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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]

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]

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]

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]

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]

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]

Enzymatic processes play an increasing role in synthetic organic chemistry which requires the access to a broad and diverse set of enzymes. Metagenome mining is a valuable and efficient way to discover novel enzymes with unique properties for biotechnological applications. Here, we report the discovery and biocatalytic characterization of six novel metagenomic opine dehydrogenases from a hot spring environment (mODHs) (EC 1.5.1.X). These enzymes catalyze the asymmetric reductive amination between an amino acid and a keto acid resulting in opines which have defined biochemical roles and represent promising building blocks for pharmaceutical applications. The newly identified enzymes exhibit unique substrate specificity and higher thermostability compared to known examples. The feature that they preferably utilize negatively charged polar amino acids is so far unprecedented for opine dehydrogenases. We have identified two spatially correlated positions in their active sites that govern this substrate specificity and demonstrated a switch of substrate preference by site-directed mutagenesis. While they still suffer from a relatively narrow substrate scope, their enhanced thermostability and the orthogonality of their substrate preference make them a valuable addition to the toolbox of enzymes for reductive aminations. Importantly, enzymatic reductive aminations with highly polar amines are very rare in the literature. Thus, the preparative-scale enzymatic production, purification, and characterization of three highly functionalized chiral secondary amines lend a special significance to our work in filling this gap. Key points: • Six new opine dehydrogenases have been discovered from a hot spring metagenome • The newly identified enzymes display a unique substrate scope • Substrate specificity is governed by two correlated active-site residues [ABSTRACT FROM AUTHOR]

In this work the green synthesis of gold nanoparticles (Au-NPs) using the oxidoreductive enzymes Myriococcum thermophilum cellobiose dehydrogenase (Mt CDH), Glomerella cingulata glucose dehydrogenase (Gc GDH), and Aspergillus niger glucose oxidase (An GOX)) as bioreductants was investigated. The influence of reaction conditions on the synthesis of Au-NPs was examined and optimised. The reaction kinetics and the influence of Au ions on the reaction rate were determined. Based on the kinetic study, the mechanism of Au-NP synthesis was proposed. The Au-NPs were characterized by UV–Vis spectroscopy and transmission electron microscopy (TEM). The surface plasmon resonance (SPR) absorption peaks of the Au-NPs synthesised with Mt CDH and Gc GDH were observed at 535 nm, indicating an average size of around 50 nm. According to the image analysis performed on a TEM micrograph, the Au-NPs synthesized with Gc GDH have a spherical shape with an average size of 2.83 and 6.63 nm after 24 and 48 h of the reaction, respectively. Key points: • The Au NPs were synthesised by the action of enzymes CDH and GDH. • The synthesis of Au-NPs by CDH is related to the oxidation of cellobiose. • The synthesis of Au-NPs by GDH was not driven by the reaction kinetic. [ABSTRACT FROM AUTHOR]

Diabetic cardiomyopathy describes heart disease in patients with diabetes who have no other cardiac conditions but have a higher risk of developing heart failure. Specific therapies to treat the diabetic heart are limited. A key mechanism involved in the progression of diabetic cardiomyopathy is dysregulation of cardiac energy metabolism. The aim of this study was to determine if increasing the expression of medium-chain acyl-coenzyme A dehydrogenase (MCAD; encoded by Acadm), a key regulator of fatty acid oxidation, could improve the function of the diabetic heart. Male mice were administered streptozotocin to induce diabetes, which led to diastolic dysfunction 8 weeks post-injection. Mice then received cardiac-selective adeno-associated viral vectors encoding MCAD (rAAV6:MCAD) or control AAV and were followed for 8 weeks. In the non-diabetic heart, rAAV6:MCAD increased MCAD expression (mRNA and protein) and increased Acadl and Acadvl, but an increase in MCAD enzyme activity was not detectable. rAAV6:MCAD delivery in the diabetic heart increased MCAD mRNA expression but did not significantly increase protein, activity, or improve diabetes-induced cardiac pathology or molecular metabolic and lipid markers. The uptake of AAV viral vectors was reduced in the diabetic versus non-diabetic heart, which may have implications for the translation of AAV therapies into the clinic. Key messages: The effects of increasing MCAD in the diabetic heart are unknown. Delivery of rAAV6:MCAD increased MCAD mRNA and protein, but not enzyme activity, in the non-diabetic heart. Independent of MCAD enzyme activity, rAAV6:MCAD increased Acadl and Acadvl in the non-diabetic heart. Increasing MCAD cardiac gene expression alone was not sufficient to protect against diabetes-induced cardiac pathology. AAV transduction efficiency was reduced in the diabetic heart, which has clinical implications. [ABSTRACT FROM AUTHOR]

Bacterial polyhydroxyalkanoates (PHAs) are promising bio-based biodegradable polyesters. It was recently reported that novel PHA block copolymers composed of (R)-3-hydroxybutyrate (3HB) and (R)-2-hydroxybutyrate (2HB) were synthesized by Escherichia coli expressing PhaCAR, a chimeric enzyme of PHA synthases derived from Aeromonas caviae and Ralstonia eutropha. In this study, the sequence-regulating PhaCAR was applied in the natural PHA-producing bacterium, R. eutropha. During the investigation, (R/S)-2HB was found to exhibit strong growth inhibitory effects on the cells of R. eutropha. This was probably due to formation of excess 2-ketobutyrate (2KB) from (R/S)-2HB and the consequent l-valine depletion caused by dominant l-isoleucine synthesis attributed to the excess 2KB. Deletion analyses for genes of lactate dehydrogenase homologs identified cytochrome-dependent d-lactate dehydrogenase (Dld) and [Fe-S] protein-dependent l-lactate dehydrogenase as the enzymes responsible for sensitivity to (R)-2HB and (S)-2HB, respectively. The engineered R. eutropha strain (phaCAR+, ldhACd-hadACd+ encoding clostridial (R)-2-hydroxyisocaproate dehydrogenase and (R)-2-hydoroxyisocaproate CoA transferase, ∆dld) synthesized PHA containing 10 mol% of 2HB when cultivated on glucose with addition of sodium (RS)-2HB, and the 2HB composition in PHA increased up to 35 mol% by overexpression phaCAR. The solvent fractionation and NMR analyses showed that the resulting PHAs were most likely to be block polymers consisting of P(3HB-co-3HV) and P(2HB) segments, suggesting that PhaCAR functions as the sequence-regulating PHA synthase independently from genetic and metabolic backgrounds of the host cell. Key points: (R/S)-2-hydroxubutyrates (2HB) caused l-valine deletion in Ralstonia eutropha (R)- and (S)-lactate/2HB dehydrogenases functional in R. eutropha were identified The engineered R. eutropha synthesized block copolymers of 2HB-containing polyhydroxyalkanoates on glucose and 2HB [ABSTRACT FROM AUTHOR]

