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Aldehydes are attractive chemical targets both as end products in the flavors and fragrances industry and as synthetic intermediates due to their propensity for C–C bond formation. Here, we identify and address unexpected oxidation of a model collection of aromatic aldehydes, including many that originate from biomass degradation. When diverse aldehydes are supplemented to E. coli cells grown under aerobic conditions, as expected they are either reduced by the wild-type MG1655 strain or stabilized by a strain engineered for r educed aromatic a ldehyde re duction (the E. coli RARE strain). Surprisingly, when these same aldehydes are supplemented to resting cell preparations of either E. coli strain, under many conditions we observe substantial oxidation. By performing combinatorial inactivation of six candidate aldehyde dehydrogenase genes in the E. coli genome using multiplexed automatable genome engineering (MAGE), we demonstrate that this oxidation can be substantially slowed, with greater than 50% retention of 6 out of 8 aldehydes when assayed 4 h after their addition. Given that our newly engineered strain exhibits r educed o xidation a nd r eduction of aromatic aldehydes, we dubbed it the E. coli ROAR strain. We applied the new strain to resting cell biocatalysis for two kinds of reactions – the reduction of 2-furoic acid to furfural and the condensation of 3-hydroxybenzaldehyde and glycine to form a non-standard β-hydroxy-α-amino acid. In each case, we observed substantial improvements in product titer 20 h after reaction initiation (9-fold and 10-fold, respectively). Moving forward, the use of this strain to generate resting cells should allow aldehyde product isolation, further enzymatic conversion, or chemical reactivity under cellular contexts that better accommodate aldehyde toxicity. • When genes encoding aldehyde reductases are knocked out in Escherichia coli , supplemented aldehydes can undergo oxidation. • For the aromatic aldehydes tested, oxidation is more substantial with resting cells than during aerobic fermentation. • We use multiplex genome engineering to identify the genes responsible for aldehyde oxidation. • The strain we engineered exhibits Reduced Oxidation And Reduction (ROAR), enabling more efficient aldehyde biocatalysis. [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]

The reductive glycine pathway was described as the most energetically favorable synthetic route of aerobic formate assimilation. Here we report the successful implementation of formatotrophy in Escherichia coli by means of a stepwise adaptive evolution strategy. Medium swap and turbidostat regimes of continuous culture were applied to force the channeling of carbon flux through the synthetic pathway to pyruvate establishing growth on formate and CO 2 as sole carbon sources. Labeling with 13C-formate proved the assimilation of the C1 substrate via the pathway metabolites. Genetic analysis of intermediate isolates revealed a mutational path followed throughout the adaptation process. Mutations were detected affecting the copy number (gene ftfL) or the coding sequence (genes folD and lpd) of genes which specify enzymes implicated in the three steps forming glycine from formate and CO 2 , the central metabolite of the synthetic pathway. The mutation R191S present in methylene-tetrahydrofolate dehydrogenase/cyclohydrolase (FolD) abolishes the inhibition of cyclohydrolase activity by the substrate formyl-tetrahydrofolate. The mutation R273H in lipoamide dehydrogenase (Lpd) alters substrate affinities as well as kinetics at physiological substrate concentrations likely favoring a reactional shift towards lipoamide reduction. In addition, genetic reconstructions proved the necessity of all three mutations for formate assimilation by the adapted cells. The largely unpredictable nature of these changes demonstrates the usefulness of the evolutionary approach enabling the selection of adaptive mutations crucial for pathway engineering of biotechnological model organisms. • E. coli was adapted to grow on formate as sole carbon source in continuous culture. • 13C-tracing proved that formate is assimilated via the reductive glycine pathway. • Point mutations in the genes folD and lpd encoding the two dehydrogenases of the pathway are essential for growth on formate. • Activity analysis of mutated FolD and Lpd revealed altered inhibition patterns. [ABSTRACT FROM AUTHOR]

The objective of this study was to implement direct sunlight-driven conversion of CO 2 into a naturally excreted ready-to-use fuel. We engineered four different synthetic metabolic modules for biosynthesis of short-to medium-chain length hydrocarbons in the model cyanobacterium Synechocystis sp. PCC 6803. In module 1, the combination of a truncated clostridial n-butanol pathway with over-expression of the native cyanobacterial aldehyde deformylating oxygenase resulted in small quantities of propane when cultured under closed conditions. Direct conversion of CO 2 into propane was only observed in strains with CRISPRi-mediated repression of three native putative aldehyde reductases. In module 2, three different pathways towards pentane were evaluated based on the polyunsaturated fatty acid linoleic acid as an intermediate. Through combinatorial evaluation of reaction ingredients, it was concluded that linoleic acid undergoes a spontaneous non-enzymatic reaction to yield pentane and hexanal. When Synechocystis was added to the reaction, hexanal was converted into 1-hexanol, but there was no further stimulation of pentane biosynthesis even in the Synechocystis strains expressing Gm LOX1. For modules 3 and 4, several different acyl-ACP thioesterases were evaluated in combination with two different decarboxylases. Small quantities of 1-heptene and 1-nonene were observed in strains expressing the desaturase-like enzyme UndB from Pseudomonas mendocina in combination with C8–C10 preferring thioesterases ('Ca FatB3.5 and 'Cho FatB2.2). When UndB instead was combined with a C12-specific 'Uc FatB1 thioesterase, this resulted in a ten-fold increase of alkene biosynthesis. When UndB was replaced with the light-dependent FAP decarboxylase, both undecane and tridecane accumulated, albeit with a 10-fold drop in productivity. Preliminary optimization of the RBS, promoter and gene order in some of the synthetic operons resulted in improved 1-alkene productivity, reaching a titer of 230 mg/L after 10 d with 15% carbon partitioning. In conclusion, the direct bioconversion of CO 2 into secreted and ready-to-use hydrocarbon fuel was implemented with several different metabolic systems. Optimal productivity was observed with UndB and a C12 chain-length specific thioesterase, although further optimization of the entire biosynthetic system is still possible. • Repression of endogenous aldehyde reductases/dehydrogenases by CRISPRi enabled propane biosynthesis • Biosynthesis of short-medium chain hydrocarbons (C7–C11) in a cyanobacterium was demonstrated for the first time • UndB catalyzed the biosynthesis of propene from butyric acid • All target products were naturally secreted and accumulated outside of the cell [ABSTRACT FROM AUTHOR]

l -Arabinose 1-dehydrogenase (AraDH) catalyzes the NAD(P)+-dependent oxidation of l -arabinose to L-arabinono-1,4-lactone in the non-phosphorylative l -arabinose pathway, and is classified into glucose-fructose oxidoreductase and short-chain dehydrogenase/reductase (SDR). We herein report the crystal structure of a SDR-type AraDH (from Herbaspirillum huttiense) for the first time. The interactions between Asp49 and the 2′- and 3′-hydroxyl groups of NAD+ were consistent with strict specificity for NAD+. In a binding model for the substrate, Ser155 and Tyr168, highly conserved in the SDR superfamily, interacted with the C1 and/or C2 hydroxyl(s) of l -arabinose, whereas interactions between Asp107, Arg109, and Gln206 and the C2 and/or C3 hydroxyl(s) were unique to AraDH. Trp200 significantly contributed to the selectivities of the C4 hydroxyl and C6 methyl of substrates. • l -Arabinose 1-dehydrogenase belongs to short-chain dehydrogenase/reductase (SDR) protein superfamily. • Crystal structure in complex with NAD+ was herein reported. • Active sites including a catalytic base differed from those of other SDR members. • The novel structure is helpful for understanding of catalysis and molecular evolution. [ABSTRACT FROM AUTHOR]

Organotins have been widely used in various industrial applications. This study investigated the structure-activity relationship as inhibitors of human, pig, and rat gonadal 3β-hydroxysteroid dehydrogenases (3β-HSD). Human KGN cell, pig, and rat testis microsomes were utilized to assess the inhibitory effects of 18 organotins on the conversion of pregnenolone to progesterone. Among them, diphenyltin, triethyltin, and triphenyltin exhibited significant inhibitory activity against human 3β-HSD2 with IC 50 values of 114.79, 106.98, and 5.40 μM, respectively. For pig 3β-HSD, dipropyltin, diphenyltin, triethyltin, tributyltin, and triphenyltin demonstrated inhibitory effects with IC 50 values of 172.00, 100.19, 87.00, 5.75, and 1.65 μM, respectively. Similarly, for rat 3β-HSD1, dipropyltin, diphenyltin, triethyltin, tributyltin, and triphenyltin displayed inhibitory activity with IC 50 values of 81.35, 43.56, 55.55, 4.09, and 0.035 μM, respectively. They were mixed inhibitors of pig and rat 3β-HSD, while triphenyltin was identified as a competitive inhibitor of human 3β-HSD2. The mechanism underlying the inhibition of organotins on 3β-HSD was explored, revealing that they may disrupt the enzyme activity by binding to cysteine residues in the catalytic sites. This proposition was supported by the observation that the addition of dithiothreitol reversed the inhibition caused by all organotins except for triethyltin, which was partially reversed. In conclusion, this study provides valuable insights into the structure-activity relationship of organotins as inhibitors of human, pig, and rat gonadal 3β-HSD. The mechanistic investigation suggests that these compounds likely exert their inhibitory effects through binding to cysteine residues in the catalytic sites. [Display omitted] • Some organotins inhibit human, pig, and rat gonadal 3β-HSDs. • Tri-organotins are more potent to inhibit 3β-HSD2 than other organotins. • TPT is the most potent inhibitor of gonadal 3β-HSDs among all species. • DTT can partially or fully reverse organotins-mediated inhibition 3β-HSD activity. [ABSTRACT FROM AUTHOR]

p -Terphenyls represent a unique family of aromatic natural products generated by nonribosomal peptide synthetase-like (NRPS-like) enzyme. After formation of p -terphenyl skeleton, tailoring modifications will give rise to structural diversity and various biological activities. Here we demonstrated a two-enzyme (EchB, a short-chain dehydrogenase/reductase (SDR), and EchC, a nuclear transport factor 2 (NTF2)-like dehydratase) participated transformation from dihydroxybenzoquinone core to 2′,3′,5′-trihydroxy-benzene in the biosynthesis of echosides. Beginning with polyporic acid as substrate, successive steps of reduction-dehydration-reduction cascade catalyzed by EchB-EchC-EchB were concluded after in vivo gene disruption and in vitro bioassay experiments. These findings demonstrated a conserved synthesis pathway of 2′,3′,5′-trihydroxy- p -terphenyls in bacteria, such as Actinomycetes and Burkholderia. The parallel pathway in fungi has yet to be explored. [Display omitted] • Isolated intermediates for 2′,3′,5′-trihydroxy- p -terphenyls biosynthesis. • Characterized a new short-chain dehydrogenase/reductase (SDR) EchB in vitro. • Characterized a novel NTF2-like dehydratase EchC with high substrate specificity in vitro. • Successive steps of reduction-dehydration-reduction catalyzed by EchB-EchC-EchB. [ABSTRACT FROM AUTHOR]

