveal the impacts of the wheat species and the sowing density, together with the biochemical properties of the soil, on the accumulation of bioactive ingredients in the crop plants, and the subsequent impacts on the occurrence of phytophagic entomofauna in various management systems. Explaining these processes creates an opportunity for more sustainable development of agriculture. The study aimed to determine the effect of wheat species and sowing density on the biochemical properties of the soil, concentrations of biologically active compounds in the plant and the occurrence of insect pests in organic (OPS) and conventional (CPS) production systems. The research was conducted on spring wheat species (Indian dwarf wheat—Triticum sphaerococcum Percival and Persian wheat—Triticum persicum Vavilov) grown in OPS and CPS at sowing densities 400, 500, 600 (seeds m−2). The following analyzes were performed: (i) soil analysis: the activity of catalases (CAT), dehydrogenases (DEH), peroxidases (PER); (ii) plant analysis: total phenolic compounds (TP), chlorogenic acid (CA), antioxidant capacity (FRAP); (iii) entomological analysis of the number of insects—Oulema spp. adults and larvae. Performing analyzes in such a wide (interdisciplinary) scope will allow for a comprehensive understanding of the soil-plant-insect biological transformation evaluation. Our results showed that an increase in soil enzyme activity caused a decrease in TP contents in the wheat grown the OPS. Despite this, both the content of TP and the anti-oxidative activity of the ferric reducing ability of plasma (FRAP) were higher in these wheats. Bioactive compound contents and FRAP were most favoured by the lowest sowing density. Regardless of the production system, the occurrence of the Oulema spp. adults on T. sphaerococcum was the lowest at a sowing density of 500 seeds m−2. The occurrence of this pest’s larvae was lowest at a sowing density of 400 seeds m−2. Research on bioactive compounds in plants, biochemical properties of soil and the occurrence of pests make it possible to comprehensively assess the impact of the sowing density of ancient wheat in the ecological and conventional production system, which is necessary for the development of environmentally sustainable agriculture.

Native amine dehydrogenases (nat-AmDHs) catalyze the (S)-stereoselective reductive amination of various ketones and aldehydes in the presence of high concentrations of ammonia. Based on the structure of CfusAmDH from Cystobacter fuscus complexed with Nicotinamide adenine dinucleotide phosphate (NADP+) and cyclohexylamine, we previously hypothesized a mechanism involving the attack at the electrophilic carbon of the carbonyl by ammonia followed by delivery of the hydride from the reduced nicotinamide cofactor on the re-face of the prochiral ketone. The direct reduction of carbonyl substrates into the corresponding alcohols requires a similar active site architecture and was previously reported as a minor side reaction of some native amine dehydrogenases and variants. Here we describe the ketoreductase (KRED) activity of a set of native amine dehydrogenases and variants, which proved to be significant in the absence of ammonia in the reaction medium but negligible in its presence. Conducting this study on a large set of substrates revealed the heterogeneity of this secondary ketoreductase activity, which was dependent upon the enzyme/substrate pairs considered. In silico docking experiments permitted the identification of some relationships between ketoreductase activity and the structural features of the enzymes. Kinetic studies of MsmeAmDH highlighted the superior performance of this native amine dehydrogenases as a ketoreductase but also its very low activity towards the reverse reaction of alcohol oxidation. [ABSTRACT FROM AUTHOR]

Metabolic syndrome is considered the precursor of type 2 diabetes mellitus. Tuberculosis is a leading infection that constitutes a global threat remaining a major cause of morbi-mortality in developing countries. People with type 2 diabetes mellitus are more likely to suffer from infection with Mycobacterium tuberculosis. For both type 2 diabetes mellitus and tuberculosis, there is pulmonary production of anti-inflammatory glucocorticoids mediated by the enzyme 11b-hydroxysteroid dehydrogenase type 1 (11b-HSD1). The adrenal hormone dehydroepiandrosterone (DHEA) counteracts the glucocorticoid effects of cytokine production due to the inhibition of 11b-HSD1. Late advanced tuberculosis has been associated with the suppression of the Th1 response, evidenced by a high ratio of cortisol/DHEA. In a murine model of metabolic syndrome, we determined whether DHEA treatment modifies the pro-inflammatory cytokines due to the inhibition of the 11b-HSD1 expression. Since macrophages express 11b-HSD1, our second goal was incubating them with DHEA and Mycobacterium tuberculosis to show that the microbicide effect was increased by DHEA. Enoyl-acyl carrier protein reductase (InhA) is an essential enzyme of Mycobacterium tuberculosis involved in the mycolic acid synthesis. Because 11b-HSD1 and InhA are members of a short-chain dehydrogenase/reductase family of enzymes, we hypothesize that DHEA could be an antagonist of InhA. Our results demonstrate that DHEA has a direct microbicide effect against Mycobacterium tuberculosis; this effect was supported by in silico docking analysis and the molecular dynamic simulation studies between DHEA and InhA. Thus, DHEA increases the production of proinflammatory cytokines in the lung, inactivates GC by 11b-HSD1, and inhibits mycobacterial InhA. The multiple functions of DHEA suggest that this hormone or its synthetic analogs could be an efficient co-adjuvant for tuberculosis treatment. [ABSTRACT FROM AUTHOR]

Background and objective: dihydropyridine dehydrogenase (DPD) deficiency has been associated with an increased risk of toxicity after exposure to fluoropyrimidines (FP). Determination of endogenous uracil (U) plasma concentrations is the recommended test to identify DPD deficiency. However, the value of U can be affected by various factors. The objective was to determine the serum concentration of U in a population candidate to receive treatment with FP and to verify if its distribution was compatible with the prevalence of partial DPD deficiency estimated in the Caucasian population. Material and methods: prospective observational study in which cancer patients candidates for FP treatment were included. For the analytical determination, a Dionex Ultimate 3000 UHPLC system coupled to a Q-exactive hybrid quadrupole-orbitrap mass spectrometer was used. Results: 77 patients, with a mean age of 71 years, were included. The mean and median serum U concentrations were 30.4 and 24.0 ng/ml, respectively. The range was from 7.1 to 139.7 ng/ml. 79.2% of the patients presented a U level between 16 and 150ng/ml, showing a statistically significant difference when compared to the estimated prevalence in the Caucasian population (8%) (p-value <0.0001). The analytical method used has a correlation coefficient R2 > 0.99 and a detection limit <0.2 ng/ml. Conclusions: it is necessary to carry out more studies with a design aimed at establishing the optimal conditions related to the pretreatment of the samples in order to avoid or minimize the influence of these factors on the analyte values. [ABSTRACT FROM AUTHOR]

Gene fusion or co-immobilization are key tools to optimize enzymatic reaction cascades by modulating catalytic features, stability and applicability. Achieving a defined spatial organization between biocatalysts by site-specific applications is complicated by the involvement of oligomeric enzymes. It can lead to activity losses due to disturbances of the quaternary structures and difficulties in stoichiometric control. Thus, a toolkit of active and robust monomeric enzymes is desirable for such applications. In this study, we engineered one of the rare examples of monomeric alcohol dehydrogenases for improved catalytic characteristics by site-directed mutagenesis. The enzyme from the hyperthermophilic archaeon Thermococcus kodakarensis naturally exhibits high thermostability and a broad substrate spectrum, but only low activity at moderate temperatures. The best enzyme variants showed an ~5-fold (2-heptanol) and 9-fold (3-heptanol) higher activity while preserving enantioselectivity and good thermodynamic stability. These variants also exhibited modified kinetic characteristics regarding regioselectivity, pH dependence and activation by NaCl. [ABSTRACT FROM AUTHOR]