Anthropogenic carbon dioxide (CO2) levels are rising to alarming concentrations in earth's atmosphere, causing adverse effects and global climate changes. In the last century, innovative research on CO2 reduction using chemical, photochemical, electrochemical and enzymatic approaches has been addressed. In particular, natural CO2 conversion serves as a model for many processes and extensive studies on microbes and enzymes regarding redox reactions involving CO2 have already been conducted. In this review we focus on the enzymatic conversion of CO2 to carbon monoxide (CO) as the chemical conversion downstream of CO production render CO particularly attractive as a key intermediate. We briefly discuss the different currently known natural autotrophic CO2 fixation pathways, focusing on the reversible reaction of CO2, two electrons and protons to CO and water, catalyzed by carbon monoxide dehydrogenases (CODHs). We then move on to classify the different type of CODHs, involved catalyzed chemical reactions and coupled metabolisms. Finally, we discuss applications of CODH enzymes in photochemical and electrochemical cells to harness CO2 from the environment transforming it into commodity chemicals. [ABSTRACT FROM AUTHOR]

Flavin-dependent dehydrogenases and oxidases are enzymes that play an important role in living organisms and are widely used in practice. Despite the different reactivity with respect to oxygen, the similarity of the structures of the active sites of a number of dehydrogenases and oxidases, up to the possibility of interconversion via the substitution of one amino acid, makes it possible to consider these classes as related. The vanillyl-alcohol oxidase family, one of the best-known families of oxidases, too, includes both classes of enzymes. However, in the literature, the relationship between oxidases and dehydrogenases is presented as a comparison of specific representatives of these classes or, more rarely, structural features of individual subclasses. In view of the aforesaid, consideration of the functional properties of oxidases beyond the context of their comparison with dehydrogenases seems to be incomplete. In addition, the search for and study of effectors, electron acceptors and inhibitors of oxidases and dehydrogenases, should be carried out taking account of the similarity of these classes, including the development of drugs that target these enzymes (for example, antitrypanosomal or antifungal drugs). This review analyzes the main structural differences between oxidases and dehydrogenases and considers the practical application of these classes, with particular attention to their role as drug targets. [ABSTRACT FROM AUTHOR]

Main conclusion: Genome-wide screening of short-chain dehydrogenases/reductases (SDR) family reveals functional diversification of borneol dehydrogenase (BDH) in Wurfbainia villosa. Wurfbainia villosa is an important medicinal plant, the fruits of which accumulate abundant terpenoids, especially bornane-type including borneol and camphor. The borneol dehydrogenase (BDH) responsible for the conversion of borneol to camphor in W. villosa remains unknown. BDH is one member of short-chain dehydrogenases/reductases (SDR) family. Here, a total of 115 classical WvSDR genes were identified through genome-wide screening. These WvSDRs were unevenly distributed on different chromosomes. Seven candidate WvBDHs based on phylogenetic analysis and expression levels were selected for cloning. Of them, four BDHs can catalyze different configurations of borneol and other monoterpene alcohol substrates to generate the corresponding oxidized products. WvBDH1 and WvBDH2, preferred (+)-borneol to (−)-borneol, producing the predominant (+)-camphor. WvBDH3 yielded approximate equivalent amount of (+)-camphor and (−)-camphor, in contrast, WvBDH4 generated exclusively (+)-camphor. The metabolic profiles of the seeds showed that the borneol and camphor present were in the dextrorotatory configuration. Enzyme kinetics and expression pattern in different tissues suggested WvBDH2 might be involved in the biosynthesis of camphor in W. villosa. All results will increase the understanding of functional diversity of BDHs. [ABSTRACT FROM AUTHOR]

Previously, the gene of formate dehydrogenase (FDH, EC 1.2.1.2) from the thermotolerant methylotrophic yeast Ogataea parapolymorpha DL 1 (OpaFDH) was cloned in our laboratory. Recombinant enzyme with additional glycine amino acid residue (OpaFDH_GK) was obtained in Escherichia coli cells in active and soluble form with a yield of more than 1 g per liter of the medium. In the present work, a detailed comparison of this enzyme with FDHs from other sources was carried out. Among eukaryotic formate dehydrogenases, OpaFDH has the highest thermal stability. To elucidate effect of N-terminal residue on the properties of the enzyme, OpaFDH_K (identical to natural) and OpaFDH_AK variants containing an additional Ala residue at the N-terminus were also obtained. It was shown that addition of an Ala residue to the N-terminus reduces four-fold the rate constant of thermal inactivation compared with the addition of a Gly residue. Addition of six more histidine residues to the N-terminus of OpaFDH_AK leads to acceleration of purification, practically does not affect kinetic parameters, but somewhat reduces thermal stability, which, however, can be restored to the level of OpaFDH_AK stability by adding 0.5 M NaCl. [ABSTRACT FROM AUTHOR]

Chemotherapy-induced cardiac damage remains a leading cause of death amongst cancer survivors. Anthracycline-induced cardiotoxicity is mediated by severe mitochondrial injury, but little is known about the mechanisms by which cardiomyocytes adaptively respond to the injury. We observed the translocation of selected mitochondrial tricarboxylic acid (TCA) cycle dehydrogenases to the nucleus as an adaptive stress response to anthracycline-cardiotoxicity in human induced pluripotent stem cell-derived cardiomyocytes and in vivo. The expression of nuclear-targeted mitochondrial dehydrogenases shifts the nuclear metabolic milieu to maintain their function both in vitro and in vivo. This protective effect is mediated by two parallel pathways: metabolite-induced chromatin accessibility and AMP-kinase (AMPK) signaling. The extent of chemotherapy-induced cardiac damage thus reflects a balance between mitochondrial injury and the protective response initiated by the nuclear pool of mitochondrial dehydrogenases. Our study identifies nuclear translocation of mitochondrial dehydrogenases as an endogenous adaptive mechanism that can be leveraged to attenuate cardiomyocyte injury. Chemotherapy can cause severe damage to cardiomyocytes in some patients but it is unclear how cardiomyocytes protect themselves against such stress. Here the authors show that cardiomyocytes initiate an endogenous protective response when exposed to chemotherapeutic agents by translocating mitochondrial enzymes to the nucleus. [ABSTRACT FROM AUTHOR]

