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At least 217 records · Page 12

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NASA NTRS1992

Nucleotide-protectable labeling of sulfhydryl groups in subunit I of the ATPase from Halobacterium saccharovorum

The membrane ATPase from Halobacterium saccharovorum was purified as described by Hochstein et al. (1987) and was incubated with C-14 labeled N-ethylmaleimide (NEM), with and without adenine nucleotides, to determine the effect of nucleotides on the enzyme labeling. It was found that NEM incorporates into the 87,000-Da subunit (subunit I) of the enzyme and that the conditions for enzyme modification are similar to those which result in the inhibition of the enzyme activity. The presence of ATP, ADP, and AMP was found to reduce both the inhibitor incorporation and enzyme inhibition. It was shown that the reaction involves a modification of thiol groups.

Sulzner, Michael↗

DOE OSTI2024

Reduced ribonucleotide reductase RRM2 subunit expression increases DNA damage and mitochondria dysfunction in woody breast chickens

Abstract OBJECTIVE The aim of this study was to investigate the roles of ribonucleotide reductase subunit M2 (RRM2; subunit of ribonucleotide reductase) in severe woody breast (WB) and normal breast muscles. ANIMALS 40 8-week-old male Ross-708 broiler chickens. METHODS Quantitative PCR was performed to determine gene expression, and commercial ELISA/assay kits were used to obtain several enzymatic activities. RESULTS Results showed that RRM2 activity (P= .0002) and RRM2 (P= .05) and hydroxymethylbilane synthase expression (impaired oxygen transport and metabolism,P= .002) were reduced in WB, while caveolin-3 (defected membrane integrity,P= .09), endoglin (increased fibrosis,P= .06), and secreted protein acidic rich in cysteine (metabolic dysregulation,P= .09) expression tended to increase in WB. WB tended to have increased levels of homocysteine (P= .06), aspartate aminotransferase mitochondria (P= .02), pyruvate kinase (P= .04), DNA damage (P= .06), creatine kinase (P= .05), and triglyceride (P= .002) but decreased ATPase activity (P= .01), all indicating mitochondria dysfunction and tissue damage. CLINICAL RELEVANCE In this study, differences in various enzyme activities and increased DNA damage suggest that RRM2-mediated mitochondrial abnormalities may play a role in WB myopathy.

Veterinary Sciences↗

DOE OSTI2022

Exogenous Hemin alleviated cadmium stress in maize (Zea mays L.) by enhancing leaf photosynthesis, AsA-GSH cycle and polyamine metabolism

Cadmium (Cd) stress is one of the principal abiotic stresses that inhibit maize growth. The research was to explore (hemin chloride) Hemin (100 μmol L −1 ) on photosynthesis, ascorbic acid (AsA)-glutathione (GSH) cycle system, and polyamine metabolism of maize under Cd stress (85 mg L −1 ) using nutrient solution hydroponics, with Tiannong 9 (Cd tolerant) and Fenghe 6 (Cd sensitive) as experimental materials. The results showed that Hemin can increase leaf photosynthetic pigment content and ameliorate the ratio of Chlorophyll a/chlorophyll b ( Chla / Chlb ) under Cd stress. The values of ribose 1, 5-diphosphate carboxylase/oxygenase (RuBPcase) and phosphoenolpyruvate carboxylase (PEPCase), and total xanthophyll cycle pool [(violoxanthin (V), antiflavin (A) and zeaxanthin (Z)] increased, which enhancing xanthophyll cycle (DEPS) de-epoxidation, and alleviating stomatal and non-stomatal limitation of leaf photosynthesis. Hemin significantly increased net photosynthetic rate ( P n ), stomatal conductance ( g s ), transpiration rate ( T r ), photochemical quenching coefficient ( qP ), PSII maximum photochemical efficiency ( F v /F m ), and electron transfer rate ( ETR ), which contributed to the improvement of the PSII photosynthetic system. Compared with Cd stress, Hemin can reduce thiobartolic acid reactant (TBARS) content, superoxide anion radical (O 2 − ) production rate, hydrogen peroxide (H 2 O 2 ) accumulation, and the extent of electrolyte leakage (EL); decreased the level of malondialdehyde (MDA) content and increased the activities of superoxide dismutase (SOD), peroxidase (POD) and catalase (CAT); slowed the decrease in dehydroascorbic acid reductase (DHAR) and monodehydroascorbate reductase (MDHAR) activity and the increase in glutathione reductase (GR) and ascorbate peroxidase (APX) activity in leaves; promoted the increase in AsA and GSH content, decreased dehydroascorbic acid (DHA) and oxidized glutathione (GSSG), and increased AsA/DHA and GSH/GSSG ratios under Cd stress. Hemin promoted the increase of conjugated and bound polyamine content, and the conversion process speed of free putrescine (Put) to free spermine (Spm) and spermidine (Spd) in maize; decreased polyamine oxidase (PAO) activity and increased diamine oxidase (DAO), arginine decarboxylase (ADC), ornithine decarboxylase (ODC) and S-adenosylmethionine decarboxylase (SAMDC) enzyme activities in leaves under Cd stress.