• Eight types of Hsd17b were cloned in a clupeoid fish, Japanese sardine. • Gene expression of types 3, 12a, and 12b was higher in ovary than in testis. • Gene expression of types 3, 4, 12a, and 12b increased during ovarian development. • Hsd17b12a and Hsd17b12b converted AD to T and E1 to E2. • E2 was synthesized via AD and T, but not via E1, in the sardine ovary. 17β-hydroxysteroid dehydrogenases (Hsd17bs) play a critical role in sex steroid biosynthesis. Although multiple types of Hsd17b have been found in fish, there is limited research on their expression and function. Recently, we succeeded in identifying eight types of Hsd17b (types 3, 4, 7, 8, 10, 12a, 12b, and 14) by RNA sequencing in the Japanese sardine Sardinops melanostictus , a commercially important clupeoid fish; however, a homologous sequence of Hsd17b1, which catalyzes the key reaction of estradiol-17β (E2) synthesis, was absent. Here, we aimed to identify the Hsd17b type that plays a major role in E2 synthesis during ovarian development in Japanese sardine. The cDNAs encoding those eight types of Hsd17b were cloned and sequenced. The expressions of hsd17b3 , hsd17b12a , and hsd17b12b were higher in ovary than in testis. In particular, hsd17b12a was predominantly expressed in the ovary. Expression of hsd17b3 , hsd17b4 , hsd17b12a , and hsd17b12b in the ovary increased during ovarian development. The enzymatic activities of Hsd17b3, Hsd17b12a, and Hsd17b12b were evaluated by expressing their recombinants in human embryonic kidney 293T cells. Hsd17b12a and Hsd17b12b catalyzed the conversion of androstenedione (AD) to testosterone (T) and estrone (E1) to E2. The results of in vitro bioassays using sardine ovaries indicated that E2 is synthesized from pregnenolone via AD and T, but not E1. These results suggest that Hsd17b12a plays a major role in E2 synthesis in sardine ovary by catalyzing the conversion of AD to T. [ABSTRACT FROM AUTHOR]

Fungi-derived flavin adenine dinucleotide (FAD)-dependent glucose dehydrogenases (FADGDHs) are the most popular and advanced enzymes for SMBG sensors because of their high substrate specificity toward glucose and oxygen insensitivity. However, this type of FADGDH hardly shows direct electron transfer (DET) ability. In this study, we developed a new DET-type FADGDH by harboring Cytochrome b 562 (cyt b 562) derived from Escherichia coli as the electron transfer domain. The structural genes encoding fusion enzymes composed of cyt b 562 at either the N- or C-terminus of fungal FADGDH, (cyt b 562 -GDH or GDH-cyt b 562), were constructed, recombinantly expressed, and characteristics of the fusion proteins were investigated. Both constructed fusion enzymes were successfully expressed in E. coli , as the soluble and GDH active proteins, showing cyt b 562 specific redox properties. Thusconstructed fusion proteins showed internal electron transfer between FAD in FADGDH and fused cyt b 562. Consequently, both cyt b 562 -GDH and GDH-cyt b 562 showed DET abilities toward electrode. Interestingly, cyt b 562 -GDH showed much rapid internal electron transfer and higher DET ability than GDH-cyt b 562. Thus, we demonstrated the construction and production of a new DET-type FADGDH using E.coli as the host cells, which is advantageous for future industrial application and further engineering. • New direct electron transfer (DET) type FADGDH was constructed employing cyt b 562. • Fusion proteins were produced in E.coli showing GDH activity and cyt b 562 redox activity. • Fusion proteins showed internal electron transfer between FAD and cyt b 562. • Fusion proteins showed DET abilities with electrode. • FADGDH with cyt b 562 in N-terminal showed higher DET abilities than those in C-terminal. [ABSTRACT FROM AUTHOR]

The nutrition management guideline for very-long chain acyl-CoA dehydrogenase deficiency (VLCAD) is the fourth in a series of web-based guidelines focusing on the diet treatment for inherited metabolic disorders and follows previous publication of guidelines for maple syrup urine disease (2014), phenylketonuria (2016) and propionic acidemia (2019). The purpose of this guideline is to establish harmonization in the treatment and monitoring of individuals with VLCAD of all ages in order to improve clinical outcomes. Six research questions were identified to support guideline development on: nutrition recommendations for the healthy individual, illness management, supplementation, monitoring, physical activity and management during pregnancy. This report describes the methodology used in its development including review, critical appraisal and abstraction of peer-reviewed studies and unpublished practice literature; expert input through two Delphi surveys and a nominal group process; and external review from metabolic physicians and dietitians. It includes the summary statements of the nutrition management recommendations for each research question, followed by a standardized rating based on the strength of the evidence. Online, open access of the full published guideline allows utilization by health care providers, researchers and collaborators who advise, advocate and care for individuals with VLCAD and their families and can be accessed from the Genetic Metabolic Dietitians International (https://GMDI.org) and Southeast Regional Genetics Network (https://southeastgenetics.org/ngp) websites. [ABSTRACT FROM AUTHOR]

The biosynthesis of polyunsaturated fatty acids (PUFAs) in bacteria has been extensively studied. In contrast, studies of PUFA metabolism remain limited. Shewanella livingstonensis Ac10 is a psychrotrophic bacterium producing eicosapentaenoic acid (EPA), a long-chain ω-3 PUFA. This bacterium has the ability to convert exogenous docosahexaenoic acid (DHA) into EPA and incorporate both DHA and EPA into membrane phospholipids. Our previous studies revealed the importance of 2,4-dienoyl-CoA reductase in the conversion, suggesting that DHA is metabolized through a general β-oxidation pathway. Herein, to gain further insight into the conversion mechanism, we analyzed the role of acyl-CoA dehydrogenase (FadE), the first committed enzyme of the β-oxidation pathway, in DHA conversion. S. livingstonensis Ac10 has two putative FadE proteins (FadE1 and FadE2) that are highly homologous to Escherichia coli FadE. We found that FadE1 expression was induced by addition of DHA to the medium and fadE1 deletion reduced DHA conversion into EPA. Consistently, purified FadE1 exhibited dehydrogenase activity towards DHA-CoA. Moreover, its activity towards DHA- and EPA-CoAs was higher than that towards palmitoleoyl- and palmitoyl-CoAs. In contrast, fadE2 deletion did not impair DHA conversion, and purified FadE2 had higher activity towards palmitoleoyl- and palmitoyl-CoAs than towards DHA- and EPA-CoAs. These results suggest that FadE1 is the first enzyme of the β-oxidation pathway that catalyzes DHA conversion. • Shewanella livingstonensis Ac10 synthesizes eicosapentaenoic acid (EPA) de novo. • This strain can convert exogenous docosahexaenoic acid (DHA) into EPA. • It has two acyl-CoA dehydrogenases (FadE1 and FadE2) for fatty acid β-oxidation. • FadE1 and FadE2 have different substrate specificity and inducible nature. • FadE1 is DHA inducible and plays a major role in DHA conversion into EPA. [ABSTRACT FROM AUTHOR]

Dicarboxylic fatty acids, taken as a nutritional supplement or produced endogenously via omega oxidation of monocarboxylic fatty acids, may have therapeutic potential for rare inborn errors of metabolism as well as common metabolic diseases such as type 2 diabetes. Breakdown of dicarboxylic acids yields acetyl-CoA and succinyl-CoA as products, the latter of which is anaplerotic for the TCA cycle. However, little is known about the metabolic pathways responsible for degradation of dicarboxylic acids. Here, we demonstrated with whole-cell fatty acid oxidation assays that both mitochondria and peroxisomes contribute to dicarboxylic acid degradation. Several mitochondrial acyl-CoA dehydrogenases were tested for activity against dicarboxylyl-CoAs. Medium-chain acyl-CoA dehydrogenase (MCAD) exhibited activity with both six and 12 carbon dicarboxylyl-CoAs, and the capacity for dehydrogenation of these substrates was significantly reduced in MCAD knockout mouse liver. However, when dicarboxylic acids were fed to normal mice, the expression of MCAD did not change, while expression of peroxisomal fatty acid oxidation enzymes was greatly upregulated. In conclusion, mitochondrial fatty acid oxidation, and in particular MCAD, contributes to dicarboxylic acid degradation, but feeding dicarboxylic acids induces only the peroxisomal pathway. • Both mitochondria and peroxisomes chain-shorten dicarboxylic fatty acids (DCAs). • Medium-chain acyl-CoA dehydrogenase is active against dicarboxylyl-CoAs. • Feeding DCAs induces liver expression of peroxisomal, but mitochondrial, enzymes. [ABSTRACT FROM AUTHOR]