Cytochromes are enzymes of the dehydrogenase class with a hemoprotein structure in which the iron in these compounds undergoes oxidation and reduction reactions upon receiving or losing electrons. Quantitatively speaking, CYP3A4 is the most important isoenzyme of cytochrome P450, oxidizing foreign organic molecules such as drugs or toxins to cause them to leave the body. Many drugs and antibiotics can induce or inhibit the activity of cytochrome P450, including dexamethasone. Dexamethasone is a steroidal anti-inflammatory drug used to treat of inflammatory diseases and chronic autoimmunity. This study aimed to investigate the induction effect of dexamethasone in biotransformation pathways by in silico tools. Molegro Virtual Docker software was used to investigate the molecular docking of the enzyme and dexamethasone, which indicated the binding of the drug to the enzyme. The molecular simulation was performed in Linux with the GROMACS program. Root-mean-square distance (RMSD), and radius of gyration (Rg), were evaluated. The results were analyzed with Pymol and VMD software, and the obtained curve was plotted with GRACE software. Docking results show that a cluster with a bond energy of -60.81 was the best cluster, and the bond size between ligand and internal atoms was -23.191in the complex. In addition, the amount of bond between the ligand and water for this pose was zero. The stability of the enzyme-ligand complex and the induction effect of dexamethasone on CYP3A4 were indicated by RMSD and RG results. Results of RMSD and RG of CYP3A4 glucocorticoid obtained from the simulation showed the stability of binding of the drug to the enzyme. Also, RMSD results showed the stability of glucocorticoid and dexamethasone complex during molecular dynamics simulation. It reached relative stability at 0.8 nm after 80,000 ps until the end of the simulation. [ABSTRACT FROM AUTHOR]

Metal-dependent formate dehydrogenases (Fdh) catalyze the reversible conversion of CO2 to formate, with unrivalled efficiency and selectivity. However, the key catalytic aspects of these enzymes remain unknown, preventing us from fully benefiting from their capabilities in terms of biotechnological applications. Here, we report a time-resolved characterization by X-ray crystallography of the Desulfovibrio vulgaris Hildenborough SeCys/W-Fdh during formate oxidation. The results allowed us to model five different intermediate structures and to chronologically map the changes occurring during enzyme reduction. Formate molecules were assigned for the first time to populate the catalytic pocket of a Fdh. Finally, the redox reversibility of DvFdhAB in crystals was confirmed by reduction and reoxidation structural studies. [ABSTRACT FROM AUTHOR]

SDR (Short-chain Dehydrogenases/Reductases) are one of the oldest and heterogeneous superfamily of proteins, whose classification is problematic because of the low percent identity, even within families. To get clearer insights into SDR molecular evolution, we explored the splicing site organization of the 75 human SDR genes across their vertebrate and invertebrate orthologs. We found anomalous gene structures in members of the human SDR7C and SDR42E families that provide clues of retrogene properties and independent evolutionary trajectories from a common invertebrate ancestor. The same analyses revealed that the identity value between human and invertebrate non-allelic variants is not necessarily associated with the homologous gene structure. Accordingly, a revision of the SDR nomenclature is proposed by including the human SDR40C1 and SDR7C gene in the same family. [ABSTRACT FROM AUTHOR]

Two methanol dehydrogenases (MDHs), MxaFI and XoxF, have been characterized in methylotrophic and methanotrophic bacteria. MxaFI contains a calcium ion in its active site, whereas XoxF contains a lanthanide ion. Importantly, the expression of MxaFI and XoxF is inversely regulated by lanthanide bioavailability, i.e., the "lanthanide switch." To reveal the genetic and environmental factors affecting the lanthanide switch, we focused on two Methylosinus trichosporium OB3b mutants isolated during routine cultivation. In these mutants, MxaF was constitutively expressed, but lanthanide-dependent XoxF1 was not, even in the presence of 25 μM cerium ions, which is sufficient for XoxF expression in the wild type. Genotyping showed that both mutants harbored a loss-of-function mutation in the CQW49_RS02145 gene, which encodes a TonB-dependent receptor. Gene disruption and complementation experiments demonstrated that CQW49_RS02145 was required for XoxF1 expression in the presence of 25 μM cerium ions. Phylogenetic analysis indicated that CQW49_RS02145 was homologous to the Methylorubrum extorquens AM1 lanthanide transporter gene (lutH). These findings suggest that CQW49_RS02145 is involved in lanthanide uptake across the outer membrane. Furthermore, we demonstrated that supplementation with cerium and glycerol caused severe growth arrest in the wild type. CQW49_RS02145 underwent adaptive laboratory evolution in the presence of cerium and glycerol ions, resulting in a mutation that partially mitigated the growth arrest. This finding implies that loss-offunction mutations in CQW49_RS02145 can be attributed to residual glycerol from the frozen stock. [ABSTRACT FROM AUTHOR]

Gluconobacter is a potential strain for single-step production of 2-keto-L-gulonic acid (2-KLG), which is the direct precursor of vitamin C. Three dehydrogenases, namely, sorbitol dehydrogenase (SLDH), sorbose dehydrogenase (SDH), and sorbosone dehydrogenase (SNDH), are involved in the production of 2-KLG from D-sorbitol. In the present study, the potential SNDH/SDH gene cluster in the strain Gluconobacter cerinus CGMCC 1.110 was mined by genome analysis, and its function in transforming L-sorbose to 2-KLG was verified. Proteomic analysis showed that the expression level of SNDH/SDH had a great influence on the titer of 2-KLG, and fermentation results showed that SDH was the rate-limiting enzyme. A systematic metabolic engineering process, which was theoretically suitable for increasing the titer of many products involving membrane-bound dehydrogenase from Gluconobacter, was then performed to improve the 2-KLG titer in G. cerinus CGMCC 1.110 from undetectable to 51.9 g/L in a 5-L bioreactor after fermentation optimization. The strategies used in this study may provide a reference for mining other potential applications of Gluconobacter. Key points: • The potential SNDH/SDH gene cluster in G. cerinus CGMCC 1.110 was mined. • A systematic engineering process was performed to improve the titer of 2-KLG. • The 2-KLG titer was successfully increased from undetectable to 51.9 g/L. [ABSTRACT FROM AUTHOR]

In clear cell renal cell carcinoma (ccRCC), activation of hypoxic signaling induces NADH dehydrogenase (ubiquinone) 1 alpha subcomplex, 4-like 2 (NDUFA4L2) expression. Over 90% of ccRCCs exhibit overexpression of NDUFA4L2, which we previously showed contributes to ccRCC proliferation and survival. The function of NDUFA4L2 in ccRCC has not been fully elucidated. NDUFA4L2 was reported to reduce mitochondrial respiration via mitochondrial complex I inhibition. We found that NDUFA4L2 expression in human ccRCC cells increases the extracellular acidification rate, indicative of elevated glycolysis. Conversely, NDUFA4L2 expression in non-cancerous kidney epithelial cells decreases oxygen consumption rate while increasing extracellular acidification rate, suggesting that a Warburg-like effect is induced by NDUFA4L2 alone. We performed mass-spectrometry (MS)-based proteomics of NDUFA4L2 associated complexes. Comparing RCC4-P (parental) ccRCC cells with RCC4 in which NDUFA4L2 is knocked out by CRISPR-Cas9 (RCC4-KO-643), we identified 3,215 proteins enriched in the NDUFA4L2 immunoprecipitates. Among the top-ranking pathways were "Metabolic Reprogramming in Cancer" and "Glycolysis Activation in Cancer (Warburg Effect)." We also show that NDUFA4L2 enhances mitochondrial fragmentation, interacts with lysosomes, and increases mitochondrial-lysosomal associations, as assessed by highresolution fluorescence microscopy and live cell imaging. We identified 161 lysosomal proteins, including Niemann-Pick Disease Type C Intracellular Cholesterol Transporters 1 and 2 (NPC1, NPC2), that are associated with NDUFA4L2 in RCC4-P cells. RCC4-P cells have larger and decreased numbers of lysosomes relative to RCC4 NDUFA4L2 knockout cells. These findings suggest that NDUFA4L2 regulates mitochondrial- lysosomal associations and potentially lysosomal size and abundance. Consequently, NDUFA4L2 may regulate not only mitochondrial, but also lysosomal functions in ccRCC. [ABSTRACT FROM AUTHOR]

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The study objective was to determine the relationship of selected enzyme activities with carbon sequestration and N, P, K, Mg, Zn and Cu contents in Phaeozem soils. Soil samples were taken from a 10 ha area. A selection of their physical and chemical properties and the contents of the available forms of selected macro- and microelements were determined. The activities of dehydrogenases (DEH), catalase (CAT), peroxidases (PER), alkaline (AlP) and acid (AcP) phosphatase, β-glucosidase (BG) and proteases (PR) were also determined. The relationship between enzymatic soil fertility indices (AlP/AcP, BIF, GMea, TEI, BA12 and BA13) and selected soil parameters was also determined. The research used principal component analysis (PCA) to distinguish significantly correlated parameters of a Phaeozem used for agricultural purposes. The study area showed low TOC and K contents and average P and Mg contents. Significant positive correlations were found between the TOC content and activity of the tested enzymes, evidencing that soil enzymes are an important parameter in carbon sequestration and soil nutrient dynamics. [ABSTRACT FROM AUTHOR]