Acyl-CoA dehydrogenase (ChsE) is involved in the steroid side-chain degradation process. However, their function in vivo remains unclear. In this study, three ChsE, ChsE1-ChsE2, ChsE3, and ChsE4-ChsE5, were identified in Mycolicibacterium neoaurum, and their functions in vivo are studied and compared with those from Mycobacterium tuberculosis in vitro. By gene knockout, complementation, and the bioconversion of phytosterols, the function of ChsE was elucidated that ChsE4-ChsE5 could utilize C27, C24, and C22 steroids in vivo. ChsE3 could utilize C27 and C24 steroids in vivo. ChsE1-ChsE2 could utilize C27, C24, and C22 steroids in vivo. What is more, the production strain of a C22 steroid, 3-oxo-4,17-pregadiene-20-carboxylic acid methyl ester (PDCE), is constructed with ChsE overexpression. This study improved the understanding of the steroid bioconversion pathway and proposed a method of the production of a new C22 steroid. Key points: • Three ChsE paralogs from M. neoaurum are identified and studied. • The function of ChsE is overlapped in vivo. • A C22 steroid (PDCE) producer was constructed with ChsE overexpression. [ABSTRACT FROM AUTHOR]

Ethylene glycol is an attractive two-carbon alcohol substrate for biochemical product synthesis as it can be derived from CO2 or syngas at no sacrifice to human food stocks. Here, we disclose a five-step synthetic metabolic pathway enabling the carbon-conserving biosynthesis of the versatile platform molecule 2,4-dihydroxybutyric acid (DHB) from this compound. The linear pathway chains ethylene glycol dehydrogenase, D-threose aldolase, D-threose dehydrogenase, D-threono-1,4-lactonase, D-threonate dehydratase and 2-oxo-4-hydroxybutyrate reductase enzyme activities in succession. We screen candidate enzymes with D-threose dehydrogenase and D-threonate dehydratase activities on cognate substrates with conserved carbon-centre stereochemistry. Lastly, we show the functionality of the pathway by its expression in an Escherichia coli strain and production of 1 g L−1 and 0.8 g L−1 DHB from, respectively, glycolaldehyde or ethylene glycol. Ethylene glycol is an attractive two-carbon alcohol substrate for bioproduction as it can be derived from CO2 or syngas. Here, the authors design a five-step synthetic metabolic pathway in E. coli to enable the carbon-conserving biosynthesis of the platform chemical 2,4-dihydroxybutyric acid from ethylene glycol. [ABSTRACT FROM AUTHOR]

Marine algae produce complex polysaccharides, which can be degraded by marine heterotrophic bacteria utilizing carbohydrate-active enzymes. The red algal polysaccharide porphyran contains the methoxy sugar 6-O-methyl-d-galactose (G6Me). In the degradation of porphyran, oxidative demethylation of this monosaccharide towards d-galactose and formaldehyde occurs, which is catalyzed by a cytochrome P450 monooxygenase and its redox partners. In direct proximity to the genes encoding for the key enzymes of this oxidative demethylation, genes encoding for zinc-dependent alcohol dehydrogenases (ADHs) were identified, which seem to be conserved in porphyran utilizing marine Flavobacteriia. Considering the fact that dehydrogenases could play an auxiliary role in carbohydrate degradation, we aimed to elucidate the physiological role of these marine ADHs. Although our results reveal that the ADHs are not involved in formaldehyde detoxification, a knockout of the ADH gene causes a dramatic growth defect of Zobellia galactanivorans with G6Me as a substrate. This indicates that the ADH is required for G6Me utilization. Complete biochemical characterizations of the ADHs from Formosa agariphila KMM 3901T (FoADH) and Z. galactanivorans DsijT (ZoADH) were performed, and the substrate screening revealed that these enzymes preferentially convert aromatic aldehydes. Additionally, we elucidated the crystal structures of FoADH and ZoADH in complex with NAD+ and showed that the strict substrate specificity of these new auxiliary enzymes is based on a narrow active site. Key points: • Knockout of the ADH-encoding gene revealed its role in 6-O-methyl-D-galactose utilization, suggesting a new auxiliary activity in marine carbohydrate degradation. • Complete enzyme characterization indicated no function in a subsequent reaction of the oxidative demethylation, such as formaldehyde detoxification. • These marine ADHs preferentially convert aromatic compounds, and their strict substrate specificity is based on a narrow active site. [ABSTRACT FROM AUTHOR]

Xylitol dehydrogenase (XDH) catalyzes the NAD+-dependent oxidization of xylitol into d-xylulose, and belongs to a zinc-dependent medium-chain dehydrogenase/reductase family. This protein family consists of enzymes with one or two zinc atoms per subunit, among which catalytic zinc is necessary for the activity. Among many XDHs from yeast and fungi, XDH from Pichia stipitis is one of the key enzymes for bioethanol production by lignocellulosic biomass, and possesses only a catalytic zinc atom. Despite its importance in bioindustry, a structural data of XDH has not yet been available, and little insight into the role of a second zinc atom in this protein family is known. We herein report the crystal structure of XDH from P. stipitis using a thermostabilized mutant. In the refined structure, a second zinc atom clearly coordinated with four artificially introduced cysteine ligands. Homologous mutations in XDH from Saccharomyces cerevisiae also stabilized and enhanced activity. The substitution of each of the four cysteine ligands with an aspartate in XDH from Schizosaccharomyces pombe contributed to the significantly better maintenance of activity and thermostability than their substitution with a serine, providing a novel hypothesis for how this zinc atom was eliminated. [ABSTRACT FROM AUTHOR]

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]

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]

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]

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]

Using Escherichia coli strain MG1655 lacIQ, ∆ackA-pta, ∆poxB, ∆ldhA, ∆adhE, ∆fadE, PL‑SDφ10-atoB, Ptrc-ideal-4-SDφ10-fadB, PL-SDφ10-tesB, ∆yciA as a core strain, the efficiency of the reversal of fatty acid β-oxidation upon the action of native cellular enzymes capable of serving as acyl-CoA dehydrogenases was examined. Increased expression of fadE, fabI, and ydiO/ydiQRST genes encoding the corresponding enzymes was ensured in derivatives of the core strain by substituting their native regulatory regions with artificial regulatory element Ptrc-ideal-4-SDφ10. A three-turn reversal of the cycle in the engineered recombinants was demonstrated that was accompanied by considerable secretion of butyric, caproic, and caprylic acids. The highest level of six- and eight-carbon carboxylates production was achieved upon the overexpression of the fabI gene, while the lowest levels of secretion of the corresponding compounds were demonstrated by the strain with the enhanced expression of the ydiO and ydiQRST genes. The recombinant with the individually enhanced expression of ydiO did not produce detectable amounts of the derivatives of the complete and successful β-oxidation reversal. [ABSTRACT FROM AUTHOR]