Pseudomonas aeruginosa can metabolize acyclic monoterpenoids (such as citronellol and geraniol) as the only carbon and energy sources. A total of seven proteins (AtuA, AtuB, AtuCF, AtuD, AtuE, AtuG, AtuH) have been identified in Pseudomonas aeruginosa as participating in the acyclic terpene utilization pathway. AtuB is a dehydrogenase enzyme responsible for citronellol and geraniol catabolism in the acyclic terpene utilization (Atu) pathway, although its structure and function have not been characterized to date. Here we report the crystal structure of AtuB from Pseudomonas aeruginosa PAO1 (PaAtuB) to 1.8 Å resolution. PaAtuB crystallizes in the space group F222 with a single monomer in the asymmetric unit. Analytical ultracentrifugation data shows that PaAtuB forms a stable tetramer in solution, which is consistent with the structure. Structural analysis confirms that AtuB belongs to the short-chain dehydrogenase/reductase (SDR) family. AtuB is predicted to bind NADP(H) from the crystal structure, which is confirmed by MicroScale Thermophoresis analysis that shows PaAtuB binds NADP(H) with a Kd value of 258 μM. This work provides a starting point to explore potential biotechnology and pharmaceutical applications of AtuB. • A crystal structure of P. aeruginosa dehydrogenase AtuB was determined. • P. aeruginosa AtuB was confirmed to be a member of the short-chain dehydrogenase/reductase (SDR) family. • P. aeruginosa AtuB forms a stable tetramer in solution. • P. aeruginosa AtuB is confirmed to bind NADP(H). • The AtuB structure should provide a starting point to explore industrial applications in citronellol and geraniol catabolism. [ABSTRACT FROM AUTHOR]

The long-term consequences and need for therapy in children with short-chain acyl-CoA dehydrogenase deficiency (SCADD) or isobutyryl-CoA dehydrogenase deficiency (IBDD) identified via newborn screening (NBS) remains controversial. Initial clinical descriptions were severe; however, while most cases identified through NBS have remained asymptomatic, clinical concerns have been raised in these populations. It is not clear whether these children are asymptomatic because of the success of NBS, or because the normal clinical course of these disorders is relatively benign. To evaluate these possibilities in our program, we evaluated the clinical outcomes of children with SCADD or IBDD identified by the Georgia NBS compared to the health status of a healthy age-matched control group. We also assessed parental anxiety during a phone interview both subjectively and objectively using the Pediatric Inventory for Parents (PIP), a validated measure of illness-related parental stress. The general health of 52 SCADD and nine IBDD cases from 2007 to 2016 were compared to the general health of unaffected control children obtained through the Centers for Disease Control and Prevention (CDC) parent listserv. We also collected statements from parents who participated in a phone survey regarding events they experienced during and after their diagnostic process. Overall, the children with SCADD and IBDD had no major health problems. There was no significant difference in cognitive development (p =.207). We identified a slightly higher incidence of reported neonatal hypoglycemia in the SCADD group; two of these occurred in the context of maternal diabetes. All interviewed parents reported extreme anxiety during the diagnostic period and current feelings of uncertainty about their child's future. PIP scores for all six caregivers who responded to that portion of the survey were consistent with some degree of parental stress. The greatest reported stressor was the unknown long-term impact of the illness. All children with SCADD and IBDD had no significant long-term sequelae. The phone interviews revealed substantial parental anxiety about the identification and follow-up of SCADD and IBDD. Based on our findings, the anxiety parents experience may be unwarranted given that we see no disease-associated morbidity or mortality in these children. Consideration should be given to the removal of these conditions from NBS panels, or if that is not possible, clinicians could educate parents on the benign nature of these diagnoses and release them from follow-up without treatment. [ABSTRACT FROM AUTHOR]

Alcohol dehydrogenases (ADHs) are popular catalysts for synthesizing chiral synthons a vital step for active pharmaceutical intermediate (API) production. They are grouped into three superfamilies namely, medium-chain (MDRs), short-chain dehydrogenase/reductases (SDRs), and iron-containing alcohol dehydrogenases. The former two are used extensively for producing various chiral synthons. Many studies screen multiple enzymes or engineer a specific enzyme for catalyzing a substrate of interest. These processes are resource-intensive and intricate. The current study attempts to decipher the ability to match different ADHs with their ideal substrates using machine learning algorithms. We explore the catalysis of 284 antibacterial ketone intermediates, against MDRs and SDRs to demonstrate a unique pattern of activity. To facilitate machine learning we curated a dataset comprising 33 features, encompassing 4 descriptors for each compound. Subsequently, an ensemble of machine learning techniques viz. Partial Least Squares (PLS) regression, k-Nearest Neighbors (kNN) regression, and Support Vector Machine (SVM) regression, was harnessed. Moreover, the assimilation of Principal Component Analysis (PCA) augmented precision and accuracy, thereby refining and demarcating diverse compound classes. As such, this classification is useful for discerning substrates amenable to diverse alcohol dehydrogenases, thereby mitigating the reliance on high-throughput screening or engineering in identifying the optimal enzyme for specific substrate. • New, complex molecules need fresh strategies beyond traditional enzyme libraries. The current study proposes a method based on consistent patterns in different enzymes. • Some ADHs are more sensitive to certain molecules than others, which the current methods may not fully capture. • To address this, deep learning with additional chemical information is proposed. Past research show using datasets helps understand molecule properties. The current study aims to predict new enzyme-substrate pairs for making pharmaceuticals. [ABSTRACT FROM AUTHOR]

Acinetobacter baumannii (A. baumannii) is a clinically relevant, highly drug-resistant pathogen of global concern. An attractive approach to drug design is to specifically target the type II fatty acid synthesis (FASII) pathway which is critical in Gram negative bacteria and is significantly different to the type I fatty acid synthesis (FASI) pathway found in mammals. Enzymes involved in FASII include members of the short-chain dehydrogenase/reductase (SDR) superfamily. SDRs are capable of performing a diverse range of biochemical reactions against a broad spectrum of substrates whilst maintaining conserved structural features and sequence motifs. Here, we use X-ray crystallography to describe the structure of an SDR from the multi-drug resistant bacteria A. baumannii, previously annotated as a putative FASII FabG enzyme. The protein was recombinantly expressed, purified, and crystallized. The protein crystals diffracted to 2.0 Å and the structure revealed a FabG-like fold. Functional assays revealed, however, that the protein was not active against the FabG substrate, acetoacetyl-CoA. This study highlights that database annotations may show the necessary structural hallmarks of such proteins, however, they may not be able to cleave substrates that are typical of FabG enzymes. These results are important for the selection of target enzymes in future drug development. • Structural characterization of an annotated FabG enzyme from A. baumannii. • Functional data shows the enzyme is not active against acetoacetyl-CoA substrate. • We suggest annotation as a putative short chain dehydrogenase/reductase. [ABSTRACT FROM AUTHOR]

The NAD(P)-dependent malate dehydrogenases (MalDHs) and NAD-dependent lactate dehydrogenases (LDHs) are homologous enzymes involved in central metabolism. They display a common protein fold and the same catalytic mechanism, yet have a stringent capacity to discriminate between their respective substrates. The MalDH/LDH superfamily is divided into several phylogenetically related groups. It has been shown that the canonical LDHs and LDH-like group of MalDHs are primarily tetrameric enzymes that diverged from a common ancestor. In order to gain understanding of the evolutionary history of the LDHs and MalDHs, the biochemical properties and crystallographic structure of the LDH-like MalDH from the hyperthermophilic archaeon Ignicoccus islandicus (I. isl) were determined. I. isl MalDH recognizes oxaloacetate as main substrate, but it is also able to use pyruvate. Surprisingly, with pyruvate, the enzymatic activity profile looks like that of allosteric LDHs, suggesting a hidden allosteric capacity in a MalDH. The I. isl MalDH tetrameric structure in the apo state is considerably different from those of canonical LDH-like MalDHs and LDHs, representing an alternative oligomeric organization. A comparison with MalDH and LDH counterparts provides strong evidence that the divergence between allosteric and non-allosteric members of the superfamily involves homologs with intermediate, atypical properties. [ABSTRACT FROM AUTHOR]

Recently, we demonstrated that the expression of 3β-hydroxysteroid dehydrogenase type 1 (HSD3B1) in breast cancer is associated with shorter recurrence-free survival, and genetic or pharmacologic inhibition of HSD3B1 reduced colony formation and xenograft growth. However, the mechanisms are unclear. Triple-negative MDA-MB-231 and BT-20 breast cancer cells underwent HSD3B1 silencing. Microarray and bioinformatic analysis were performed. The interleukin-6 (IL-6) expression and secretion were evaluated using real-time quantitative polymerase chain reaction and enzyme-linked immunosorbent assay. Clonogenic ability and cell viability were determined in the absence or presence of recombinant IL-6. Functional and pathway enrichment analyses showed that HSD3B1 silencing modulates the expression of several growth factors and cytokines. Cells transfected with HSD3B1-targeting small interfering RNA or treated with an HSD3B1 inhibitor (trilostane) had decreased IL-6 expression and secretion. HSD3B1 inhibition reduced colony formation, which was partially rescued by IL-6 supplementation. The HSD3B1 knockdown enhanced paclitaxel sensitivity, and IL-6 treatment partially reversed the augmented cytotoxicity. Our findings suggest that the therapeutic potential of targeting HSD3B1 is in part mediated by IL-6 suppression. [ABSTRACT FROM AUTHOR]