The liver maintains important homeostatic functions such as metabolism and detoxification. Failure to remove toxic intermediates can cause hepatic damage, liver fibrosis, and even cancer development. This review focuses on acetaldehyde dehydrogenases (ALDHs), a group of key enzymes within the ALDH superfamily with the ability to convert highly reactive aldehyde substrates to the corresponding carboxylic acids in NAD(P)-dependent manners. These enzymes participate in a diverse array of biological processes such as detoxification, biosynthesis, antioxidant, and regulatory functions. ALDH dysfunction can disrupt homeostasis, leading to toxic buildup, tissue damage, and cancer. Here, we examine the expression patterns of hepatic ALDHs in adult normal human livers and two types of liver cancers--hepatocellular carcinoma and cholangiocarcinoma. We also investigated their distributions related to liver zonation. These observations provide deep insights into previously unrecognized spatial and temporal regulation of ALDHs in liver zonation. [ABSTRACT FROM AUTHOR]

A minimal light‐driven approach was established for studying enzymatic CO2 conversion spectroscopically. The system consists of a photosensitizer Eosin Y, EDTA as a sacrificial electron donor and substrate source, and formate dehydrogenase from Rhodobacter capsulatus (RcFDH) as a biocatalyst. This simplified three‐component system provides a photo‐triggered control for in situ characterization of the entire catalytic reaction. Direct reduction of RcFDH by the photosensitizer without additional electron carriers was confirmed via UV‐Vis spectroscopy, while GC‐MS and IR spectroscopy were used to follow photoinduced CO2 generation from EDTA and its subsequent enzymatic reduction, yielding the product formate. Photo‐driven and in vitro, dye‐based CO2 reduction was inhibited by azide under a mixed (competitive‐non‐competitive) inhibition mode. IR spectroscopy reveals displacement of the competitively‐bound azide by CO2, reflecting an interaction of both with the active site cofactor. This work comprises a proof‐of‐concept for a new approach to employ light for regulating the reaction of formate dehydrogenases and other CO2 reductases. [ABSTRACT FROM AUTHOR]

Glucose-6-phosphate dehydrogenase (G6PDH) catalyzes a metabolic hub between glycolysis and the pentose phosphate pathway (PPP), which is the oxidation of glucose-6-phosphate (G6P) to 6-phosphogluconolactone concomitantly with the production of nicotinamide adenine dinucleotide phosphate (NADPH), a reducing power. It is considered to be the rate-limiting step that governs carbon flow through the oxidative pentose phosphate pathway (OPPP). The OPPP is the main supplier of reductant (NADPH) for several "reducing" biosynthetic reactions. Although it is involved in multiple physiological processes, current knowledge on its exact role and regulation is still piecemeal. The present review provides a concise and comprehensive picture of the diversity of plant G6PDHs and their role in seed germination, nitrogen assimilation, plant branching, and plant response to abiotic stress. This work will help define future research directions to improve our knowledge of G6PDHs in plant physiology and to integrate this hidden player in plant performance. [ABSTRACT FROM AUTHOR]

Succinate dehydrogenase (SDH) plays an important role in reverse electron transfer during hypoxia/anoxia, in particular, in ischemia, when blood supply to an organ is disrupted, and oxygen is not available. It was detected in the voltammetry studies about three decades ago that the SDHA/SDHB subcomplex of SDH can have such a strong nonlinear property as a "tunnel-diode" behavior in reverse quinol-fumarate reductase direction. The molecular and kinetic mechanisms of this phenomenon, that is, a strong drop in the rate of fumarate reduction as the driving force is increased, are still unclear. In order to account for this property of SDH, we developed and analyzed a mechanistic computational model of reverse electron transfer in the SDHA/SDHB subcomplex of SDH. It was shown that a decrease in the rate of succinate release from the active center during fumarate reduction quantitatively explains the experimentally observed tunnel-diode behavior in SDH and threshold values of the electrode potential of about −80 mV. Computational analysis of ROS production in the SDHA/SDHB subcomplex of SDH during reverse electron transfer predicts that the rate of ROS production decreases when the tunnel-diode behavior appears. These results predict a low rate of ROS production by the SDHA/SDHB subcomplex of SDH during ischemia. [ABSTRACT FROM AUTHOR]

The consumption of high-fructose corn syrup (HFCS) has been increasing in recent decades, especially among children. Some reports suggest that children and adolescents are more sensitive to the adverse effects of fructose intake than adults. However, the underlying mechanism of the difference in vulnerability between adolescence and adulthood have not yet been elucidated. In this study, we attempted to elucidate the different effects of HFCS intake at different growth stages in rats: childhood and adolescence (postnatal day (PD) 21–60), young adulthood (PD60–100), and adulthood (PD100–140). Since alterations in hepatic glucocorticoid (GC) metabolism can cause diseases including insulin resistance, we focused on GC metabolizing enzymes such as 11 beta-hydroxysteroid dehydrogenase 1 and 2 (Hsd11b1 and Hsd11b2) and steroid 5 alpha-reductase 1 (Srd5a1). Western blotting showed an increase in Hsd11b1 expression and a decrease in Hsd11b2 expression in childhood and adolescence but not in adulthood. We also observed changes in Hsd11b1 and Hsd11b2 activities only in childhood and adolescence, consistent with the results of mRNA and protein expression analysis. The effect of high-fructose intake with regards to GC metabolism may therefore vary with developmental stage. This study provides insight into the adverse effects of fructose on GC metabolism in children in the context of increasing rates of HFCS consumption. [ABSTRACT FROM AUTHOR]

Patulin is a fatal mycotoxin that is widely detected in drinking water and fruit-derived products contaminated by diverse filamentous fungi. Cg SDR from Candida guilliermondii represents the first NADPH-dependent short-chain dehydrogenase/reductase that catalyzes the reduction of patulin to the nontoxic E -ascladiol. To elucidate the catalytic mechanism of Cg SDR, we solved its crystal structure in complex with cofactor and substrate. Structural analyses indicate that patulin is situated in a hydrophobic pocket adjacent to the cofactor, with the hemiacetal ring orienting toward the nicotinamide moiety of NADPH. In addition, we conducted structure-guided engineering to modify substrate-binding residue V187 and obtained variant V187F, V187K and V187W, whose catalytic activity was elevated by 3.9-, 2.2- and 1.7-fold, respectively. The crystal structures of Cg SDR variants suggest that introducing additional aromatic stacking or hydrogen-bonding interactions to bind the lactone ring of patulin might account for the observed enhanced activity. These results illustrate the catalytic mechanism of SDR-mediated patulin detoxification for the first time and provide the upgraded variants that exhibit tremendous potentials in industrial applications. • Crystal structure of Cg SDR in complex with NADPH and patulin was solved. • Cg SDR-mediated patulin-degrading mechanism was illustrated. • Structure-based rational design yielded Cg SDR variants with higher catalytic activity. [ABSTRACT FROM AUTHOR]

Comparative phylogenetic analysis of NAD+-dependent formate dehydrogenases (NAD+-FDH) genes, which have been detected in all available genomes of methylotrophs of the genera Ancylobacter, Starkeya, and Angulomicrobium, as well as in other members of the family Xanthobacteraceae (Xanthobacter, Aquabacter, Azorhizobium), was carried out. The position of Xanthobacteraceae on the tree constructed based on comparison of NAD+−FDH amino acid sequences was found to correlate with the 16S rRNA gene-based phylogeny. The sequences of the NAD+−FDH proteins of the genera Ancylobacter, Starkeya, and Angulomi-crobium exhibited 87.8–98.3% identity, indicating that this protein is very conservative within this group of methylotrophs. For the first time, analysis of the NAD+−FDH functional genes is recommended as a supplementary criterion for interspecies differentiation between methylotrophic bacteria of the genus Ancylobacter. [ABSTRACT FROM AUTHOR]