Oxidative DNA damage is closely related to the occurrence and progression of cancer. Oxidative stress plays an important role in alcohol-induced hepatocellular carcinoma (HCC). Aldehyde dehydrogenase (ALDH) is a family of enzymes that plays an essential role in the reducing oxidative damage. However, how ALDHs family affects alcohol-related HCC remains obscure. We aimed to explore the correlation between the differential expression of ALDHs in patients with HCC and pathological features, as well as the relationship between ALDHs and prognosis, and finally analyze the possible mechanism of ALDHs in targeted therapy of HCC. The data of HCC were downloaded from The Cancer Genome Atlas (TCGA) database. This research explored the expression and prognostic values of ALDHs in HCC using Oncomine, UALCAN, Human Protein Atlas, cBioPortal, Kaplan–Meier plotter, GeneMANIA, Tumor Immune Estimation Resource, GEPIA databases, and WebGestalt. Low mRNA and protein expressions of ALDHs were found to be significantly associated with tumor grade and clinical cancer stages in HCC patients. In particular, the loss of ALDH expression is more obvious in Asians, and its effect on prognosis is far more significant than that in the White race. Our findings play an important role in the study of prognostic markers and anti-liver cancer therapeutic targets for the members of the ALDHs family, especially in patients with liver cancer in Asia. [ABSTRACT FROM AUTHOR]

The function and mechanism of nitric oxide (NO) in regulating Pleurotus eryngii biological response to cadmium (Cd) stress was evaluated by using anti-oxidation and short-chain dehydrogenase/reductase (SDR) family analysis. The fresh biomass of P. eryngii mycelia sharply decreased after treatment with 50 µM Cd; the lipid peroxidation and H2O2 accumulation in P. eryngii were found responsible for it. Proper exogenous supply of NO (150 µM SNP) alleviated the oxidative damage induced by Cd stress in P. eryngii, which reduced the accumulation of thiobarbituric acid reactive substances (TBARS) and H2O2. The activities of antioxidant enzymes (superoxide dismutase, peroxidase) were significantly increased to deal with Cd stress when treated with SNP (150 µM), and the content of proline was also closely related to NO-mediated reduction of Cd toxicity. Moreover, SDR family members were widely involved in the response to Cd stress, especially PleSCH70 gene was observed for the first time in participating in NO-mediated enhancement of Cd tolerance in P. eryngii. Taken together, this study provides new insights in understanding the tolerance mechanisms of P. eryngii to heavy metal and lays a foundation for molecular breeding of P. eryngii to improve its tolerance to environmental stress. [ABSTRACT FROM AUTHOR]

Three disposable stochastic sensors designed using nanolayer deposition of copper (Cu), graphene (GR), and copper-graphene (Cu-GR) composite on the silk textile, as substrate, were modified with chitosan (n=371–744), for biomedical analysis. Isocitrate dehydrogenase 1 (IDH1) and isocitrate dehydrogenase 2 (IDH2) served as model analytes for molecular recognition and quantification in biological samples such as whole blood and brain tumor tissue samples. The best sensitivities (3.77×107s μg mL−1 for IDH1, and 1.88×107s μg mL−1 for IDH2) and the lowest limits of quantification (10−2fg mL−1 for IDH1, and 5×10−2fg mL−1 for IDH2) for both IDH1 and IDH2 were recorded for the disposable stochastic sensors based on chitosan/graphene nanolayer. Very good correlations between the screening method based on disposable stochastic sensors and enzyme-linked immunosorbent assay (ELISA) were obtained; this was also proved by the results obtained using the paired t-test. [ABSTRACT FROM AUTHOR]

Cancer cells often change their metabolism to support uncontrolled proliferation. Proline is the only proteogenic secondary amino acid that is abundant in the body. Recent studies have shown that proline metabolism plays an important role in metabolic reprogramming and affects the occurrence and development of cancer. Proline metabolism is related to ATP production, protein and nucleotide synthesis, and redox homeostasis in tumor cells. Proline can be synthesized by aldehyde dehydrogenase family 18 member A1 (ALDH18A1) and delta1-pyrroline-5-carboxylate reductase (PYCR), up-regulating ALDH18A1 and PYCR can promote the proliferation and invasion of cancer cells. As the main storage of proline, collagen can influence cancer cells proliferation, invasion, and metastasis. Its synthesis depends on the hydroxylation of proline catalyzed by prolyl 4-hydroxylases (P4Hs), which will affect the plasticity and metastasis of cancer cells. The degradation of proline occurs in the mitochondria and involves an oxidation step catalyzed by proline dehydrogenase/proline oxidase (PRODH/POX). Proline catabolism has a dual role in cancer, linking apoptosis with the survival and metastasis of cancer cells. In addition, it has been demonstrated that the regulation of proline metabolic enzymes at the genetic and post-translational levels is related to cancer. This article reviews the role of proline metabolic enzymes in cancer proliferation, apoptosis, metastasis, and development. Research on proline metabolism may provide a new strategy for cancer treatment. [ABSTRACT FROM AUTHOR]

Bioleaching of rare earth elements (REEs), using microorganisms such as Gluconobacter oxydans, offers a sustainable alternative to environmentally harmful thermochemical extraction, but is currently not very efficient. Here, we generate a whole-genome knockout collection of single-gene transposon disruption mutants for G. oxydans B58, to identify genes affecting the efficacy of REE bioleaching. We find 304 genes whose disruption alters the production of acidic biolixiviant. Disruption of genes underlying synthesis of the cofactor pyrroloquinoline quinone (PQQ) and the PQQ-dependent membrane-bound glucose dehydrogenase nearly eliminates bioleaching. Disruption of phosphate-specific transport system genes enhances bioleaching by up to 18%. Our results provide a comprehensive roadmap for engineering the genome of G. oxydans to further increase its bioleaching efficiency. Bioleaching of rare earth elements using microorganisms offers an environmentally friendly alternative to thermochemical extraction. Here, Schmitz et al. generate a whole-genome knockout collection of mutants for one such microorganism, Gluconobacter oxydans, and identify genes affecting the production of acidic biolixiviant and thus bioleaching efficacy. [ABSTRACT FROM AUTHOR]

Fusion of multiple enzymes to multifunctional constructs has been recognized as a viable strategy to improve enzymatic properties at various levels such as stability, activity and handling. In this study, the genes coding for cytochrome P450 BM3 from B. megaterium and formate dehydrogenase from Pseudomonas sp. were fused to enable both substrate oxidation catalyzed by P450 BM3 and continuous cofactor regeneration by formate dehydrogenase within one construct. The order of the genes in the fusion as well as the linkers that bridge the enzymes were varied. The resulting constructs were compared to individual enzymes regarding substrate conversion, stability and kinetic parameters to examine whether fusion led to any substantial improvements of enzymatic properties. Most noticeably, an activity increase of up to threefold was observed for the fusion constructs with various substrates which were partly attributed to the increased diflavin reductase activity of the P450 BM3. We suggest that P450 BM3 undergoes conformational changes upon fusion which resulted in altered properties, however, no NADPH channeling was detected for the fusion constructs. [ABSTRACT FROM AUTHOR]