Fluorescent proteins, such as the green fluorescent protein, are used for detection of cellular components and events. However, green fluorescent protein and its derivatives have limited usage under anaerobic conditions and require a long maturation time. On the other hand, the NADPH-dependent blue fluorescent protein (BFP) without oxidative modification of residues is instantly functional in both aerobic and anaerobic systems. BFP proteins belong to a short-chain dehydrogenase/reductase (SDR) protein family, and their fluorescent property changes with reaction time in the presence of a substrate. With the aim of developing a better fluorescent reporter independent of redox state, we elucidated the crystal structure of a tetrameric mBFP from soil metagenomes with and without NADPH. Apart from the previously known regions, structure-guided mutational studies have identified several residues that contribute to the fluorescence of mBFP, including two aromatic residues (F97 and Y157) near the nicotinamide moiety of the bound NADPH. A single histidine mutation at Y157 (Y157H) has conferred more stabilized, time-independent fluorescence even in the presence of substrates. Furthermore, we discovered another SDR protein that can also emit blue fluorescence. These results open a new possibility for the development of BFP as a stable cellular reporter for widespread use, independent of subcellular environments. Unlabelled Image • mBFP is an NADPH-dependent blue fluorescent protein. • Its fluorescence changes by oxidizing or reducing putative substrates. • Structure-guided mBFP derivative emits a constant fluorescence with substrates. • mBFP can be used as a stable cellular reporter regardless of redox states. [ABSTRACT FROM AUTHOR]

Adipose tissue plays a role in energy storage and metabolic balance and is composed of different cell types. The metabolic activity of the tissue itself has been a matter of research for a long time, but comparative data about the energy metabolism of different cell types of human subcutaneous adipose tissue are sparse. Therefore, we compared the activity of major energy metabolic pathways of adipocytes and CD34+ cells from the stromal vascular fraction (SVF) separated from the same tissue. This CD34+ cell fraction is enriched with adipose tissue-derived mesenchymal progenitors, as they account for the largest proportion of CD34+ cells of the SVF. Adipocytes displayed significantly higher mitochondrial enzyme capacities compared to CD34+ SVF-cells, as shown by the higher activities of isocitrate dehydrogenase and ß-hydroxyacyl-CoA dehydrogenase. Inversely, the CD34+ SVF-cells showed higher capacities for cytosolic carbohydrate metabolism, represented by the activity of glycolysis and the pentose phosphate pathway. Thus, the CD34+ SVF-cells may ensure the provision of pentose phosphates and reduction equivalents for the replication of DNA during proliferation. The data indicate that these two cell fractions of the human adipose tissue vary in their metabolic configuration adapted to their physiological demands regarding proliferation and differentiation in vivo. [ABSTRACT FROM AUTHOR]

• Short-chain acyl-CoA dehydrogenases initiate the first enzymatic mitochondria β-oxidation. • The contribution of short-chain acyl-CoA dehydrogenase to the pathogenesis of M. oryzae. • The ER likely functions as an additional site for β-oxidation. • Mitochondrial, peroxisome, and ER β-oxidation pathways promote stress tolerance in M. oryzae. • Mitochondrial localized Scad (MoScad2) is essential for appressorium morphogenesis in M. oryzae. Short-chain acyl-CoA dehydrogenase (Scad) mediated β-oxidation serves as the fastest route for generating essential energies required to support the survival of organisms under stress or starvation. In this study, we identified three putative SCAD genes in the genome of the globally destructive rice blast pathogen Magnaporthe oryzae , named as MoSCAD1 , MoSCAD2 , and MoSCAD3. To elucidate their function, we deployed targeted gene deletion strategy to investigate individual and the combined influence of MoSCAD genes on growth, stress tolerance, conidiation and pathogenicity of the rice blast fungus. First, localization and co-localization results obtained from this study showed that MoScad1 localizes to the endoplasmic reticulum (ER), MoScad2 localizes exclusively to the mitochondria while MoScad3 partially localizes to the mitochondria and peroxisome at all developmental stages of M. oryzae. Results obtained from this investigation showed that the deletion of MoSCAD1 and MoSCAD2 caused a minimal but significant reduction in the growth of Δ Moscad1 and Δ Moscad2 strains, while, growth characteristics exhibited by the Δ Moscad3 strain was similar to the wild-type strain. Furthermore, we observed that deletion of MoSCAD2 resulted in drastic reduction in conidiation, delayed conidia germination, triggered the development of abnormal appressorium and suppressed host penetration and colonization efficiencies of the Δ Moscad1 strain. This study provides first material evidence confirming the possible existence of ER β-oxidation pathway in M. oryzae. We also infer that mitochondria β-oxidation rather than peroxisomal and ER β-oxidation play an essential role in the vegetative growth, conidiation, appressorial morphogenesis and progression of pathogenesis in M. oryzae. [ABSTRACT FROM AUTHOR]

Abstract A Pseudomonas sp. DNE-S1 (GenBank accession number MF803832), able to degrade DEP in a wide range of acid-base conditions, was isolated from landfill soil. The growth kinetics of DNE-S1 on DEP followed the inhibition model. Fe3+ could promote the degradation ability of DNE-S1 to DEP probably by over-expression of the gene phthalate dihydrodiol dehydrogenase (ophB) and phthalate dioxygenase ferredoxin reductase (ophA4). The degradation rate of DEP (500 mg L−1 at 12 h) increased by 14.5% in the presence of Fe3+. Cu2+, Zn2+, and Mn2+ showed an inhibiting effect on the degradation performance of the strain and could alter the cellular morphology, surface area and volume of DNE-S1. Three degradation intermediates, namely ethyl methyl phthalate (EMP), dimethyl phthalate (DMP), and phthalic acid (PA), were detected in the biodegradation of DEP, and the biochemical pathway of DEP degradation was proposed. This study provides new information on the biochemical pathways and the responsible genes involved in DEP degradation. Highlights • Pseudomonas sp. DNE-S1 was able to degrade DEP in a wide range of acid-base conditions. • Two phthalate degrading genes ophB and ophA4 were identified in this bacterium. • Cellular morphology, surface area and volume were altered upon exposure to trace metal. • Fe3+ could promote the DEP degradation of DNE-S1. [ABSTRACT FROM AUTHOR]

Abstract The novel isolated Rhizobium sp. S10 was identified as d -glucoside 3-dehydrogenase (G3DH) producing microbe. Therefore, the gene encoding for G3DH from Rhizobium sp. S10 was cloned and overexpressed in Escherichia coli strain JM109 as a soluble enzyme complex. The recombinant G3DH (rG3DH) was purified with relatively high specific activity of 38.54 U/mg compared to the previously characterized and cloned G3DHs. The purified rG3DH showed the highest activity at pH 7.0, 40 °C toward cellobiose. It can also oxidize a broad range of mono-disaccharides including saccharide derivatives. The glycosides oxidizing activity combined with chemical reaction, could produce d -gulose from lactitol via 3-ketolactitol. Highlights • Novel d -glucoside 3-dehydrogenase identified in Rhizobium sp. S10 • d -glucoside 3-dehydrogenasecloned in E. coli was expressed as heteromeric enzyme with 38.54 U/mg. • d -glucoside 3-dehydrogenase activity was highest at pH 7.0 and 40 °C toward cellobiose. • Recombinant d -glucoside 3-dehydrogenase showed over 95% conversion rate and suitable for keto-sugar production. [ABSTRACT FROM AUTHOR]

Abstract High hydrostatic pressure (HHP) is an interesting hurdle in minimal food processing that aims to synergistically combine different stresses to improve food microbiological safety and stability without compromising quality. For a proper understanding and design of hurdle technology, the cellular impact of the applied stresses on foodborne pathogens should be well-established. To study the mechanism of HHP-mediated cell injury and death, we screened for loss-of-function mutations in E. coli MG1655 that affected HHP sensitivity. More specifically, ca. 6000 random transposon insertion mutants were individually exposed to HHP, after which the phenotype of the most resistant or sensitive mutations was confirmed by de novo gene deletions in the parental strain. We found that disruption of rbsK , rbsR , hdfR and crl decreased HHP resistance, while disruption of sucC and sucD (encoding subunits of the succinyl-CoA synthetase) increased HHP resistance. More detailed study of the tricarboxylic acid cycle enzymes encoded by the sdhCDAB-sucABCD operon surprisingly showed that disruption of the sucA or sucB gene (encoding subunits of the 2-oxoglutarate dehydrogenase complex) notably decreased HHP survival. We also found that the increased HHP resistance of a Δ sucC and Δ sucD mutant was mediated by increased basal RpoS activity levels, although it did not correlate with their heat resistance. Our results reveal that compromising TCA cycle enzymes can profoundly affect HHP resistance in E. coli. Highlights • Disruption of rbsK , rbsR , hdfR and crl gene sensitized E. coli to HHP resistance. • Deletion of sucC/D and sucA/B increased and decreased HHP resistance, respectively. • Increased HHP resistance of Δ sucC/D mutants was mediated by increased RpoS activity. • HHP resistance of Δ sucA/B/C/D mutants did not correlate with their heat resistance. [ABSTRACT FROM AUTHOR]