Piao, Lin↗

NASA NTRS1973

Studies of a Halophilic NADH Dehydrogenase. 1: Purification and Properties of the Enzyme

An NADH dehydrogenase obtained from an extremely halophilic bacterium was purified 570-fold by a combination of gel filtration, chromatography on hydroxyapatite, and ion-exchange chromatography on QAE-Sephadex. The purified enzyme appeared to be FAD-linked and bad an apparent molecular weight of 64000. Even though enzyme activity was stimulated by NaCl, considerable activity (430 % of the maximum activity observed in the presence of 2.5 M NaCl) was observed in the absence of added NaCl. The enzyme was unstable when incubated in solutions of low ionic strength. The presence of NADH enhanced the stability of the enzyme.

Hochstein, Lawrence I.↗

NASA NTRS1971

Tissue enzyme studies in Macaca nemestrina monkeys.

Total enzyme activities in fresh tissue specimens from major organs of Macaca nemestrina were analyzed for lactic dehydrogenase (LDH), creatine phosphokinase (CPK), and aldolase. The concentration of these enzymes varied among the different tissue with skeletal muscle, heart, and brain having the highest activities. LDH isozymes determinations for the various tissues were also made. The spectrum of LDH isozyme distribution appears to be quite specific and characteristic for at least some of the tissues analyzed.

Hubbard, R. W.↗

NASA NTRS1993

Monoclonal Antibody Testing for Cancer Metastasis

Malignant cells are characterized by the ability to invade surrounding normal tissues. Tumor invasion is abetted by proteolytic enzymes that have been correlated with recurrent disease and metastasis. These enzymes are involved in a cascade of proteolytic interactions with other enzymes and inhibitors which allow cancer cells to dissolve surrounding extracellular matrix, thereby enabling the cells to rapidly invade adjacent tissues and migrate to metastatic sites distant from the primary tumor. Among these proteases are the plasminogen activators (PA), collagenase IV, faminase, and in some cases cathepsin D, which together mediate key steps in the invasion process of metastasis. Cells which have the selective advantage for invasion and metastasis are those capable of regulating their proteolytic activity and proliferation. Cells in the process of invasion would be probably down-regulated for proliferation, but subsequent to attachment and adhesion at a distant site, would then be in a proliferative mode, up-regulating DNA replication. Urokinase (uPA) can be present in the tissues in several molecular forms. The inactive proenzyme is a single chain protein (scuPA) that is cleaved at Lys. 158 to form the double chain, high molecular weight active form (HMW-uPA) of 54 kD. A low molecular weight form (LMW-uPA) can also be produced by cleavage of the HMW-U PA at Lys. 135 - Lys. 136 giving a 35 kD active enzyme. Recently, it has been shown that the HMW active form of urokinase, bound to the tumor cell membrane, is responsible for the local lysis of the extracellular matrix, hence the tissue invasion mechanism for metastasis (Andreasen et al, 19861. Receptor- (membrane) bound uPA is twice as efficient (catalytically) as free fluid-phase uPA. Tho unbound uPA and the LMW form is not responsible for most of the local dissolution of extracellular matrix in the immediate vicinity of the metastatic tumor cell. High levels of urokinase (greater than 3.49 ng/mg of total protein) extracted from breast tumor tissues have recently been shown, together with plasminogen activator inhibitor 1 (PAI-1), to be a good prognostic indicator for high risk of recurrence and shorter patient survival times. In this project, we have attempted to develop immunocytochemical methodologies for the clinical assessment of the expression of urokinase plasminogen activator, which has been implicated to be important for initial steps in tumor invasion, and to relate it to cell proliferation and DNA replication at the single-cell level.

Source record↗

DOE OSTI2022

Enzyme Discovery in Anaerobic Fungi (Neocallimastigomycetes) Enables Lignocellulosic Biorefinery Innovation

Lignocellulosic biorefineries require innovative solutions to realize their full potential, and the discovery of novel lignocellulose-active enzymes could improve biorefinery deconstruction processes. Enzymatic deconstruction of plant cell walls is challenging, as noncarbohydrate linkages in hemicellulosic sidechains and lignin protect labile carbohydrates from hydrolysis.