Objectives: 3,4-Dihydroxybutyric acid (3,4-DHBA) is a multifunctional C4 platform compound widely used for the synthesis of various materials, including pharmaceuticals. Although, a biosynthetic pathway for 3,4-DHBA production has been developed, its low yield still precludes large-scale use. Here, a heterologous four-step biosynthetic pathway was established in recombinant Escherichia coli (E. coli) using a combinatorial strategy. Results: Several aldehyde dehydrogenases (ALDHs) were screened, using in vitro enzyme assays, to identify suitable catalysts for the dehydrogenation of 3,4-dihydroxybutanal (3,4-DHB) to 3,4-DHBA. A pathway containing glucose dehydrogenase (BsGDH) from Bacillus subtilis, d-xylonate dehydratase (YagF) from E. coli, benzoylformate decarboxylase (PpMdlC) from Pseudomonas putida and ALDH was introduced into E. coli, generating 3.04 g/L 3,4-DHBA from d-xylose (0.190 g 3,4-DHBA/g d-xylose). Disruption of competing pathways by deleting xylA, ghrA, ghrB and adhP contributed to an 87% increase in 3,4-DHBA accumulation. Expression of a fusion construct containing PpMdlC and YagF enhanced the 3,4-DHBA titer, producing the highest titer and yield reported thus far (7.71 g/L; 0.482 g 3,4-DHBA/g d-xylose). Conclusions: These results showed that deleting genes from competing pathways and constructing fusion proteins significantly improved the titer and yield of 3,4-DHBA in engineered E. coli. [ABSTRACT FROM AUTHOR]

Clostridium acetobutylicum DSM 792 has been characterized as a native acetone-butanol-ethanol (ABE) fermenting bacterium. C. acetobutylicm is used as a model organism to improve solvent production by metabolic engineering strategies. In this study, the ABE pathway in C. acetobutylicum was modified by overexpressing the pyruvate decarboxylase gene from Zymomonas mobilis under the control of 2 promoters, namely Pferr and Ppta (promoters of genes encoding pyruvate ferredoxin oxidoreductase and phosphotransacetylase) and the recombinant strains were designated as DSM 792(pPDC1) and DSM 792(pPDC2). The specific activity of the alcohol dehydrogenases in the pdc-expressing strains was higher than that in the wild-type strain. In batch fermentation experiments, the total ABE production from the recombinant strains DSM 792 (pPDC1) and DSM 792 (pPDC2) was 1.57 and 7.9 g/L, respectively, which was higher when compared to the wild type ABE production (0.64 g/L) under uncontrolled pH. The alcohol to acetone ratio (BE/A) was found to be 2.28, 2.77, and 5.26 in wild type, DSM 792 (pPDC1), and DSM 792 (pPDC2), respectively. This suggests that the re-routing of pyruvate by overexpression of the pdc could play a role in the generation of more reducing equivalents towards ethanol and butanol production. [ABSTRACT FROM AUTHOR]

"Bug as drug" is a concept recognized over a century ago and has gained significant research attention recently for fighting diseases such as immune disorders and others. Bacteria and viruses are constantly studied for this purpose, but the use of parasitic organisms is still rare. Recently, we found that Toxoplasma gondii mutants lacking two lactate dehydrogenases (ME49 Δldh1-Δldh2) were avirulent in mice but able to stimulate high levels of Th1 immunity. This outcome prompted us to determine whether Δldh mutants also displayed antitumor activities. Using a mouse melanoma model, we showed that intratumoral administration of Δldh1-Δldh2 repressed the growth of established tumors and helped to inhibit lethal tumor development in the mice. The sera of parasite-treated mice had high levels of TNF-α and INF-γ, which likely contributed to the tumor-repressing activity. We also found that chronic Toxoplasma infection, which is common in animals and humans, also led to antitumor activity. In addition, pre-existing chronic infections did not affect the antitumor efficiency of the Δldh1-Δldh2 mutant. Together, these results suggest that the attenuated T. gondii mutant Δldh1-Δldh2 has the potential to be a good antitumor therapy and provide new insights into the development of novel tumor therapeutics. [ABSTRACT FROM AUTHOR]

Mitochondrial abnormalities and redox imbalance are major pathogenic factors in progression of Huntington's disease (HD), manifested clinically by affective, motor, cognitive, and psychiatric incompetence. Antioxidants behold much promise in mitigation of several pathological facets in HD. Ellagic acid (EA) is a naturally derived polyphenol acknowledged for potent neuroprotective abilities that enabled its significance amongst popular brain tonics. The present study is aimed to examine the outcome of EA pre-treatment in 3-nitropropionic acid (3-NP) rat prototype of HD. Separate rat groups were pre-treated with EA (25, 50, and 100 mg/kg, p.o.) for 21 days and 3-NP (10 mg/kg, i.p.) was given for 14 days alongside to induce symptoms of HD. The physical/motor functions (narrow beam paradigm, footprint study, hanging-wire assessment) and cognitive abilities using elevated plus maze and novel object recognition task were evaluated. Entire brain was isolated and succinate dehydrogenase activity and parameters of oxido-nitrosative stress were assessed in mitochondrial fraction. 3-NP accrued oxido-nitrosative stress and significant decrease in succinate dehydrogenase activity caused motor and cognitive deficits in rats. EA pre-treatment resurrected succinate dehydrogenase activity in 3-NP treated rats that indicated preservation of mitochondrial function. A significant decrease in thiobarbituric acid reactive substances and nitrite levels and increase in glutathione and catalase activity by EA in 3-NP treated rats was noted. EA protected the rats against 3-NP triggered cognitive insufficiency and motor disturbances. It can be inferred that ellagic acid protects against 3-NP induced mitochondrial dysfunction and oxido-nitrosative stress in the brain. EA supplements or nutraceuticals might possess protective potential against symptoms of HD. [ABSTRACT FROM AUTHOR]