The chlorophyll cycle is a metabolic pathway in plants and green algae that interconverts chlorophyll a and chlorophyll b. The chlorophyll a-to-b conversion is catalyzed by a Rieske-type oxygenase, chlorophyll(ide) a oxygenase, while the chlorophyll b-to-a conversion is catalyzed in two steps by chlorophyll b reductase (a short-chain dehydrogenase) and 7-hydroxymethyl chlorophyll a reductase (a flavoprotein). The level of chlorophyll(ide) a oxygenase is regulated in a feedback loop by the accumulation of chlorophyll b in plants. In contrast, chlorophyll b reductase levels are regulated by a pool of light-harvesting complexes (LHC) that are energetically uncoupled to the core complexes of photosystems. Experimental evidence suggests that LHC levels in plants are regulated by chlorophyll b biosynthesis. LHC levels, however, appear to be independent of chlorophyll b in green algae. Instead, Prasinophytes, a group of green algae, have a chlorophyllide a oxygenase protein possessing structural alterations that differ dramatically from the rest of the green algae, which appear to boost chlorophyll b biosynthesis in these organisms. Hypothetical scenarios for the evolution of the chlorophyll cycle are presented. [ABSTRACT FROM AUTHOR]

Abstract l -Histidine analysis is essential in physiological research and clinical applications because l -histidine concentrations in biofluids are associated with various diseases. However, an enzymatic method for l -histidine quantitation has not yet been established. Here, we describe a novel l -histidine quantitation assay using a combination of histidine decarboxylase (HDC) and histamine dehydrogenase (HDH) enzymes. Wild-type HDC is unstable and completely lost its activity within 50 days of storage at 4 °C in solution. We rationally designed a HDC C57S mutant with markedly improved stability (storage at 4 °C for over 200 days) without altering the enzyme's substrate specificity. Together with HDH, the HDC C57S mutant was applied to quantify l -histidine concentrations in human plasma. The assay showed high precision (<2.0% inter-assay variation) and high accuracy (<5.8% deviation from the results of LC/MS). Graphical abstract Image 1 Highlights • A novel colorimetric assay for quantitating l -histidine using two types of enzymes is proposed. • A rationally designed l -histidine decarboxylase C57S mutant was stable for over 200 days. • Using this assay, l -histidine can be quantitated in human plasma with high precision and accuracy. [ABSTRACT FROM AUTHOR]

Abstract Increasing the availability of NADPH is commonly used to improve lysine production by Corynebacterium glutamicum since 4 mol of NADPH are required for the synthesis of 1 mol of lysine. Alternatively, engineering of enzymes in lysine synthesis pathway to utilize NADH directly can also be explored for cofactor balance during lysine overproduction. To achieve such a goal, enzyme mining was used in this study to quickly identify a full set of NADH-utilizing dehydrogenases, namely aspartate dehydrogenase from Pseudomonas aeruginosa (PaASPDH), aspartate-semialdehyde dehydrogenase from Tistrella mobilis (TmASADH), dihydrodipicolinate reductase from Escherichia coli (EcDHDPR), and diaminopimelate dehydrogenase from Pseudothermotoga thermarum (PtDAPDH). This allowed us to systematically perturb cofactor utilization of lysine synthesis pathway of C. glutamicum for the first time. Individual overexpression of PaASPDH, TmASADH, EcDHDPR, and PtDAPDH in C. glutamicum LC298, a basic lysine producer, increased the production of lysine by 30.7%, 32.4%, 17.4%, and 36.8%, respectively. Combinatorial replacement of NADPH-dependent dehydrogenases in C. glutamicum ATCC 21543, a lysine hyperproducer, also resulted in significantly improved lysine production. The highest increase of lysine production (30.7%) was observed for a triple-mutant strain (27.7 g/L, 0.35 g/g glucose) expressing PaASPDH, TmASADH, and EcDHDPR. A quadruple-mutant strain expressing all of the four NADH-utilizing enzymes allowed high lysine production (24.1 g/L, 0.30 g/g glucose) almost independent of the oxidative pentose phosphate pathway. Collectively, our results demonstrated that a combination of enzyme mining and cofactor engineering was a highly efficient approach to improve lysine production. Similar strategies can be applied for the production of other amino acids or their derivatives. Highlights • NADH-utilizing dehydrogenases were identified by enzyme mining for cofactor perturbation of lysine synthesis pathways. • Individual overexpression of any NADH-utilizing dehydrogenase increased lysine production. • Simultaneous replacement of three native enzymes by NADH-utilizing counterparts significantly improved lysine production. • A fully NADH-utilizing lysine synthesis pathway was constructed, building the basis for anaerobic lysine fermentation. [ABSTRACT FROM AUTHOR]

Abstract Succinate-CoA ligase (SUCL) is a heterodimer consisting of an alpha subunit encoded by SUCLG1 , and a beta subunit encoded by either SUCLA2 or SUCLG2 catalyzing an ATP- or GTP-forming reaction, respectively, in the mitochondrial matrix. The deficiency of this enzyme represents an encephalomyopathic form of mtDNA depletion syndromes. We describe the fatal clinical course of a female patient with a pathogenic mutation in SUCLG1 (c.626C > A, p.Ala209Glu) heterozygous at the genomic DNA level, but homozygous at the transcriptional level. The patient exhibited early-onset neurometabolic abnormality culminating in severe brain atrophy and dystonia leading to death by the age of 3.5 years. Urine and plasma metabolite profiling was consistent with SUCL deficiency which was confirmed by enzyme analysis and lack of mitochondrial substrate-level phosphorylation (mSLP) in skin fibroblasts. Oxygen consumption- but not extracellular acidification rates were altered only when using glutamine as a substrate, and this was associated with mild mtDNA depletion and no changes in ETC activities. Immunoblot analysis revealed no detectable levels of SUCLG1, while SUCLA2 and SUCLG2 protein expressions were largely reduced. Confocal imaging of triple immunocytochemistry of skin fibroblasts showed that SUCLG2 co-localized only partially with the mitochondrial network which otherwise exhibited an increase in fragmentation compared to control cells. Our results outline the catastrophic consequences of the mutated SUCLG1 leading to strongly reduced SUCL activity, mSLP impairment, mislocalization of SUCLG2, morphological alterations in mitochondria and clinically to a severe neurometabolic disease, but in the absence of changes in mtDNA levels or respiratory complex activities. [ABSTRACT FROM AUTHOR]

Abstract The most important mode of enzyme inactivation is thermal inactivation. Immobilization technology is an efficient approach to elongate the life-time of enzymes. d -lactate dehydrogenase (D-LDH) was stabilized at high temperatures with immobilization on CNT and fCNT. The kinetic and thermodynamic parameters, optimum temperature and pH, and the intrinsic fluorescence of free and immobilized enzymes were examined in the present study. Also, an attempt was made to investigate the effect of CNT and fCNT on the adsorption and conformation of d -lactate dehydrogenase using molecular dynamics (MD) simulations. In comparison with free enzyme, the immobilized enzyme displayed an improved stability at high temperatures and, therefore, the immobilized enzyme is suitable for use in the industry because most reactions in the industry happen at high temperatures. Results of the present study showed that the adsorption of enzyme on CNT is mediated through the van der Waals and π-π stacking interactions, whereas in the adsorption of enzyme on fCNT in addition to hydrophobic interactions, the hydrogen bonding between enzyme and functional groups of fCNT is involved. Moreover, RMSD, RMSF and secondary structure analysis indicate that the fCNT protects the conformation of enzyme more than CNT. Therefore, D-LDH can be efficiently immobilized upon the fCNT compared to the pristine CNT. Graphical abstract Image 1 Highlights • Immobilization of D-LDH enzyme on pristine and functionalized CNT are examined by experimental and theoretical methods. • The immobilization of the D-LDH upon CNT and fCNT increased the enzymatic catalytic efficiency. • Thermodynamic parameters show the high thermo-stability of immobilized enzyme during the heat inactivation. • RMSD, RMSF and secondary structure analysis indicate that the fCNT protects the conformation of enzyme more than CNT. [ABSTRACT FROM AUTHOR]

Abstract Invariant natural killer T (iNKT) cells exhibit potent antitumor effects upon activation by recognizing a specific glycolipid antigen. We previously performed phase I–II clinical studies to utilize iNKT cells using α-galactosylceramide-pulsed dendritic cells and identified leukotriene B4 12-hydroxydehydrogenase (LTB4DH) as a biomarker highly expressed in T cells derived from non-small cell lung cancer (NSCLC) patients who showed prolonged survival in respond to the iNKT cell immunotherapy. Because LTB4DH expression correlated with prolonged survival of NSCLC patients, we considered LTB4DH to play a role in iNKT cell immunotherapy. We herein demonstrate that the overexpression of LTB4DH in CD4+ or CD8+ T cells increases interferon-γ production and tumoricidal activity in the presence of prostaglandin E 2. Moreover, the expression of granzyme a , granzyme b , and perforin mRNA was increased in LTB4DH-overexpressing cells. Highlights • LTB4DH in CD4+ T cells rescued IFN-γ production with PGE 2. • LTB4DH in CD8+ T cells rescued tumoricidal activity with PGE 2. • LTB4DH in CD8+ T cells increased the expression of cytotoxic molecules with PGE 2. [ABSTRACT FROM AUTHOR]

Abstract Catabolism of the branched-chain amino acids (BCAAs: leucine, isoleucine, and valine) is regulated by the branched-chain α-ketoacid dehydrogenase (BCKDH) complex, which in turn is regulated by phosphorylation catalyzed by BCKDH kinase (BDK). Thiamine pyrophosphate (TPP) is required as a coenzyme for the E1 component of the BCKDH complex and can also bring about activation of the complex by inhibiting BDK. The present study shows that free Ca2+ in the physiological range greatly increases the sensitivity of BDK to inhibition by TPP (IC 50 of 2.5 μM in the presence of 1 μM free Ca2+). This novel mechanism may be responsible for the stimulation of BCAA oxidation by conditions that increase mitochondrial free Ca2+ levels, e.g. in skeletal muscle during exercise. [ABSTRACT FROM AUTHOR]