The enzymatic transformation of various chemicals, especially using NADPH-dependent hydroxylase, into more soluble and/or high value-added products has steadily garnered increasing attention. However, the industrial application of these NADPH-dependent hydroxylases has been limited due to the high cost of the cofactor NADPH. As an alternative, enzymatic NADPH-regeneration systems have been developed and are frequently used in various fields. Here, we expressed and compared two recombinant isocitrate dehydrogenases (IDHs) from Corynebacterium glutamicum and Azotobacter vinelandii in Escherichia coli. Both enzymes were hyper-expressed in the soluble fraction of E. coli and were single-step purified to apparent homogeneity with yields of more than 850 mg/L. These enzymes also functioned well when paired with NADPH consumption systems. Specifically, NADPH was regenerated from NADP+ when an NADPH-consuming cytochrome P450 BM3 from Bacillus megaterium was incorporated. Therefore, both enzymes could be used as alternatives to the commonly used regeneration system for NADPH. These enzymes also have promising potential as genetic fusion partners with NADPH-dependent enzymes due to the monomeric nature of their quaternary structure, thereby resulting in self-sufficient biocatalysts via NADPH regeneration in a single polypeptide with NADPH-dependent activity. [ABSTRACT FROM AUTHOR]

Application of thermoplastic fiber-reinforced lightweight composite materials provides a wide range of advantages that are of particular importance for the mobility sector. UD tapes composed of unidirectionally (UD) oriented inorganic fibers embedded in a thermoplastic matrix represent light-weight materials with high tensile strength. This publication addresses recycling aspects of novel UD tape made of a combination of basalt fibers and different PLA (polylactic acid) formulations. The kinetics of enzyme-based separation of polymer from the fiber were investigated. Different types of UD tapes with a thickness of 270-290 μm reinforced with basalt fiber weight ratios ranging between 51 and 63% were incubated at 37 °C in buffer solution (pH 7.4) containing proteinase K. The influence of enzyme concentration, tape weight per incubation tube, proteinase K activators, and tape types on the rate of enzymatic decomposition was investigated. Enzyme activity was measured by analyzing lactate concentration with lactate dehydrogenase and by measuring weight loss of the composite material. The rate of lactate release increased in the first 30 min of incubation and remained stable for at least 90 min. Weight loss of 4% within 4 h was achieved for a tape with 56% (w/w) fiber content. For a sample with a surface area of 3 cm² in a buffer volume of 10 mL, the rate of lactate release as a function of enzyme concentration reached saturation at 300 μg enzyme/mL. With this enzyme concentration, the rate of lactate release increased in a linear manner for tape surface areas between 1 and 5 cm². Four tapes with different PLA types were treated with the enzyme for 17 h. Weight loss ranged between 7 and 24%. Urea at a concentration of 0.5% (w/v) increased lactate release by a factor of 9. Pretreatment of tapes in alkaline medium before enzymatic degradation increased weight loss to 14% compared to 5% without pretreatment. It is concluded that enzymatic PLA hydrolysis from UD tapes is a promising technology for the release of basalt fibers after alkaline pretreatment or for the final cleaning of basalt fibers. [ABSTRACT FROM AUTHOR]

Microbial degradation of natural rubber and synthetic poly(cis-1,4-isoprene) is expected to become an alternative treatment system for waste from poly(cis-1,4-isoprene) products including scrap tires. Nocardia farcinica NBRC 15,532, a gram-positive rubber-degrading bacterium, can utilize poly(cis-1,4-isoprene) as the sole source of carbon and energy to produce oligo-isoprene metabolites containing aldehyde and keto end groups. A homology-based search of the genome revealed a gene encoding a latex-clearing protein (Lcp). Gene disruption analysis indicated that this gene is essential for the utilization of poly(cis-1,4-isoprene) in this strain. Further analysis of the genome sequence identified aldehyde dehydrogenase (ALDH) genes as potential candidates for oxidative degradation of oligo-isoprene aldehydes. Based on the enzymatic activity of the ALDH candidates, NF2_RS14000 and NF2_RS14385 may be involved in the degradation of oligo-isoprene aldehydes. Analysis of the reaction products revealed that these ALDHs oxidized tri- to penta-isoprene aldehydes, which were generated by the reaction of Lcp. Based on the inability of ALDH gene deletion mutants, we concluded that NF2_RS14000 is mainly involved in the utilization of poly(cis-1,4-isoprene) and the oxidative degradation of oligo-isoprene aldehydes in Nocardia farcinica NBRC 15,532. [ABSTRACT FROM AUTHOR]

The aim of the study was to assess the quality of agricultural soils, which accumulate additional amounts of heavy metals from fertilization and modify their bioavailability, with the use of interdependencies between their biological and chemical properties conditioned by the distance from communication routes. Our results indicated that heavy metals had an impact on enzyme activity in soils and their accumulation was significantly related to the distance from the edge of the road, location of sampling sites, date of soil sampling, and years of research. It was found that the greatest amounts of zinc, cadmium, lead, and copper were accumulated at a distance of 5–20 m from the edge of the road. The highest enrichment factor and geoaccumulation index values were recorded for Pb, followed by Zn, Cu, and Cd. Principal component analysis and regression models showed that the activities of phosphatase and dehydrogenases seem to be the best bioindicators of contamination of roadside soils used for agricultural purposes. Since their activity is related to soil's organic carbon content, inputs of organic fertilizers and crop residues should be ensured in the agroecosystems along roadsides. [ABSTRACT FROM AUTHOR]

Medium‐chain alcohol dehydrogenases (ADHs) comprise a highly conserved enzyme family that catalyse the reversible reduction of aldehydes. However, recent discoveries in plant natural product biosynthesis suggest that the catalytic repertoire of ADHs has been expanded. Here we report the crystal structure of dihydroprecondylocarpine acetate synthase (DPAS), an ADH that catalyses the non‐canonical 1,4‐reduction of an α,β‐unsaturated iminium moiety. Comparison with structures of plant‐derived ADHs suggest the 1,4‐iminium reduction does not require a proton relay or the presence of a catalytic zinc ion in contrast to canonical 1,2‐aldehyde reducing ADHs that require the catalytic zinc and a proton relay. Furthermore, ADHs that catalysed 1,2‐iminium reduction required the presence of the catalytic zinc and the loss of the proton relay. This suggests how the ADH active site can be modified to perform atypical carbonyl reductions, providing insight into how chemical reactions are diversified in plant metabolism. [ABSTRACT FROM AUTHOR]

Native Amine Dehydrogenases (nat‐AmDHs) are NAD(P)H‐enzymes performing reductive amination, mainly active towards small aliphatic aldehydes and cyclic ketones, due to active site volumes limited by the presence of several bulky amino acids. Herein, inspired by the diversity of residues at these positions among the family, we report the implementation of mutations F140A and W145A in CfusAmDH and their transposition in nine other members. Moderate to high conversions were obtained with substrates not accepted by the native enzymes, notably n‐alkylaldehydes (44.6 %–99.5 % for hexanal to nonanal) and n‐alkylketones (16.0 %–53.7 % for hexan‐2‐one to nonan‐2‐one) with retention of excellent (S)‐enantioselectivity (>99 % ee). Complementary to the reported (R)‐selective AmDHs, the promising mutant CfusAmDH−W145A was further characterized for its synthetic potential. Crystal structure resolution and molecular dynamics gave insights into the cofactor and substrate specificity and the whole structural dynamics, thus providing keys for mutagenesis work on this enzyme family. [ABSTRACT FROM AUTHOR]

Upgrading of the bio-based platform chemical 5-hydroxymethylfurfural (HMF) into high-value derivatives is an important research topic, particularly in green and sustainable chemistry. Herein, we applied a new, highly HMF-tolerant strain, Enterobacter ludwigii YYP3, as a whole-cell biocatalyst for efficient reduction of HMF to 2,5-bis(hydroxymethyl)furan (BHMF). Upon process optimization, within only 3 h, BHMF was produced with a yield >99% and 98.5% selectivity using 100 mM HMF, resulting in the highest space time yield (4.2 g L−1 h−1) among the currently reported HMF bioreduction processes. In a fed-batch conversion, E. ludwigii YYP3 achieved large-scale production of 290 mM BHMF within 9 h and retained its high catalytic activity for three runs (27 h), suggesting an excellent cycling stability. Based on genome and transcriptome analysis, the molecular mechanism underlying the high HMF tolerance of E. ludwigii YYP3 was explored, primarily through the downregulation of genes related to amino acid biosynthetic and metabolic processes and upregulation of genes associated with DNA replication, recombination, and repair; biofilm formation; and redox homeostasis. Meanwhile, two novel short-chain dehydrogenase/reductase family oxidoreductases ElSDR-ykvO and ElSDR-SSP1627 were identified as target enzymes responsible for conversion of HMF to less toxic BHMF in E. ludwigii YYP3. Combined with structure and mutation analysis, the catalytic mechanisms of target enzymes were determined to be based on the active sites Ser, Tyr, and Lys. Our work not only confirms that E. ludwigii YYP3 has promising application prospects in large-scale production of BHMF, but also provides novel insights into understanding the molecular mechanism of HMF reduction. [ABSTRACT FROM AUTHOR]