The red imported fire ant (RIFA), Solenopsis invicta Buren is native to South America and is known as a global problematic invasive species. This study focused on the molecular response of RIFA by comparing gene expression profiles after exposing ants to low (10 °C) and high (40 °C) temperature stress and comparing them to untreated controls (30 °C). A total of 99,085 unigenes (the clustered non-redundant transcripts that are filtered from the longest assembled contigs) were obtained, of which 19,154 were annotated with gene descriptions, gene ontology terms, and metabolic pathways. 86 gene ontology (GO) functional sub-groups and 23 EggNOG terms resulted. Differentially expressed genes (DEGs) with log2FC ≥ 10 were screened and were compared at different temperatures. We found 203, 48, and 66 specific DEGs co-regulated at 10, 20, and 40 °C. Comparing transcriptome profiles for differential gene expression resulted in various DE genes, including cytochrome P450, NADH dehydrogenase subunit 1, cuticle protein and heat shock protein (HSP), which have previously been reported to be involved in cold and high temperature resistance. GO analysis revealed that antioxidant activity is up-regulated under high temperature stress. We verified the RNA-seq data by qPCR on 20 up- and down-regulated DEGs. These findings provide a basis for future understanding of the adaptation mechanisms of RIFA and the molecular mechanisms underlying the response to low and high temperatures. [ABSTRACT FROM AUTHOR]

Here, we characterized a gene expression system induced by 3-hydroxypropionic acid (3-HP) in Pseudomonas denitrificans. The system consists of a putative LysR-type transcriptional regulator (LTTR) encoded by hpdR and a LTTR-responsive promoter that positively controls the expression of hpdH, encoding 3-HP dehydrogenase in the presence of 3-HP. In the hpdH-responsive promoter region, two operators exhibiting dyad symmetry and designated O1 and O2 and centered at the −73 and −30 positions, respectively, were located upstream of the hpdH transcription start site. When either O1, O2, or both regions were mutated, the inducibility by the HpdR-3-HP complex was significantly reduced or completely removed, indicating that both sites are required for transcriptional activation. The HpdR protein and its operator sites on hpdH were highly specific for each other, and did not engage in cross-talk with another similar 3-HP-inducible system that is also present in P. denitrificans. This 3-HP-inducible promoter system should be useful in developing biosensors for 3-HP, and/or dynamic gene expression systems for metabolic engineering purposes. [ABSTRACT FROM AUTHOR]

Pyrazines are typically formed from amino acids and sugars in chemical reactions such as the Maillard reaction. In this study, we demonstrate that 3-ethyl-2,5-dimethylpyrazine can be produced from L-Thr by a simple bacterial operon. We conclude that EDMP is synthesized chemoenzymatically from L-Thr via the condensation reaction of two molecules of aminoacetone and one molecule of acetaldehyde. Aminoacetone is supplied by L-threonine 3-dehydrogenase using L-Thr as a substrate via 2-amino-3-ketobutyrate. Acetaldehyde is supplied by 2-amino-3-ketobutyrate CoA ligase bearing threonine aldolase activity from L-Thr when CoA was at low concentrations. Considering the rate of EDMP production, the reaction intermediate is stable for a certain time, and moderate reaction temperature is important for the synthesis of EDMP. When the precursor was supplied from L-Thr by these enzymes, the yield of EDMP was increased up to 20.2%. Furthermore, we demonstrate that this reaction is useful for synthesizing various alkylpyrazines. Threonine is a biosynthetic precursor to dimethylpyrazine derivatives, but the pathway by which this occurs is not fully established. Here l-throenine-3-dehydrogenase and 2-amino-3-ketobutyrate CoA ligase together are shown to convert l-threonine to dimethylpyrazine derivatives as a byproduct of glycine metabolism. [ABSTRACT FROM AUTHOR]

Kluyveromyces marxianus is a yeast that could be identified from kefir and can use a broad range of substrates, such as glucose and lactate, as carbon sources. The lactate produced in kefir culture can be a substrate for K. marxianus. However, the complexity of the kefir microbiota makes the traits of K. marxianus difficult to study. In this research, we focused on K. marxianus cultured with lactate as the sole carbon source. The optimal growth and released protein in lactate culture were determined under different pH conditions, and the LC–MS/MS-identified proteins were associated with the tricarboxylic acid cycle, glycolysis pathway, and cellular stress responses in cells, indicating that autolysis of K. marxianus had occurred under the culture conditions. The abundant glyceraldehyde-3-phosphate dehydrogenase 1 (GAP1) was cocrystallized with other proteins in the cell-free fraction, and the low transcription level of the GAP1 gene indicated that the protein abundance under autolysis conditions was dependent on protein stability. These results suggest that lactate induces the growth and autolysis of K. marxianus, releasing proteins and peptides. These findings can be fundamental for K. marxianus probiotic and kefir studies in the future. [ABSTRACT FROM AUTHOR]

Glutamate dehydrogenases (GDHs) are widespread metabolic enzymes that play key roles in nitrogen homeostasis. Large glutamate dehydrogenases composed of 180 kDa subunits (L-GDHs180) contain long N- and C-terminal segments flanking the catalytic core. Despite the relevance of L-GDHs180 in bacterial physiology, the lack of structural data for these enzymes has limited the progress of functional studies. Here we show that the mycobacterial L-GDH180 (mL-GDH180) adopts a quaternary structure that is radically different from that of related low molecular weight enzymes. Intersubunit contacts in mL-GDH180 involve a C-terminal domain that we propose as a new fold and a flexible N-terminal segment comprising ACT-like and PAS-type domains that could act as metabolic sensors for allosteric regulation. These findings uncover unique aspects of the structure-function relationship in the subfamily of L-GDHs. Lázaro et. al. report the first 3D structure of a large glutamate dehydrogenase (L-GDH), the one corresponding to the Mycobacterium smegmatis enzyme composed of 180 kDa subunits (mL-GDH180), obtained by X-ray crystallography and cryo-electron microscopy. This structure reveals that mL-GDH180 assembles as tetramers with the N- and C-terminal domains being involved in inter-subunit contacts and unveils unique features of the subfamily of L-GDHs. [ABSTRACT FROM AUTHOR]

A novel alanine dehydrogenase (AlaDH; EC.1.4.1.1) was isolated from Amycolatopsis sulphurea and the AlaDH gene was cloned into a pET28a(+) plasmid and expressed in E. coli BL21 (DE3). The molecular mass of this enzyme was calculated as 41.09 kDa and the amino acid residues of the pure protein indicated the presence of N terminus polyhistidine tags. Its enzyme kinetic values were Km 2.03 mM, kcat 13.24 (s−1), and kcat/Km 6.53 (s−1 mM−1). AlaDH catalyzes the reversible conversion of l-alanine and pyruvate, which has an important role in the TCA energy cycle. Maximum AlaDH activity occurred at about pH 10.5 and 25 °C for the oxidative deamination of l-alanine. AlaDH retained about 10% of its relative activity at 55 °C and it remained about 90% active at 50 °C. These findings show that the AsAlaDH from A. sulphurea has the ability to produce valuable molecules for various industrial purposes and could represent a new potential biocatalyst for biotechnological applications after further characterization and improvement of its catalytic properties. [ABSTRACT FROM AUTHOR]