Graphical abstract Highlights • Bulk MoS 2 is demonstrated as an active catalyst for ethanol to acetates. • A high surface area co-precipitated sample shows excellent activity. • High specific activity is correlated with low S- to Mo-edge ratio from CO DRIFTS. • Acetaldehyde is dominant at low conversion and may form at CUS on the Mo edge. Abstract Gas phase acceptorless dehydrogenative coupling (ADC) of ethanol to ethyl acetate is investigated over bulk MoS 2. Yields of up to 56% are achieved at gas hourly space velocity of 1700 h−1 and 260 °C using a high surface area co-precipitated MoS 2 material. Addition of water to the ethanol feed decreases overall conversion but increases selectivity to acetic acid. After a short activation period, the catalyst is stable over reaction times exceeding 13 h. Diffuse reflectance infrared Fourier transform spectroscopy (DRIFTS) with CO as a probe molecule and X-ray absorption spectroscopy (XAS) are used to measure structural disorder within MoS 2 samples. Samples with larger amounts of structural disorder are demonstrated to have enhanced dehydrogenation activity per total surface area. [ABSTRACT FROM AUTHOR]

Certain agricultural practices, such as the repeated application of herbicides or organic amendments to soil, can influence herbicide dissipation. This research has studied the effects of two repeated applications of mesotrione, pethoxamid, and triasulfuron on their dissipation rates in unamended soil (S) and soil amended with green compost (S+GC) or sewage sludge (S+SS). The dissipation experiment has also included an evaluation of the adsorption of the three herbicides by soils and of changes in soil dehydrogenase activity (DHA). The adsorption of the three herbicides by amended soils (Kf range 0.83–2.98) was higher than by unamended soil (Kf range 0.20–0.64). The adsorption coefficients (Kd) of mesotrione and triasulfuron were higher for S+SS, while that of pethoxamid was higher for S+GC, but no relationship between values for the time to 50% degradation (DT 50 ) and adsorption coefficients could be determined. The repeated application of mesotrione decreased its dissipation rate in unamended soil (DT 50 increased from 4.75 to 8.15 days) and amended soils (DT 50 increased from 11.7 to 28.2 days in S+GC and from 17.7 to 37.9 in S+SS), whereas the repeated application of pethoxamid increased its dissipation rate in all the treatments, and the rate for triasulfuron increased only in amended soils. The highest DT 50 values for pethoxamid (12.3 days) and triasulfuron (57.1 days) were in S+GC, and the lowest in S+SS (8.35 and 24.7 days). Soil DHA was stimulated by the presence of GC in the soil and by the first application of mesotrione. The second application of mesotrione and pethoxamid positively affected soil DHA, but this did not occur for triasulfuron. The repeated applications of herbicides and soil organic amendments have different effects on herbicide dissipation, adsorption, and soil DHA, and they should be taken into account when assessing soil quality and other potential environmental implications of pesticide use. [ABSTRACT FROM AUTHOR]

Acute lung injury (ALI) is an acute failure of the respiratory system with unacceptably high mortality, for which effective treatment is urgently necessary. Infiltrations by immune cells, such as leukocytes and macrophages, are responsible for the inflammatory response in ALI, which is characterized by excessive production of pro-inflammatory mediators in lung tissues exposed to various pathogen-associated molecules such as lipopolysaccharide (LPS) from microbial organisms. α-Ketoglutarate (α-KG) is a key metabolic intermediate and acts as a pro-inflammatory metabolite, which is responsible for LPS-induced proinflammatory cytokine production through NF-κB signaling pathway. Mitochondrial NADP + -dependent isocitrate dehydrogenase (IDH2) has been reported as an essential enzyme catalyzing the conversion of isocitrate to α-KG with concurrent production of NAPDH. Therefore, we evaluated the role of IDH2 in LPS-induced ALI using IDH2-deficient mice. We observed that LPS-induced inflammation and lung injury is attenuated in IDH2-deficient mice, leading to a lengthened life span of the mice. Our results also suggest that IDH2 disruption suppresses LPS-induced proinflammatory cytokine production, resulting from an inhibition of the NF-κB signaling axis in an α-KG-dependent manner. In conclusion, disruption of IDH2 leads to a decrease in α-KG levels, and the activation of NF-κB in response to LPS is attenuated by reduction of α-KG levels, which eventually reduces the inflammatory response in the lung during LPS-induced ALI. The present study supports the rationale for targeting IDH2 as an important therapeutic strategy for the treatment of systemic inflammatory response syndromes, particularly ALI. [ABSTRACT FROM AUTHOR]

Parasitic nematodes infect more than two billion people worldwide particularly in developing countries. We previously reported nematicidal activity of natural honey using model nematode Caenorhabditis elegans. In this study, characterization of nematicidal effects of natural honey and its glycoproteins has been carried out. Chromatographically separated honey glycoproteins showed potent anti- C. elegans activity (LD 50  = 100 ng proteins/μL). Honey glycoproteins with molecular masses of ∼260 kD and ∼160 kD comprised of ‘major royal jelly protein-1’-containing complexes. In these complexes, MRJP1 was present in different glycosylation forms. Quantitative PCR based gene expression assays described molecular functions of C. elegans affected by honey and honey glycoproteins. Expression of 14 gene transcripts associated with key cellular and molecular functions including energy metabolism, cytoskeleton, cell division, transcription and translation was analyzed. Acacia honey exerted a concentration-dependent alteration of gene transcripts involved in the citric acid cycle ( mdh-1 and idhg-1 ) and cytoskeleton ( act-1 , act-2, and arp6 ). Likewise, MRJP1-containing glycoproteins caused down-regulation of arp-6 and idhg-1 ; and up-regulation of act-1 and mdh-1 gene transcripts. Consistent down-regulation of isocitrate dehydrogenase encoding idhg-1 gene which is among the rate-controlling enzymes of the citric acid cycle was considered as main biochemical factor involved in the nematicidal activity of honey and MRJP-containing glycoproteins. Acacia honey suppressed the expression of gene transcripts encoding actin-2, while honey glycoproteins did not. Hence, honey partly exerted anti- C. elegans activity by decreasing the transcription of actin-2 gene transcripts, demonstrated by a defect in the movement and egg laying. Moreover, arp-6 gene transcripts encoding actin-related protein 6 was significantly and constantly down-regulated by honey and honey proteins. [ABSTRACT FROM AUTHOR]

Glu-267 is highly conserved in alcohol dehydrogenases and buried as a negatively-charged residue in a loop of the NAD coenzyme binding domain. Glu-267 might have a structural role and contribute to a rate-promoting vibration that facilitates catalysis. Substitutions of Glu-267 with histidine or asparagine residues increase the dissociation constants for the coenzymes (NAD + by ∼40-fold, NADH by ∼200-fold) and significantly decrease catalytic efficiencies by 16–1200-fold various substrates and substituted enzymes. The turnover numbers modestly change with the substitutions, but hydride transfer is at least partially rate-limiting for turnover for alcohol oxidation. X-ray structures of the E267H and E267 N enzymes are similar to the apoenzyme (open) conformation of the wild-type enzyme, and the substitutions are accommodated by local changes in the structure. Surprisingly, the E267H and E267 N enzymes have endogenous (from the expression in E. coli ) 3′-dephosphocoenzyme A bound in the active site with the ADP moiety in the NAD binding site and the pantethiene sulfhydryl bound to the catalytic zinc. The kinetics and crystallography show that the substitutions of Glu-267 hinder the conformational change, which occurs when wild-type enzyme binds coenzymes, and affect productive binding of substrates. [ABSTRACT FROM AUTHOR]

Glutamate is a key metabolite and major excitatory neurotransmitter, degraded through transformation to 2-oxoglutarate which is further catabolized by 2-oxoglutarate dehydrogenase complex (OGDHC). Both the glutamate excitotoxicity and impaired OGDHC activity are hallmarks of neurodegeneration. This work quantifies a relationship between the brain OGDHC activity and glutamate levels, assessing its diagnostic value to characterize (patho)physiology. A moderate to strong positive correlation of the two parameters determined under varied physiological settings (brain regions, seasons, gender, pregnancy, rat line), is revealed. Mitochondrial impairment (OGDHC inhibition or acute hypobaric hypoxia) decreases the interdependence, even when the parameter means do not change significantly. Compared to the cortex, the cerebellum exhibits a lower inter-individual glutamate variation and a weaker glutamate-OGDHC interdependence. Specific metabolism of the brain regions is also characterized by a positive correlation between glutamate and γ-aminobutyric acid (GABA) concentrations in the cortex but not in the cerebellum. In contrast, a strong positive correlation between glutamate and glutamine is present in both the cortex and cerebellum. The differences in metabolic correlations are in line with transcriptomics data which suggest that glutamate distribution between competitive pathways contributes to the brain-region-specific features of the interdependences of glutamate and OGDHC or GABA. [ABSTRACT FROM AUTHOR]