Background: Data on the microbial community and functional proteins associated with degumming in kenaf remains scant. Here, we analyzed the microbial communities associated with kenaf (Hibiscus cannabinus) bast fibers during retting to identify potential candidate degumming bacteria. Retting liquids were collected and analyzed at 0 days, 10 days, and 34 days and then evaluated the yield and quality of kenaf fiber at the different retting times. Besides, the microbial communities were characterized using metagenomic and proteomic analysis by LC–MS/MS technology. Results: The data showed that increase in the retting time significantly improves the softness, dispersion, and fiber whiteness of the kenaf fiber. The relative abundance of Acinetobacter increased from 2.88% at the baseline to 6.64% at the 34th retting. On the other hand, some members of Clostridium were reduced from 3% at the baseline to 2% at the 34th retting. Analysis of carbohydrate active enzymes showed constant changes in the utilization of carbohydrates. Besides, benzoquinone reductase, cellobiose dehydrogenase, glucose 1-oxidase, aryl alcohol oxidase and alcohol oxidase were the top five most abundant enzymes in the retting liquids. This present results demonstrated that the expressions of B7GYR8, Q6RYW5 and Q6FFK2 proteins were suppressed in Acinetobacter with the retting time. On the contrary, P05149 was upregulated with the retting time. In Clostridium, P37698, P52040 and P54937 proteins were upregulated with the retting time. Conclusion: In addition, bacteria Acinetobacter and Clostridium might be playing important roles in the kenaf degumming process. Similarly, up-regulation of P37698, P52040 and P54937 proteins is an important manifestation and mediates important roles in the degumming process. Highlights: The growth of Prevotella was initially inhibited with the kenaf retting. Acinetobacter can promote the degumming process of kenaf. The upregulation of P37698, P52040 and P54937 proteins is a sign in the degumming. [ABSTRACT FROM AUTHOR]

This study presents the immobilization with aldehyde groups (glyoxyl carbon felt) of alcohol dehydrogenase (ADH) and formate dehydrogenase (FDH) on carbon‐felt‐based electrodes. The compatibility of the immobilization method with the electrochemical application was studied with the ADH bioelectrode. The electrochemical regeneration process of nicotinamide adenine dinucleotide in its oxidized form (NAD+), on a carbon felt surface, has been deeply studied with tests performed at different electrical potentials. By applying a potential of 0.4 V versus Ag/AgCl electrode, a good compromise between NAD+ regeneration and energy consumption was observed. The effectiveness of the regeneration of NAD+ was confirmed by electrochemical oxidation of ethanol catalyzed by ADH in the presence of NADH, which is the no active form of the cofactor for this reaction. Good reusability was observed by using ADH immobilized on glyoxyl functionalized carbon felt with a residual activity higher than 60 % after 3 batches. [ABSTRACT FROM AUTHOR]

Gluconobacter sphaericus has not yet been used in biotransformation studies. In this study, G. sphaericus SJF2-1, which produces a diffusible pigment, was isolated from grape. The spent culture medium became dark black when the cells were grown in medium containing glucose and then autoclaved. This bacterium produced 2,5-diketo-D-gluconate (2,5-DKG) from D-glucose and D-gluconate. When 5% D-glucose was used, the conversion efficiency was approximately 52.4% in a flask culture. 2,5-DKG is a precursor of 2-keto-L-gulonic acid, which is a key intermediate in the industrial production of L-ascorbic acid. The complete genome sequence of G. sphaericus SJF2-1 was determined for the first time in the G. sphaericus species. The total size was 3,198,086 bp, with 2867 protein-coding sequences; one chromosome and six plasmids were identified. From the genome of SJF2-1, multiple genes homologous to those involved in the conversion of D-glucose to 2,5-DKG were identified. In particular, six different genes encoding membrane-associated flavoprotein-cytochrome c complexed dehydrogenase were identified and divided into two different lineages. This study suggests the potential of G. sphaericus SJF2-1 to mass-produce 2,5-DKG and other D-glucose oxidation products. [ABSTRACT FROM AUTHOR]

Mitochondrial 2-oxoglutarate dehydrogenase complex (2-OGDH) that consists of multiple copies of 3 catalytic subunits (E1, E2, E3) is a regulatory enzyme of the tricarboxylic acid cycle. 2-OGDH together with branched-chain a-ketoacid dehydrogenase (BCKDH) and pyruvate dehydrogenase (PDH) belongs to the 2-oxoacid dehydrogenases family. It was shown that in protein-restricted rats simvastatin stimulated liver BCKDH, whereas it exerted no effect on BCKDH in rats fed a standard diet. We hypothesized that a combination of simvastatin and a low-protein diet could have an impact on renal 2-OGDH. The purpose of the study was to determine the effect of the combination of simvastatin and a low-protein diet on renal 2-OGDH in rats. Simvastatin (80 mg/kg b.wt/day) or the vehicle (0.3% methylcellulose) were administered orally (for 14 days) to rats fed a low-protein (8% protein) or standard (23% protein) diet. 2-OGDH activity, protein levels, and mRNA levels for E1 and E2 subunits were determined. In addition, serum creatinine level was measured. Results: The combination of simvastatin and a low-protein diet elicited an increase in renal 2-OGDH activity that corresponded to the increase of E1 protein, but not of E1 mRNA level. In contrast, simvastatin treatment did not affect 2-OGDH activity, nor protein and mRNA levels of E1 in rats fed a standard diet. Serum creatinine levels were not changed upon simvastatin administration in any group. In conclusion, the results of the present study indicate that the combination of simvastatin and a low-protein diet induces stimulation of the renal 2-OGDH complex probably at a post-transcriptional level. [ABSTRACT FROM AUTHOR]

Dithiocarbamates have been widely used in various industrial applications, such as insecticides (ferbam) or drug (disulfiram). This study explored the inhibitory effects of dithiocarbamates on human and rat gonadal 3β-hydroxysteroid dehydrogenases (3β-HSD) and investigated the structure-activity relationship and mechanistic insights. The inhibitory activity of six dithiocarbamates and thiourea on the conversion of pregnenolone to progesterone was evaluated using human KGN cell and rat testicular microsomes, with subsequent progesterone measurement using HPLC-MS/MS. The study found that among the tested compounds disulfiram, ferbam, and thiram exhibited significant inhibitory activity against human 3β-HSD2 and rat 3β-HSD1, with ferbam demonstrating the highest potency. The mode of action for these compounds was characterized, showing mixed inhibition for human 3β-HSD2 and mixed/noncompetitive inhibition for rat 3β-HSD1. Additionally, it was observed that dithiothreitol dose-dependently reversed the inhibitory effects of dithiocarbamates on both human and rat gonadal 3β-HSD enzymes. The study also delved into the penetration of these dithiocarbamates through the human KGN cell membrane and their impact on progesterone production, highlighting their potency in inhibiting human 3β-HSD2. Furthermore, bivariate correlation analysis revealed a positive correlation of LogP (lipophilicity) with IC 50 values for both enzymes. Docking analysis indicated that dithiocarbamates bind to NAD+ and steroid-binding sites, with some interactions with cysteine residues. In conclusion, this study provides valuable insights into the structure-activity relationship and mechanistic aspects of dithiocarbamates as inhibitors of human and rat gonadal 3β-HSDs, suggesting that these compounds likely exert their inhibitory effects through binding to cysteine residues. [Display omitted] • Some dithiocarbamates inhibit human and rat gonadal 3β-HSDs. • Ferbam is the most potent inhibitor of both human and rat gonadal 3β-HSD. • Dithiocarbamates are mixed/noncompetitive inhibitors of gonadal 3β-HSDs. • DTT can reverse dithiocarbamate-mediated inhibition on 3β-HSDs. • Lipophilicity determines the inhibitory potency of dithiocarbamates on 3β-HSDs. [ABSTRACT FROM AUTHOR]