Among the several alcohol dehydrogenases, PQQ-dependent enzymes are mainly found in the α, β, and γ-proteobacteria. These proteins are classified into three main groups. Type I ADHs are localized in the periplasm and contain one Ca2+-PQQ moiety, being the methanol dehydrogenase (MDH) the most representative. In recent years, several lanthanide-dependent MDHs have been discovered exploding the understanding of the natural role of lanthanide ions. Type II ADHs are localized in the periplasm and possess one Ca2+-PQQ moiety and one heme c group. Finally, type III ADHs are complexes of two or three subunits localized in the cytoplasmic membrane and possess one Ca2+-PQQ moiety and four heme c groups, and in one of these proteins, an additional [2Fe-2S] cluster has been discovered recently. From the bioinorganic point of view, PQQ-dependent alcohol dehydrogenases have been revived recently mainly due to the discovery of the lanthanide-dependent enzymes. Here, we review the three types of PQQ-dependent ADHs with special focus on their structural features and electron transfer processes. [ABSTRACT FROM AUTHOR]

Aerobic methanotrophic bacteria utilize methane as a growth substrate but are unable to grow on any sugars. In this study we have shown that two obligate methanotrophs, Methylotuvimicrobium alcaliphilum 20Z and Methylobacter luteus IMV-B-3098, possess functional glucose dehydrogenase (GDH) and gluconate kinase (GntK). The recombinant GDHs from both methanotrophs were homotetrameric and strongly specific for glucose preferring NAD+ over NADP+. GDH from Mtm. alcaliphilum was most active at pH 10 (Vmax = 95 U/mg protein) and demonstrated very high Km for glucose (91.8 ± 3.8 mM). GDH from Mb. luteus was most active at pH 8.5 (Vmax = 43 U/mg protein) and had lower Km for glucose (16 ± 0.6 mM). The cells of two Mtm. alcaliphilum double mutants with deletions either of the genes encoding GDH and glucokinase (gdh─/glk─) or of the genes encoding gluconate kinase and glucokinase (gntk─/glk─) had the lower glycogen level and the higher contents of intracellular glucose and trehalose compared to the wild type strain. The gntk─/glk─ knockout mutant additionally accumulated gluconic acid. These data, along with bioinformatics analysis, demonstrate that glycogen derived free glucose can enter the Entner–Doudoroff pathway or the pentose phosphate cycle in methanotrophs, bypassing glycolysis via the gluconate shunt. [ABSTRACT FROM AUTHOR]

Oxidative plant cell-wall processing enzymes are of great importance in biology and biotechnology. Yet, our insight into the functional interplay amongst such oxidative enzymes remains limited. Here, a phylogenetic analysis of the auxiliary activity 7 family (AA7), currently harbouring oligosaccharide flavo-oxidases, reveals a striking abundance of AA7-genes in phytopathogenic fungi and Oomycetes. Expression of five fungal enzymes, including three from unexplored clades, expands the AA7-substrate range and unveils a cellooligosaccharide dehydrogenase activity, previously unknown within AA7. Sequence and structural analyses identify unique signatures distinguishing the strict dehydrogenase clade from canonical AA7 oxidases. The discovered dehydrogenase directly is able to transfer electrons to an AA9 lytic polysaccharide monooxygenase (LPMO) and fuel cellulose degradation by LPMOs without exogenous reductants. The expansion of redox-profiles and substrate range highlights the functional diversity within AA7 and sets the stage for harnessing AA7 dehydrogenases to fine-tune LPMO activity in biotechnological conversion of plant feedstocks. Microbial oxidoreductases are key in biomass breakdown. Here, the authors expand the specificity and redox scope within fungal auxiliary activity 7 family (AA7) enzymes and show that AA7 oligosaccharide dehydrogenases can directly fuel cellulose degradation by lytic polysaccharide monooxygenases. [ABSTRACT FROM AUTHOR]

NAD+-dependent formate dehydrogenases (FDHs) are extensively used in the regeneration of NAD(P)H and the reduction of CO2 to formate. In addition to their industrial importance, FDHs also play a crucial role in the maintenance of a reducing environment to combat oxidative stress in plants. Therefore, it is important to investigate the response of NAD+-dependent FDH against both temperature and H2O2, to understand the defense mechanisms, and to increase its stability under oxidative stress conditions. In the present study, we characterized the oxidative and thermal stability of NAD+-dependent FDH isolated from cotton, Gossypium hirsutum (GhFDH), by investigating the effect of Met/Leu substitutions in the positions of 225, 234, and 243. Results showed that the single mutant, M234L (0.72 s−1 mM−1), and the triple mutant, M225L/M234L/M243L (0.55 s−1 mM−1), have higher catalytic efficiency than the native enzyme. Substitution of methionine by leucine on the position of 243 increased the free energy gain by 670 J mol−1. The most remarkable results in chemical stability were seen for double and triple mutants, cumulatively. Double and triple substitution of Met to Leu (M225L/M243L and M225L/M243L/M234L) reduce the kefin by a factor of 2 (12.3×10−5 and 12.8×10−5 s−1, respectively. Key points • The closer the residue to NAD+, in which we substituted methionine to leucine, the lower the stability against H2O2we observed. • The significant gain in the Tmvalue for the M243L mutant was observed as +5°C. • Residue 234 occupies a critical position for oxidation defense mechanisms. [ABSTRACT FROM AUTHOR]

In this study, alcohol dehydrogenases (ADH) enzymes from Yarrowia lipolytica were investigated for the cloning, soluble expression, and biotransformation of 1-dodecanol to 1-dodecanal, which reaction was thermodynamically unfavorable. Sole expression of ADHs in Escherichia coli did not produce soluble form of cytosolic protein, in spite of the effort to solubilize ADH protein by optimizing IPTG concentration, temperature, and auto-induction medium. Eventually, the active form of soluble ADH proteins was successfully obtained through the co-expression of ADH with chaperone protein in pG-KJE8 vector. After analyzing the individual sets of optimization, it was determined that pET-28a(+)::adh coexpression with the pG-KJE8 molecular chaperone in LB medium with 0.1 mM IPTG, 4 mg/mL arabinose, and 2 ng/mL tetracycline achieved optimum expressions against all of the five ADH proteins. Finally, the whole cell biotransformation activity of ADH2 was determined in 1-dodecanol oxidation to 1-dodecanal, followed by further oxidation to 1-dodecanoic acid. [ABSTRACT FROM AUTHOR]