The feasibility of medical stone (MS) amendment as an innovative additive for dewatered fresh sewage sludge (DFSS) co-composting was assessed using a 130-L vessel-scale composter. To verify successful composting, five treatments were designed with four different dosages (2, 4, 6, and 10) % of MS with a 1:1 mixture (dry weight) of DFSS + wheat straw (WS). The WS was used as a bulking agent. A control without any amendment treatment was carried out for the purpose of comparison. For DFSS co-composting, the amendment with MS improved the mineralization efficiency and compost quality in terms of CO 2 emissions, dehydrogenase enzyme (DE), electrical conductivity (EC), water-solubility, and total nutrients transformation. The DTPA-extractable Cu and Zn were also estimated to confirm the immobilization ability of the applied MS. Seed germination and plant growth tests were conducted to ensure the compost stability and phytotoxicity for Chinese cabbage ( Brassica rapa chinensis L.) growth and biomass, as well as chlorophyll content. The results showed that during the bio-oxidative phase, DOC, DON, AP, NH 4 + -N, and NO 3 − -N increased drastically in all the MS-blended treatments, except the application of 2% MS and the control treatment; significantly lower water-soluble nutrients were observed in the 2% MS and control treatments. A novel additive with 6–10% MS dosages considerably enhanced the organic matter conversion in the stable end-product (compost) and reduced the maturity period by two weeks compared to the 2% MS and control treatments. Consequently, the maturity parameters (e.g., EC, SGI, NH 4 + -N, DOC, and DON) confirmed that compost with 6–10% MS became more stable and mature within four weeks of DFSS co-composting. At the end of composting, significantly higher DTPA-extractable Cu and Zn contents were observed in the control treatment, and subsequently, in the very low application (10%) of MS. Higher MS dosage lowered the pH and EC to within the permissible limit compared to the control, while increased concentrations of water-soluble nutrients diminished the DTPA-extractable Cu and Zn contents. In addition, plant growth experiments demonstrated that the addition of compost with 150 kg ha −1 TKN improved the Chinese cabbage biomass and chlorophyll level. The highest dry weight biomass (2.78 ± 0.02 g/pot) was obtained with 6% MS-blended compost while the maximum chlorophyll content was found with application of 4% MS compost (41.84 SPAD-unit) for Chinese cabbage. Therefore, 6–10% MS can be recommended to improve DFSS composting and to reduce the period to maturity by two weeks when considering its composting effect on Chinese cabbage growth, biomass yield, and chlorophyll level. However, amendment with 6% MS is a more economically feasible approach for DFSS co-composting. [ABSTRACT FROM AUTHOR]

Abstract Diaphorases are flavin-containing enzymes with potential applications in biotransfomation reactions, biosensor design and in vitro diagnostic tests. In this paper, we present recombinant expression, characterization and medium optimization of a lipoamide dehydrogenase (DLD) with NADH-dependent diaphorase activity from a Lysinibacillus sp. strain. DLD encoding sequence showed an open reading frame of 1413-bp encoding a 470 amino acid chain. Lysinibacillus sp. DLD catalyzed the NADH-dependent reduction of electron acceptors and exhibited diaphorase activity. The molecular mass of the isolated enzyme was found to be about 50 kDa, and determined to be a monomeric protein. The optimum pH and temperature for the catalytic activity of the enzyme was about pH 7.5 and 30 °C. The K m and V max values were estimated to be 0.025 mM and 1.33 μmol/min, respectively. Recombinant enzyme was optimally produced in fermentation medium containing 10 g/L sucrose, 25 g/L yeast extract, 5 g/L NaCl and 0.25 g/L MgSO 4. By Scaling up fermentation from flask to bioreactor, enzyme activity was increased to 487.5 U/ml. This study provides data on the identification, characterization and medium optimization of a NADH-dependent diaphorase from a newly isolated Lysinibacillus sp. PAD-91. Graphical abstract Image 1 Highlights • Identification of a NADH-dependent diaphorase from Lysinibacillus sp. PAD-91. • K m value for NADH (0.025 mM) was lower than other previously reported diaphorases. • Medium optimization for recombinant enzyme production was performed using RSM. • By Scaling up fermentation, enzyme activity was increased to 487.5 U/ml. [ABSTRACT FROM AUTHOR]

During research to identify fibronectin (Fn)-binding proteins (Fbps) on the surface of Clostridium perfringens cells, we identified glyceraldehyde-3-phosphate dehydrogenase (GAPDH) as a candidate Fbp. GAPDH is a glycolytic enzyme found in a wide range of prokaryotes and eukaryotes. The Fn-binding activity of recombinant C. perfringens GAPDH (rGAPDH) was investigated using both ligand blotting analysis and enzyme-linked immunosorbent assay (ELISA). rGAPDH strongly bound plasminogen but not laminin or gelatin. Although GAPDH has no signal sequence, it is expressed on the cell surface of many microorganisms. The presence of GAPDH on the surface of C. perfringens cells was analyzed using ELISA and flow cytometry analyses; purified rGAPDH bound to the surface of C. perfringens cells. As autolysin is reportedly involved in the binding of GAPDH to the cell surface, we evaluated the interaction between rGAPDH and the C. perfringens autolysin Acp by both ELISA and ligand blotting assay. These assays revealed that rGAPDH binds to the catalytic domain of Acp but not the cell wall binding domains. These results suggest that autolysin mediates expression of GAPDH on the surface of C. perfringens cells and indicate a possible moonlighting function for GAPDH in binding both Fn and plasminogen. [ABSTRACT FROM AUTHOR]

Sewage sludge composting is a profitable process economically viable and environmentally friendly. In despite of there are several kind of composting types, the use of combined system of semipermeable cover film and aeration air-floor is widely developed at industrial scale. However, the knowledge of the linkages between microbial communities structure, enzyme activities and physico-chemical factors under these conditions it has been poorly explored. Thus, the aim of this study was to investigate the bacterial dynamic and community structure using next generation sequencing coupled to analyses of microbial enzymatic activity and culturable dependent techniques in a full-scale real composting plant. Sewage sludge composting process was conducted using a semi-permeable Gore-tex cover, in combination with an air-insufflation system. The highest values of enzymatic activities such as dehydrogenase, protease and arylsulphatase were detected in the first 5 days of composting; suggesting that during this period of time a greater degrading activity of organic matter took place. Culturable bacteria identified were in agreement with the bacteria found by massive sequencing technologies. The greatest bacterial diversity was detected between days 15 and 30, with Actinomycetales and Bacillales being the predominant orders at the beginning and end of the process. Bacillus was the most representative genus during all the process. A strong correlation between abiotic factors as total organic content and organic matter and enzymatic activities such as dehydrogenase, alkaline phosphatase, and ß-glucosidase activity was found. Bacterial diversity was strongly influenced by the stage of the process, community-structure change was concomitant with a temperature rise, rendering favorable conditions to stimulate microbial activity and facilitate the change in the microbial community linked to the degradation process. Moreover, results obtained confirmed that the use of semipermeable cover in the composting of sewage sludge allow a noticeable reduction in the process-time comparing to conventional open windrows. [ABSTRACT FROM AUTHOR]

Biofilm dispersal is characterized by the cell detachment from biofilms and expected to provide novel “anti-biofilm” approaches of prevention and treatment of biofilms in clinical and industrial settings. The E.coli protein BdcA has been identified as a biofilm dispersal factor and designed to be an important component in engineered applications to control biofilm formation. It belongs to short-chain dehydrogenase/reductase (SDR) family with the specific affinity to NADPH. Here, we show the structure of BdcA in complex with NADPH and confirm that NADPH binding is requisite for BdcA facilitating cell motility and increasing biofilm dispersal. Especially, we observe a potential substrate binding pocket surrounded by hydrophobic residues upon NADPH binding and present evidences that this pocket is essential for BdcA binding NADPH and exerting its biological functions. Our study provides the clues for illuminating the molecular mechanism of BdcA regulating biofilm dispersal and better utilizing BdcA to eliminate the biofilms. [ABSTRACT FROM AUTHOR]

Background Nintedanib is a potent intracellular inhibitor of tyrosine kinases, including the receptor kinases vascular endothelial growth factor, platelet-derived growth factor and fibroblast growth factor. A previous model assessed the population pharmacokinetics of nintedanib and its main metabolite BIBF 1202 in patients with non-small cell lung cancer and idiopathic pulmonary fibrosis (IPF). The objective of this analysis was to further characterise the population pharmacokinetics of nintedanib in patients with IPF by including data from the Phase III trials. Methods We pooled data from 933 patients with IPF participating in the Phase II TOMORROW trial and the two Phase III INPULSIS trials. Plasma concentrations of nintedanib (n = 3501) were analysed using nonlinear mixed-effects modelling. Results Pharmacokinetics of nintedanib was described by a one-compartment model with linear elimination, first-order absorption and an absorption lag time. The population estimates of absorption rate, lag time, apparent total clearance and apparent volume of distribution at steady state for a typical IPF patient were 0.0814 h −1 , 0.689 h, 994 L/h, and 265 L. The model confirmed age, body weight, smoking and Asian race (with different effect sizes in different Asian subpopulations) as statistically significant covariates influencing nintedanib exposure. Serum lactate dehydrogenase levels were identified as another factor significantly influencing nintedanib plasma concentrations. No individual covariate at extreme values (5th and 95th percentiles of baseline for continuous covariates) resulted in changes in exposure of more than 50% relative to a typical patient. Conclusions The developed model provides further details about the pharmacokinetics of nintedanib in patients with IPF and can be used for simulations exploring covariate effects and exposure-response analyses in this patient population. [ABSTRACT FROM AUTHOR]