Mutations in IDH1 are commonly observed across various cancers, causing the conversion of α-KG to 2-HG. Elevated levels of 2-HG disrupt histone and DNA demethylation processes, promoting tumor development. Consequently, there is substantial interest in developing small molecule inhibitors targeting the mutant enzymes. Herein, we report a structure-based high-throughput virtual screening strategy using a natural products library, followed by hit-to-lead optimization. Through this process, we discover a potent compound, named 11s , which exhibited significant inhibition to IDH1 R132H and IDH1 R132C with IC 50 values of 124.4 and 95.7 nM, respectively. Furthermore, 11s effectively reduced 2-HG formation, with EC 50 values of 182 nM in U87 R132H cell, and 84 nM in HT-1080 cell. In addition, 11s significantly reduced U87 R132H and HT-1080 cell proliferation with GC 50 values of 3.48 and 1.38 μM, respectively. PK-PD experiments further confirmed that compound 11s significantly decreased 2-HG formation in an HT-1080 xenograft mouse model, resulting in notable suppression of tumor growth without apparent loss in body weight. Presented herein is a novel mIDH1 inhibitor, 11s , which demonstrated a reduction in 2-HG formation with EC 50 values of 182 nM in U87 R132H cells and 84 nM in HT-1080 cells, respectively. [Display omitted] • 11s displayed IDH1 R132H and IDH1 R132C inhibitory activities with IC 50 values of 124.4 and 95.7 nM. • 11s exhibited cytotoxic effects on IDH1 mutant cancer cell lines. • 11s reduced 2-HG formation with EC 50 values of 182 nM in U87 R132H cell, and 84 nM in HT-1080 cell. [ABSTRACT FROM AUTHOR]

Sex steroid hormones such as estrogen estradiol (E2) and androgen dihydrotestosterone (DHT) are involved in the development of hormone-dependent cancers. Blockade of 17β-hydroxysteroid dehydrogenase type 7 (17β-HSD7), a member of the short chain dehydrogenase/reductase superfamily, is thought to decrease E2 levels while increasing those of DHT. Therefore, its unique double action makes this enzyme as an interesting drug target for treatment of breast cancer. The chemical synthesis, molecular characterization, and preliminary biological evaluation as 17β-HSD7 inhibitors of novel carbamate derivatives 3 and 4 are described. Like previous 17β-HSD7 inhibitors 1 and 2 , compounds 3 and 4 bear a hydrophobic nonyl side chain at the C-17β position of a 4-aza-5α-androstane nucleus, but compound 3 has an oxygen atom replacing the CH 2 in the steroid A-ring C-2 position, while compound 4 has a C17-spiranic E-ring containing a carbamate function. They both inhibited the in vitro transformation of estrone (E1) into E2 by 17β-HSD7, but the introduction of a (17 R)-spirocarbamate is preferable to replacing C-2 methylene with an oxygen atom since compound 4 (IC 50 = 63 nM) is an inhibitor 14 times more powerful than compound 3 (IC 50 = 900 nM). Furthermore, when compared to the reference inhibitor 1 (IC 50 = 111 nM), the use of a C17-spiranic E-ring made it possible to introduce differently the hydrophobic nonyl side chain, without reducing the inhibitory activity. [Display omitted] • 17β-HSD7 is an interesting drug target for breast cancer treatment. • Hydrophobic nonyl steroid carbamates were synthesized as 17β-HSD7 inhibitors. • Steroid carbamates were fully characterized by nuclear magnetic resonance spectroscopy. • C17-spiranic carbamate 4 inhibited the formation of estradiol (IC 50 =63 nM) by 17β-HSD7. • Synthesis of 4 required only 4 steps starting from 4-methyl-aza-5α-androstane-3,17-dione. [ABSTRACT FROM AUTHOR]

3α-HSDHs have a crucial role in the bioconversion of steroids, and have been widely applied in the detection of total bile acid (TBA). In this study, we report a novel NADP(H)-dependent 3α-HSDH (named Sc 3α-HSDH) cloned from the intestinal microbiome of Ursus thibetanus. Sc 3α-HSDH was solubly expressed in E. coli (BL21) as a recombinant glutathione-S-transferase (GST)-tagged protein and freed from its GST-fusion by cleavage using the PreScission protease. Sc 3α-HSDH is a new member of the short-chain dehydrogenases/reductase superfamily (SDRs) with a typical α/β folding pattern, based on protein three-dimensional models predicted by AlphaFold. The best activity of Sc 3α-HSDH occurred at pH 8.5 and the temperature optima was 55 °C, indicating that Sc 3α-HSDH is not an extremozyme. The catalytic efficiencies (k cat / K m) of Sc 3α-HSDH catalyzing the oxidation reaction with the substrates, glycochenodeoxycholic acid (GCDCA) and glycoursodeoxycholic acid (GUDCA), were 183.617 and 34.458 s−1 mM−1, respectively. In addition, multiple metal ions can enhance the activity of Sc 3α-HSDH when used at concentrations ranging from 2 % to 42 %. The results also suggest that the metagenomic approach is an efficient method for identifying novel enzymes. [ABSTRACT FROM AUTHOR]

Glyceraldehyde-3-phosphate dehydrogenase (GAPDH), an essential enzyme of the glycolysis metabolic pathway, plays a vital role in carbon metabolism, plant development, and stress resistance. As a kind of woody plant, poplars are widely cultivated for afforestation. Although the whole genome data of poplars have been published, little information is known about the GAPDH family of genes in poplar. This study performed a genome-wide identification of the poplar GAPDH family, and 13 determined PtGAPDH genes were identified from poplar genome. Phylogenetic tree showed that the PtGAPDH members were divided into PtGAPA/B, PtGAPC, PtGAPCp, and PtGAPN groups. A total of 13 PtGAPDH genes were distributed on eight chromosomes, 13 gene pairs belonging to segmented replication events were detected in poplar, and 23 collinearity gene pairs were determined between poplar and willow. The PtGAPDHcis -acting elements associated with growth and development as well as stress resistance revealed that PtGAPDHs might be involved in these processes. The phosphoglycerate kinase (PGK) and triose-phosphate isomerase (TPI) were predicted as the putative interaction proteins of PtGAPDHs. Gene ontology (GO) analysis showed that PtGAPDHs play a crucial role in the oxidation and reduction processes. PtGAPDH expression levels were induced by NaCl and PEG treatments, which implied that PtGAPDHs might be involved in stress response. Overexpression of PtGAPC1 significantly changed the contents of lipid and carbohydrate metabolites, which indicated that PtGAPC1 plays an essential role in metabolic regulation. This study highlights the characterizations and profiles of PtGAPDHs and reveals that PtGAPC1 is involved in the loop of lipid and carbohydrate metabolisms. [ABSTRACT FROM AUTHOR]

Ferroptosis is a type of lipid peroxidation-dependent cell death that is emerging as a therapeutic target for cancer. However, the mechanisms of ferroptosis during the generation and detoxification of lipid peroxidation products remain rather poorly defined. Here, we report an unexpected role for the eukaryotic translation initiation factor EIF4E as a determinant of ferroptotic sensitivity by controlling lipid peroxidation. A drug screening identified 4EGI-1 and 4E1RCat (previously known as EIF4E-EIF4G1 interaction inhibitors) as powerful inhibitors of ferroptosis. Genetic and functional studies showed that EIF4E (but not EIF4G1) promotes ferroptosis in a translation-independent manner. Using mass spectrometry and subsequent protein-protein interaction analysis, we identified EIF4E as an endogenous repressor of ALDH1B1 in mitochondria. ALDH1B1 belongs to the family of aldehyde dehydrogenases and may metabolize the aldehyde substrate 4-hydroxynonenal (4HNE) at high concentrations. Supraphysiological levels of 4HNE triggered ferroptosis, while low concentrations of 4HNE increased the cell susceptibility to classical ferroptosis inducers by activating the NOX1 pathway. Accordingly, EIF4E-dependent ALDH1B1 inhibition enhanced the anticancer activity of ferroptosis inducers in vitro and in vivo. Our results support a key function of EIF4E in orchestrating lipid peroxidation to ignite ferroptosis. Ferroptosis is lipid peroxidation-dependent cell death that has potential to be harnessed as a cancer therapeutic. Here, the authors show that the translation initiation factor eIF4E can repress ALDH1B1 independent of translation, increasing lipid peroxidation levels to promote ferroptosis. [ABSTRACT FROM AUTHOR]