Microbiology↗

DOE OSTI2023

The transcriptional activator ClrB is crucial for the degradation of soybean hulls and guar gum in Aspergillus niger

Low-cost plant substrates, such as soybean hulls, are used for various industrial applications. Filamentous fungi are important producers of Carbohydrate Active enZymes (CAZymes) required for the degradation of these plant biomass substrates. CAZyme production is tightly regulated by several transcriptional activators and repressors. One such transcriptional activator is CLR-2/ClrB/ManR, which has been identified as a regulator of cellulase and mannanase production in several fungi. However, the regulatory network governing the expression of cellulase and mannanase encoding genes has been reported to differ between fungal species. Previous studies showed that Aspergillus niger ClrB is involved in the regulation of (hemi-)cellulose degradation, although its regulon has not yet been identified. To reveal its regulon, we cultivated an A. niger ΔclrB mutant and control strain on guar gum (a galactomannan-rich substrate) and soybean hulls (containing galactomannan, xylan, xyloglucan, pectin and cellulose) to identify the genes that are regulated by ClrB. Gene expression data and growth profiling showed that ClrB is indispensable for growth on cellulose and galactomannan and highly contributes to growth on xyloglucan in this fungus. Therefore, we show that A. niger ClrB is crucial for the utilization of guar gum and the agricultural substrate, soybean hulls. Moreover, we show that mannobiose is most likely the physiological inducer of ClrB in A. niger and not cellobiose, which is considered to be the inducer of N. crassa CLR-2 and A. nidulans ClrB.

59 BASIC BIOLOGICAL SCIENCES↗

DOE OSTI2025

A distinct class of ferredoxin:NADP + oxidoreductase enzymes driving thermophilic ethanol production

Biofuel production from lignocellulosic biomass offers a transformative solution to reduce global fossil fuel dependency. Certain thermophilic anaerobes, including Clostridium thermocellum, show promise for renewable ethanol production due to their ability to break down plant material at high temperatures. However, achieving commercially viable ethanol yields has proven challenging despite extensive engineering efforts. Here, we characterized 27 ferredoxin:NADP + oxidoreductase (Fnor) enzymes for their enzyme activity, nicotinamide cofactor specificity, thermotolerance, and functional expression in C. thermocellum . We identified a subset of 10 of these enzymes as a novel class of Fnor enzymes suited for metabolic pathways aimed at high-titer ethanol production. When expressed in engineered C. thermocellum , these enzymes increased ethanol production up to 2.2-fold. These findings establish a novel ethanol pathway and provide insights into physiological roles and biotechnological applications of this new class of Fnor enzymes.

09 BIOMASS FUELS↗

DOE OSTI2023

Biocatalytic Membranes for Carbon Capture and Utilization

Innovative carbon capture technologies that capture CO 2 from large point sources and directly from air are urgently needed to combat the climate crisis. Likewise, corresponding technologies are needed to convert this captured CO 2 into valuable chemical feedstocks and products that replace current fossil-based materials to close the loop in creating viable pathways for a renewable economy. Biocatalytic membranes that combine high reaction rates and enzyme selectivity with modularity, scalability, and membrane compactness show promise for both CO 2 capture and utilization. This review presents a systematic examination of technologies under development for CO 2 capture and utilization that employ both enzymes and membranes. CO 2 capture membranes are categorized by their mode of action as CO 2 separation membranes, including mixed matrix membranes (MMM) and liquid membranes (LM), or as CO 2 gas–liquid membrane contactors (GLMC). Because they selectively catalyze molecular reactions involving CO 2 , the two main classes of enzymes used for enhancing membrane function are carbonic anhydrase (CA) and formate dehydrogenase (FDH). Small organic molecules designed to mimic CA enzyme active sites are also being developed. CO 2 conversion membranes are described according to membrane functionality, the location of enzymes relative to the membrane, which includes different immobilization strategies, and regeneration methods for cofactors. Parameters crucial for the performance of these hybrid systems are discussed with tabulated examples. Progress and challenges are discussed, and perspectives on future research directions are provided.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗

NASA NTRS2021

Oncogenic Integration of Nucleotide Metabolism via Fatty Acid Synthase in Non-Hodgkin Lymphoma