The sulfoximine insecticide sulfoxaflor is regarded as a potential substitute for neonicotinoids that were recently banned in the EU due to their side effects on bees. Like neonicotinoids, sulfoxaflor acts as a competitive modulator of nicotinic acetylcholine receptors. In agricultural environments, bees are commonly exposed to combinations of pesticides, and neonicotinoids are known to interact synergistically with fungicides. The objective of our study is to assess the acute oral toxicity of sulfoxaflor alone and in combination with a single dose of fluxapyroxad, a succinate dehydrogenase inhibitor (SDHI) fungicide, in three bee species: Apis mellifera, Bombus terrestris and Osmia bicornis. Because synergism may be dose-dependent, we tested a range of sulfoxaflor doses. Synergistic effects were assessed using three different approaches: Bliss criterion of drugs independence, ratio test comparing LD50s and model deviation ratio. Osmia bicornis was the most sensitive species to sulfoxaflor and both O. bicornis and A. mellifera showed significant synergism between the insecticide and the fungicide. For the most part, these synergistic effects were weak and only occurred at early assessment times and intermediate sulfoxaflor doses. The potential ecological relevance of these effects should be confirmed in field and/or cage studies. Overall, our laboratory results demonstrate that sulfoxaflor is somewhat less toxic than the recently banned neonicotinoids imidacloprid, thiamethoxam and clothianidin, but much more toxic than other neonicotinoids (acetamiprid, thiacloprid) still in use in the EU at the time this study was conducted. [ABSTRACT FROM AUTHOR]

3-Hydroxypropionic acid (3HP), an important three carbon (C3) chemical, is designated as one of the top platform chemicals with an urgent need for improved industrial production. Halomonas bluephagenesis shows the potential as a chassis for competitive bioproduction of various chemicals due to its ability to grow under an open, unsterile and continuous process. Here, we report the strategy for producing 3HP and its copolymer poly(3-hydroxybutyrate-co-3-hydroxypropionate) (P3HB3HP) by the development of H. bluephagenesis. The transcriptome analysis reveals its 3HP degradation and synthesis pathways involving endogenous synthetic enzymes from 1,3-propanediol. Combing the optimized expression of aldehyde dehydrogenase (AldDHb), an engineered H. bluephagenesis strain of whose 3HP degradation pathway is deleted and that overexpresses alcohol dehydrogenases (AdhP) on its genome under a balanced redox state, is constructed with an enhanced 1.3-propanediol-dependent 3HP biosynthetic pathway to produce 154 g L−1 of 3HP with a yield and productivity of 0.93 g g−1 1,3-propanediol and 2.4 g L−1 h−1, respectively. Moreover, the strain could also accumulate 60% poly(3-hydroxybutyrate-co-32–45% 3-hydroxypropionate) in the dry cell mass, demonstrating to be a suitable chassis for hyperproduction of 3HP and P3HB3HP. 3-Hydroxypropionic acid (3HP) is an important platform chemical. Here, the authors engineer Halomonas bluephagenesis by deleting newly identified degradation pathway and balancing redox state to achieve high level production of 3HP and its copolymer under open and unsterile conditions. [ABSTRACT FROM AUTHOR]

Farmers are not growing diversified crops and applying huge amounts of agrochemicals and imbalanced fertilizers in the rice-wheat cropping system (RWCS), since the 1960s. The objective of this study was to evaluate the microbial and nutrient dynamics in Indian mustard (Brassica juncea L.) under various sowing environments and nutrient sources during Rabi season (October–March), 2015–2016. The experiment was laid out in the split-plot design with three sowing dates in main-plots, and eight nutrient sources in sub-plots. The maximum bacteria, fungi, and actinomycetes population, soil microbial biomass carbon (SMBC), dehydrogenase activities, and available nitrogen, phosphorus, potassium, and sulphur (NPKS) were recorded on November 17 sown crop, and the lowest was observed on December 7 sowing during both the years, and in the pooled analysis. Furthermore, applied nutrient sources, highest bacteria, fungi, and actinomycetes population, available NPKS, SMBC, and dehydrogenase activity were observed in 75% recommended dose of fertilizers (RDF) + 25% N through pressmud (PM) + Azotobacto + phosphorus solubilizing bacteria (PSB) than other nutrient sources. In conclusion, high demand and cost of chemical fertilizers can be replaced by 25% amount easily and locally available organic manures like PM compost to sustain the soil health and crop productivity. It will be helpful to restore the soil biodiversity in the RWCS and provide a roadmap for the researchers, government planners, and policymakers for the use of PM as a source of organic matter and nutrients. [ABSTRACT FROM AUTHOR]

Knowledge about biotic (plant species diversity, biomass) and/or abiotic (physicochemical substrate parameters) factors that determine enzyme activity and functional diversity of the substrate on hard coal spoil heaps is limited. Spontaneously developed vegetation patches dominated by herbaceous species commonly occurring on these spoil heaps: grasses (Poa compressa, Calamagrostis epigejos) and forbs (Daucus carota, Tussilago farfara), were examined. The activity of dehydrogenase and alkaline phosphatase was twice as high in plots dominated by grass species compared with those dominated by forbs. Significant positive correlations were found between the activity of dehydrogenase and alkaline phosphatase with pH, available P, soil moisture, and water holding capacity and negative correlations between the activity of urease and soil organic carbon. Strong positive correlations were found between values for Shannon–Wiener diversity index, evenness, species richness and soil functional diversity in plots dominated by grasses. We found that the soil physicochemical parameters had a greater impact on enzyme activity of the substrate than plant biomass and species diversity. However, grasses, through their extensive root system, more effectively increased enzyme activity and health of the substrate than other herbaceous species, and as they stabilize the substrate and form dense plant cover, they can be recommended for reclamation purposes. [ABSTRACT FROM AUTHOR]

The MoCu CO dehydrogenase enzyme not only transforms CO into CO2 but it can also oxidise H2. Even if its hydrogenase activity has been known for decades, a debate is ongoing on the most plausible mode for the binding of H2 to the enzyme active site and the hydrogen oxidation mechanism. In the present work, we provide a new perspective on the MoCu-CODH hydrogenase activity by improving the in silico description of the enzyme. Energy refinement—by means of the BigQM approach—was performed on the intermediates involved in the dihydrogen oxidation catalysis reported in our previously published work (Rovaletti, et al. "Theoretical Insights into the Aerobic Hydrogenase Activity of Molybdenum–Copper CO Dehydrogenase." Inorganics 7 (2019) 135). A suboptimal description of the H2–HN(backbone) interaction was observed when the van der Waals parameters described in previous literature for H2 were employed. Therefore, a new set of van der Waals parameters is developed here in order to better describe the hydrogen–backbone interaction. They give rise to improved binding modes of H2 in the active site of MoCu CO dehydrogenase. Implications of the resulting outcomes for a better understanding of hydrogen oxidation catalysis mechanisms are proposed and discussed. [ABSTRACT FROM AUTHOR]