Red vinasse acid has a distinct flavor and a vivid red color that are directly tied to the intricate metabolic activities of microorganisms that produce it. In this study, metagenomic technology was used to mine its functional genes and examine the microbial diversity of red vinasse acid. The findings revealed the identification of 2,609 species, 782 genera, and 63 phyla of microorganisms, and the dominant genus was Lactobacillus. Amino acid metabolism and carbohydrate metabolism were significant activities among the 16,093 and 49,652 genes that were annotated in the evolutionary genealogy of genes: Non-supervised Orthologous Groups (eggNOG) and Kyoto Encyclopedia of Genes and Genomes (KEGG) databases, respectively. In gluconeogenesis, red vinasse acid encodes 194 genes controlling the transporter protein systems of different sugars and has key enzyme genes that catalyze the conversion of intracellular sugars into glycolytic intermediates. In amino acid flavor formation, red vinasse acid contains 32 control genes for branchedchain aminotransferase (BCAT), 27 control genes for aromatic-amino-acid transaminase (ArAT), 60 control genes for keto acid invertase, 123 control genes for alcohol/aldehyde dehydrogenase, and 27 control genes for acetyl esterase, which have the basis for the formation of strong flavor substances from amino acids. [ABSTRACT FROM AUTHOR]

Application of ferric iron is conventionally considered to inhibit methanogenesis in anoxic environments. Here we show that Methanosarcina mazei zm‐15, a strain isolated from the natural wetland of Tibetan plateau, is capable of Fe(III) reduction, which significantly promotes its growth and methanogenesis. We grew Ms. mazei zm‐15 in a medium containing acetate supplemented with Fe(III) in ferric citrate or ferrihydrite and to some cultures anthraquinone‐2,6‐disulfonate (AQDS) was applied as an electron shuttle. The reduction of Fe(III) species occurred immediately. Ferric citrate was more readily reduced than ferrihydrite. The X‐ray diffraction spectra analysis showed the formation of magnetite from ferrihydrite and amorphous reduced products from ferrihydrite plus AQDS. The analysis of protein contents revealed that Fe(III) reduction contributed 36%–46% of the cell growth. The growth yield, estimated as protein increment per acetate consumed for Fe(III) reduction, increased by 20‐ to 30‐fold compared with methanogenesis, which is in consistence with the difference in free energy available by Fe(III) reduction relative to methanogenesis. We propose that the outer‐surface multiheme c‐type cytochrome predicted from Ms. mazei zm‐15 genome serves as the terminal reductase with the energy‐converting hydrogenase and F420H2 dehydrogenase involved in electron transport chain for Fe(III) reduction. The findings shed a light to better understand the ecophysiology of Methanosarcina in anaerobic environments. [ABSTRACT FROM AUTHOR]

The global climate changes and the consequent increase in the number of soil and air droughts during the vegetation period of grain crops require the development of new strategies to adapt plants to those yield-decreasing stressors. A relevant way of increasing droughttolerance of cereals is the use of biotechnological methods, particularly RNA interference, which can down-regulate the activity of plants’ genes and increase concentration of stress metabolites that perform osmoprotective functions during drought. We studied the tolerance to soil moisture shortage in transgenic plants of winter wheat with partial suppression of the proline dehydrogenase gene, obtained using the technology of short interfering RNAs. We analyzed physiological and biochemical parameters and structural elements of yield productivity of 4 wild genotypes and their transgenic lines with reduced activity of proline dehydrogenase in the conditions of 7-day drought during the late booting–ear emergence. We determined that the presence of double-stranded RNA suppressor of the proline dehydrogenase gene in transgenic lines led to increase in the level of accumulation of free proline in flag leaves. At the same time, its concentration in transgenic lines was higher than in untransformed plants of the wild genotypes in both drought conditions and conditions of sufficient moisture. We found that against the background of water deficiency, the total chlorophyll content in leaves of plants of transgenic lines was significantly higher, and the ratio of carotenoids to chlorophyll was lower than in plants of the wild genotypes, suggesting mitigation of the negative impact of drought on the plants of transgenic lines. Lacking soil moisture, genetically altered lines of wheat had significantly higher parameters of the structure of grain yield compared with untransformed genotypes. At the same time, we observed genotypic difference according to grain productivity in biotechnological plants. Therefore, the results we obtained confirm the perspectives of using the technology of short interfering RNAs to increase tolerance of winter wheat to water deficiency. [ABSTRACT FROM AUTHOR]

A 3-year-old, male neutered Cavalier King Charles Spaniel (CKCS) presented with complex focal seizures and prolonged lethargy. The aim of the study was to investigate the clinical signs, metabolic changes and underlying genetic defect. Blood and urine organic acid analysis revealed increased medium-chain fatty acids and together with the clinical findings suggested a diagnosis of medium-chain acyl-CoA dehydrogenase (MCAD) deficiency. We sequenced the genome of the affected dog and compared the data to 923 control genomes of different dog breeds. The ACADM gene encoding MCAD was considered the top functional candidate gene. The genetic analysis revealed a single homozygous private protein-changing variant in ACADM in the affected dog. This variant, XM_038541645.1:c.444_445delinsGTTAATTCTCAATATTGTCTAAGAATTATG, introduces a premature stop codon and is predicted to result in truncation of ~63% of the wild type MCAD open reading frame, XP_038397573.1:p.(Thr150Ilefs*6). Targeted genotyping of the variant in 162 additional CKCS revealed a variant allele frequency of 23.5% and twelve additional homozygous mutant dogs. The acylcarnitine C8/C12 ratio was elevated ~43.3 fold in homozygous mutant dogs as compared to homozygous wild type dogs. Based on available clinical and biochemical data together with current knowledge in humans, we propose the ACADM frameshift variant as causative variant for the MCAD deficiency with likely contribution to the neurological phenotype in the index case. Testing the CKCS breeding population for the identified ACADM variant is recommended to prevent the unintentional breeding of dogs with MCAD deficiency. Further prospective studies are warranted to assess the clinical consequences of this enzyme defect. [ABSTRACT FROM AUTHOR]

Herein, we report a multifunctional chemoenzymatic nanoreactor (NanoNOx) for the glucose‐controlled regeneration of natural and artificial nicotinamide cofactors. NanoNOx are built of glucose oxidase‐polymer hybrids that assemble in the presence of an organometallic catalyst: hemin. The design of the hybrid is optimized to increase the effectiveness and the directional channeling at low substrate concentration. Importantly, NanoNOx can be reutilized without affecting the catalytic properties, can show high stability in the presence of organic solvents, and can effectively oxidize assorted natural and artificial enzyme cofactors. Finally, the hybrid was successfully coupled with NADH‐dependent dehydrogenases in one‐pot reactions, using a strategy based on the sequential injection of a fuel, namely, glucose. Hence, this study describes the first example of a hybrid chemoenzymatic nanomaterial able to efficiently mimic NOx enzymes in cooperative one‐pot cascade reactions. [ABSTRACT FROM AUTHOR]