The single most common cause of chronic pancreatitis (CP, a serious inflammatory disease) is chronic alcohol abuse, which impairs hepatic alcohol dehydrogenase (ADH, a major ethanol oxidizing enzyme). Previously, we found ~ 5 fold greater fatty acid ethyl esters (FAEEs), and injury in the pancreas of hepatic ADH deficient (ADH − ) vs. hepatic normal ADH (ADH + ) deer mice fed 3.5 g% ethanol via liquid diet daily for two months. Therefore, progression of ethanol-induced pancreatic injury was determined in ADH − deer mice fed ethanol for four months to delineate the mechanism and metabolic basis of alcoholic chronic pancreatitis (ACP). In addition to a substantially increased blood alcohol concentration and plasma FAEEs, significant degenerative changes, including atrophy and loss of acinar cells in some areas, ultrastructural changes evident by such features as swelling and disintegration of endoplasmic reticulum (ER) cisternae and ER stress were observed in the pancreas of ethanol-fed ADH − deer mice vs. ADH + deer mice. These changes are consistent with noted increases in pancreatic injury markers (plasma lipase, pancreatic trypsinogen activation peptide, FAEE synthase and cathepsin B) in ethanol-fed ADH − deer mice. Most importantly, an increased levels of pancreatic glucose regulated protein (GRP) 78 (a prominent ER stress marker) were found to be closely associated with increased phosphorylated eukaryotic initiation factor (eIF) 2α signaling molecule in PKR-like ER kinase branch of unfolded protein response (UPR) as compared to X box binding protein 1S and activating transcription factor (ATF)6 - 50 kDa protein of inositol requiring enzyme 1α and ATF6 branches of UPR, respectively, in ethanol-fed ADH − vs. ADH + deer mice. These results along with findings on plasma FAEEs, and pancreatic histology and injury markers suggest a metabolic basis of ethanol-induced pancreatic injury, and provide new avenues to understand metabolic basis and molecular mechanism of ACP. [ABSTRACT FROM AUTHOR]

The NAD + -dependent glycerol 3-phosphate dehydrogenase (KlGpd1) is an important enzyme for maintenance of the cytosolic redox balance in the milk yeast Kluyveromyces lactis . The enzyme is localized in peroxisomes and in the cytosol, indicating its requirement for the oxidation of NADH in both compartments. Klgpd1 mutants grow more slowly on glucose than wild-type cells and do not grow on ethanol as a sole carbon source. We studied the molecular basis of the latter phenotype and found that Gpd1 is required for high expression of KlICL1 and KlMLS1 which encode the key enzymes of the glyoxylate pathway isocitrate lyase and malate synthase, respectively. This regulation is mediated by CSRE elements in the promoters of these genes and the Snf1-regulated transcription factors KlCat8 and KlSip4. To study the transactivation function of these factors we developed a modified yeast one-hybrid system for K. lactis, using the endogenous ß-galactosidase gene LAC4 as a reporter in a lac9 deletion background. In combination with ChIP analyses we discovered that Gpd1 controls both the specific binding of Cat8 and Sip4 to the target promoters and the capacity of these factors to activate the reporter gene expression. We propose a model in which KlGpd1 activity is required for maintenance of the redox balance. In its absence, genes which function in generating redox balance instabilities are not expressed. A comparison of mutant phenotypes further indicates, that this system not only operates in K. lactis , but also in Saccharomyces cerevisiae . [ABSTRACT FROM AUTHOR]

The prohormone, dehydroepiandrosterone (DHEA) circulates in vertebrate blood with the potential for actions on target tissues including the central nervous system (CNS). Many actions of DHEA require its conversion into more active products, some of which are catalyzed by the enzyme 3β-hydroxysteroid-dehydrogenase/isomerase (3β-HSD). Studies of birds show both expression and activity of 3β-HSD in brain and its importance in regulating social behavior. In oscine songbirds, 3β-HSD is expressed at reasonably high levels in brain, possibly linked to their complex neural circuitry controlling song. Studies also indicate that circulating DHEA may serve as the substrate for neural 3β-HSD to produce active steroids that activate behavior during non-breeding seasons. In the golden-collared manakin ( Manacus vitellinus ), a sub-oscine bird, low levels of courtship behavior are displayed by males when circulating testosterone levels are basal. Therefore, we asked whether DHEA circulates in blood of manakins and whether the brain expresses 3β-HSD mRNA. Given that the spinal cord is a target of androgens and likely important in regulating acrobatic movements, we also examined expression of this enzyme in the manakin spinal cord. For comparison, we examined expression levels with those of an oscine songbird, the zebra finch ( Taeniopygia guttata ), a species in which brain, but not spinal cord, 3β-HSD has been well studied. DHEA was detected in manakin blood at levels similar to that seen in other species. As described previously, 3β-HSD was expressed in all zebra finch brain regions examined. By contrast, expression of 3β-HSD was only detected in the manakin hypothalamus where levels were greater than zebra finches. In spinal cord, 3β-HSD was detected in some but not all regions in both species. These data point to species differences and indicate that manakins have the substrate and neural machinery to convert circulating DHEA into potentially active androgens and/or estrogens. [ABSTRACT FROM AUTHOR]

To study the change of the aerobic granules' microbial community in the present of antibiotics, ampicillin (AMP) was selected as a model component. With acetate as carbon source, different concentrations of AMP (5, 10 and 15 mg L −1 ) were applied to the inflow intermittently and the results showed that the stability of the aerobic granules was maintained below 10 mg L −1 AMP. Simultaneously, under exposure to 5 and 10 mg L −1 AMP, the COD removal efficiency in the batch reactors remained at 86% and AMP was degraded almost completely with a removal efficiency of 97%. However, the EPS concentration and dehydrogenase activity decreased constantly with increasing AMP dosage. High-throughput sequencing analysis revealed that Proteobacteria was the most prominent phylum in the whole experiment and contributed to the degradation of AMP. The percentages of Azoarcus and Mycoplana increased at 10 mg L −1 AMP. In addition, Hydrogenophaga and Enterococcus played a key role in the microbial metabolism. [ABSTRACT FROM AUTHOR]

Small heat shock proteins (sHsps) are ubiquitous and diverse molecular chaperones. Found in almost all organisms, they regulate protein refolding and protect cells from stress. Until now, no sHsp has been characterized in Eimeria tenella . In this study, the novel EtsHsp20.4 gene was cloned from E. tenella by rapid amplification of cDNA ends based on a previously identified expressed sequence tag. The full-length cDNA was 1019bp in length and contained an open reading frame of 558bp that encoded a 185-amino acid polypeptide with a calculated molecular weight of 20.4 kDa. The Et sHsp20.4 protein contained a distinct HSP20/alpha-crystallin domain that is the key determinant of their function as molecular chaperones and belongs to the HSP20 protein family. EtsHsp20.4 mRNA levels were higher in sporulated oocysts than in sporozoites or second-generation merozoites by real-time quantitative PCR, the transcription of EtsHsp20.4 was barely detectable in unsporulated oocysts. Immunolocalization with Et sHsp20.4 antibody showed that Et sHsp20.4 was mainly located on the surface of sporozoites, first-generation merozoites and second-generation merozoites. Following the development of parasites in DF-1 cells, Et sHsp20.4 protein was uniformly dispersed in trophozoites, immature schizonts, and mature schizonts. Malate dehydrogenase thermal aggregation assays indicated that recombinant Et sHsp20.4 had molecular chaperone activity in vitro . These results suggested that Et sHsp20.4 might be involved in sporulation in external environments and intracellular growth of the parasite in the host. [ABSTRACT FROM AUTHOR]

Phenylalanine dehydrogenase (PheDH) is a key enzyme in medical diagnostic for determining the amount of phenylalanine to detect phenylketonuria (PKU) disease. However, determination of phenylalanine can be usually disturbed in presence of tyrosine in blood samples. Position N145 of B.sphaericus PheDH, has been previously showed a crucial role in substrate binding, which corresponded by position V144 in B. badius PheDH. In this study, the PheDH of B. badius due to reasonable activity was cloned and subjected to site-directed mutagenesis at mentioned position, followed by kinetic and structural studies to find more exclusive mutants. The results showed that the V 144 L mutant considerably increases specificity toward phenylalanine and decreases toward l -tyrosine, while in V 144 N mutant, the specificity reduces toward phenylalanine and increases toward tyrosine. Moreover, concerning the mutated V 144 D, significantly reduced k cat and also decreased k m value for phenylalanine relative to that of wild type. The Phe/Tyr specificity constant in V 144 L increased more than 4-fold compared to wild type, makes it to be a suitable candidate for more specific identification of PKU. Finally, docking and molecular dynamic simulation on wild type and mutants clarified the structural basis behind more specificity of V 144 L mutant for phenylalanine substrate. [ABSTRACT FROM AUTHOR]

Aluminum phosphide (AlP) poisoning is a severe toxicity with 30–70% mortality rate. However, several case reports presented AlP-poisoned patients with glucose-6-phosphate dehydrogenase (G6PD) deficiency and extensive hemolysis who survived the toxicity. This brought to our mind that maybe G6PD deficiency could protect the patients from severe fatal poisoning by this pesticide. In this research, we investigated the protective effect of 6-aminonicotinamide (6-AN)- as a well-established inhibitor of the NADP + - dependent enzyme 6-phosphogluconate dehydrogenase- on isolated rat hepatocytes in AlP poisoning. Hepatocytes were isolated by collagenase perfusion method and incubated into three different flasks: control, AlP, and 6-AN + ALP. Cellar parameters such as cell viability, reactive oxygen species (ROS) formation, mitochondria membrane potential collapse (MMP), lysosomal integrity, content of reduced (GSH) and oxidized glutathione (GSSG) and lipid peroxidation were assayed at intervals. All analyzed cellular parameters significantly decreased in the third group (6-AN + AlP) compared to the second group (AlP), showing the fact that G6PD deficiency induced by 6-AN had a significant protective effect on the hepatocytes. It was concluded that G6PD deficiency significantly reduced the hepatotoxicity of AlP. Future drugs with the power to induce such deficiency may be promising in treatment of AlP poisoning. [ABSTRACT FROM AUTHOR]