Simple Summary: Hyperthermophilic Thermotoga spp. are promising candidates for the metabolic engineering of cellulosic ethanol producing strains. However, Thermotoga spp. are hydrogen producers, and none of them have been found to produce significant amount of ethanol. In this study, we focused on the enzymes responsible for the part of the ethanol production pathway from ac-CoA to ethanol. Aldehyde dehydrogenases (Aldhs) and alcohol dehydrogenases (Adhs) are the vital enzymes involved in the metabolism of ethanol in Thermotoga spp.. However, the biochemical properties of a number of Adhs related to ethanol formation and consumption have not been identified until now. The aim of this study was to determine the role of putative Adhs in T. neapolitana (Tne) in the pathway from ac-CoA to ethanol. We found that the Fe-AAdh (encoded by CTN_0580) and Fe-Adh2 (encoded by CTN_1756) were the main enzymes responsible for ethanol production in Tne. While Zn-Adh (encoded by CTN_0257) was identified as the main protein responsible for ethanol consumption in the acid environment and Fe-Adh1 (encoded by CTN_1655) played a minor role in ethonal production and consumption in Tne. Basing on these results, it will be helpful to construct and assemble a novel hyperthermophilic strain to produce cellulosic ethanol. Hyperthermophilic Thermotoga spp. are candidates for cellulosic ethanol fermentation. A bifunctional iron-acetaldehyde/alcohol dehydrogenase (Fe-AAdh) has been revealed to catalyze the acetyl-CoA (Ac-CoA) reduction to form ethanol via an acetaldehyde intermediate in Thermotoga neapolitana (T. neapolitana). In this organism, there are three additional alcohol dehydrogenases, Zn-Adh, Fe-Adh1, and Fe-Adh2, encoded by genes CTN_0257, CTN_1655, and CTN_1756, respectively. This paper reports the properties and functions of these enzymes in the fermentation pathway from Ac-CoA to ethanol. It was determined that Zn-Adh only exhibited activity when oxidizing ethanol to acetaldehyde, and no detectable activity for the reaction from acetaldehyde to ethanol. Fe-Adh1 had specific activities of approximately 0.7 and 0.4 U/mg for the forward and reverse reactions between acetaldehyde and ethanol at a pHopt of 8.5 and Topt of 95 °C. Catalyzing the reduction of acetaldehyde to produce ethanol, Fe-Adh2 exhibited the highest activity of approximately 3 U/mg at a pHopt of 7.0 and Topt of 85 °C, which were close to the optimal growth conditions. These results indicate that Fe-Adh2 and Zn-Adh are the main enzymes that catalyze ethanol formation and consumption in the hyperthermophilic bacterium, respectively. [ABSTRACT FROM AUTHOR]

Malate dehydrogenase, which facilitates the reversible conversion of malate to oxaloacetate, is essential for energy balance, plant growth, and cold and salt tolerance. However, the genome-wide study of the MDH family has not yet been carried out in tomato (Solanum lycopersicum L.). In this study, 12 MDH genes were identified from the S. lycopersicum genome and renamed according to their chromosomal location. The tomato MDH genes were split into five groups based on phylogenetic analysis and the genes that clustered together showed similar lengths, and structures, and conserved motifs in the encoded proteins. From the 12 tomato MDH genes on the chromosomes, three pairs of segmental duplication events involving four genes were found. Each pair of genes had a Ka/Ks ratio < 1, indicating that the MDH gene family of tomato was purified during evolution. Gene expression analysis exhibited that tomato MDHs were differentially expressed in different tissues, at various stages of fruit development, and differentially regulated in response to abiotic stresses. Molecular docking of four highly expressed MDHs revealed their substrate and co-factor specificity in the reversible conversion process of malate to oxaloacetate. Further, co-localization of tomato MDH genes with quantitative trait loci (QTL) of salt stress-related phenotypes revealed their broader functions in salt stress tolerance. This study lays the foundation for functional analysis of MDH genes and genetic improvement in tomato. [ABSTRACT FROM AUTHOR]

The short-chain dehydrogenase/reductase (SDR) from Empedobacter brevis ZJUY-1401 (EbSDR8, GenBank: ALZ42979.1) is a promising biocatalyst for the reduction of acetophenone to (R)-1-phenylethanol, but its industrial application is restricted by its insufficient tolerance to acetophenone. In this paper, we developed a chromogenic reaction-based high-throughput screening method and employed directed evolution to enhance the acetophenone tolerance of EbSDR8. The resulting variant, M190V, showed 74.8% improvement over the wild-type in specific activity when catalyzing the reduction of 200 mM acetophenone. Kinetic analysis revealed a 70% enhancement in its catalytic efficiency (kcat/Km). Molecular docking was conducted to reveal the possible mechanism behind the improved acetophenone tolerance, and the result implied that the M190V mutation is conducive to the binding and release of coenzyme. Aside from the improved catalytic performance when dealing with a high concentration of acetophenone, other features of M190V, such as a broad pH range (6.0 to 10.5), low optimal cosubstrate concentration (1% isopropanol), and a temperature optimum close to that of E. coli cells (35 °C), also contribute to its practical application as a whole-cell catalyst. In this study, we first designed a directed evolution means to engineer the enzyme and obtained the positive variant which has a high activity under high concentrations of acetophenone. After that, we optimized the catalytic performance of the variant to adapt to industrial applications. [ABSTRACT FROM AUTHOR]

The evolutionary benefit accounting for widespread conservation of oligomeric structures in proteins lacking evidence of intersubunit cooperativity remains unclear. Here, crystal and cryo‐EM structures, and enzymological data, demonstrate that a conserved tetramer interface maintains the active‐site structure in one such class of proteins, the short‐chain dehydrogenase/reductase (SDR) superfamily. Phylogenetic comparisons support a significantly longer polypeptide being required to maintain an equivalent active‐site structure in the context of a single subunit. Oligomerization therefore enhances evolutionary fitness by reducing the metabolic cost of enzyme biosynthesis. The large surface area of the structure‐stabilizing oligomeric interface yields a synergistic gain in fitness by increasing tolerance to activity‐enhancing yet destabilizing mutations. We demonstrate that two paralogous SDR superfamily enzymes with different specificities can form mixed heterotetramers that combine their individual enzymological properties. This suggests that oligomerization can also diversify the functions generated by a given metabolic investment, enhancing the fitness advantage provided by this architectural strategy. Synopsis: Many proteins form evolutionarily conserved oligomers in the absence of obvious functional interactions between subunits. Structural and phylogenetic analyses demonstrate that these interactions maintain active‐site structure in the short‐chain dehydrogenase/reductase superfamily. A cryo‐EM structure of a short‐chain dehydrogenase/reductase (SDR) superfamily enzyme shows dynamic opening of its tetramerization interface coupled to disordering of its active site.The same homotetramer structure is conserved in the vast majority of SDR superfamily enzymes with structures deposited in the PDB.The maintenance of active‐site structure using homooligomeric interactions enables a shorter protein subunit to form an active enzyme.Increased evolutionary fitness can therefore be conferred by homooligomeric interactions due to reduced metabolic investment. [ABSTRACT FROM AUTHOR]

Embryo development is essential for seed yield and post-germination growth. Glucose-6-phosphate dehydrogenase (G6PD), the rate-limiting enzyme in oxidative pentose phosphate pathway (OPPP), is widely involved in plant development and stress tolerance by providing nicotinamide adenine dinucleotide phosphate (NADPH). In this study, the double mutant (g6pd5/6), overexpression line (G6PD5/6OE) and complementation line (g6pd5/6 Comp) of cytosolic glucose-6-phosphate dehydrogenases (Cyt-G6PD) were used to investigate Cyt-G6PD roles in embryo development of Arabidopsis. The results showed that the germination rate of g6pd5/6 seeds was delayed in comparison with that of Col-0; moreover, 11.5% of g6pd5/6 seeds did not germinate. The dysfunction of Cyt-G6PD resulted in decreased fresh weight and primary root length of g6pd5/6 seedlings. The height and silique length of g6pd5/6 plants were also decreased. Moreover, the abortion rate of siliques and seeds of g6pd5/6 plants were increased compared with those of Col-0, G6PD5/6OE and g6pd5/6 Comp lines. However, the dysfunction of Cyt-G6PD did not affect pollen activity; but in g6pd5/6 , the embryo development was partially delayed or inhibited. The contents of fatty acids and storage proteins, two main storage materials in Arabidopsis seeds, were decreased in g6pd5/6 seeds. Exogenous application of fatty acids (C18:2; C18:3) alleviated the delayed germination of g6pd5/6 seeds. RT-qPCR results further demonstrated that the early embryo development genes were down-regulated in g6pd5/6. Taken together, Cyt-G6PD plays a pivotal role in plant seed development by regulating the transcriptions of early embryo development genes and the accumulation of storage materials (especially fatty acids). • Cyt-G6PD mutation affects the transcription of genes involved in embryonic development in Arabidopsis. • Cyt-G6PD mutation decreases the contents of fatty acids and storage proteins in g6pd5/6 seeds. • Cyt-G6PD dysfunction reduces seed yield and delays post-embryonic development of Arabidopsis seedlings. [ABSTRACT FROM AUTHOR]