Metabolic dysfunctions enabling increased nucleotide biosynthesis are necessary for supporting malignant proliferation. Our investigations indicate that upregulation of fatty acid synthase (FASN) and de novo lipogenesis, commonly observed in many cancers, are associated with nucleotide metabolic dysfunction in lymphoma. The results from our experiments showed that ribonucleotide and deoxyribonucleotide pool depletion, suppression of global RNA/DNA synthesis, and cell cycle inhibition occurred in the presence of FASN inhibition. Subsequently, we observed that FASN inhibition caused metabolic blockade in the rate-limiting step of the oxidative branch of the pentose phosphate pathway (oxPPP) catalyzed by phosphogluconate dehydrogenase (PGDH). Furthermore, we determined that FASN inhibitor treatment resulted in NADPH accumulation and inhibition of PGDH enzyme activity. NADPH is a cofactor utilized by FASN, also a known allosteric inhibitor of PGDH. Through cell-free enzyme assays consisting of FASN and PGDH, we delineated that the PGDH-catalyzed ribulose-5-phosphate synthesis is enhanced in the presence of FASN and is suppressed by increasing concentrations of NADPH. Additionally, we observed that FASN and PGDH were colocalized in the cytosol. The results from these experiments led us to conclude that NADP–NADPH turnover and the reciprocal stimulation of FASN and PGDH catalysis are involved in promoting oxPPP and nucleotide biosynthesis in lymphoma. Finally, a transcriptomic analysis of non-Hodgkin’s lymphoma (n = 624) revealed the increased expression of genes associated with metabolic functions interlinked with oxPPP, while the expression of genes participating in oxPPP remained unaltered. Together we conclude that FASN–PGDH enzymatic interactions are involved in enabling oxPPP and nucleotide metabolic dysfunction in lymphoma tumors.

Non-Hodgkin lymphoma↗

DOE OSTI2024

The crystal structure of Grindelia robusta 7,13-copalyl diphosphate synthase reveals active site features controlling catalytic specificity

Diterpenoid natural products serve critical functions in plant development and ecological adaptation and many diterpenoids have economic value as bioproducts. The family of class II diterpene synthases catalyzes the committed reactions in diterpenoid biosynthesis, converting a common geranylgeranyl diphosphate precursor into different bicyclic prenyl diphosphate scaffolds. Enzymatic rearrangement and modification of these precursors generate the diversity of bioactive diterpenoids. We report the crystal structure of Grindelia robusta 7,13-copalyl diphosphate synthase, GrTPS2, at 2.1 Å of resolution. GrTPS2 catalyzes the committed reaction in the biosynthesis of grindelic acid, which represents the signature metabolite in species of gumweed (Grindelia spp., Asteraceae). Grindelic acid has been explored as a potential source for drug leads and biofuel production. The GrTPS2 crystal structure adopts the conserved three-domain fold of class II diterpene synthases featuring a functional active site in the γβ-domain and a vestigial α-domain. Substrate docking into the active site of the GrTPS2 apo protein structure predicted catalytic amino acids. Biochemical characterization of protein variants identified residues with impact on enzyme activity and catalytic specificity. Specifically, mutagenesis of Y457 provided mechanistic insight into the position-specific deprotonation of the intermediary carbocation to form the characteristic 7,13 double bond of 7,13-copalyl diphosphate.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗

DOE OSTI2026

Mutation of active site glutamate in serine hydroxymethyltransferase allows trapping a reactive intermediate: a combined neutron and X-ray crystallography study

Serine hydroxymethyltransferase (SHMT) is a pyridoxal-5′-phosphate (PLP) dependent enzyme that catalyzes a chemical transformation essential for the one-carbon (1C) metabolism. SHMT reversibly converts L-Ser into Gly and transfers a 1C unit to tetrahydrofolate (THF) to give 5,10-methylene-THF (5,10-MTHF). 5,10-MTHF, a 1C-unit donor, plays a crucial role in the downstream biomolecular syntheses required for the cell homeostasis and proliferation. SHMT is a prominent target for the drug discovery to battle bacterial and parasitic infections, and to treat various types of cancer. SHMT-catalyzed chemistry is governed by the general acid-base catalysis. Knowledge of the catalytic mechanism can aid drug design but can only be achieved when the atomic details of each reaction step are mapped, including accurate determination of hydrogen atom positions. Here we utilized the inactive E53Q mutant of Thermus thermophilus ( Tth ) SHMT to directly determine protonation states with room-temperature neutron crystallography and to capture a reactive intermediate containing the PLP-L-Ser external aldimine and THF in the enzyme active site. We observed protonation of the Schiff base nitrogen (N SB ) in the PLP internal aldimine but no change in the protonation states of other ionizable PLP groups and active site residues compared to wild-type Tth SHMT. X-ray structural analysis of the ternary intermediate complex E53Q-Ser-THF that eluded previous structural characterization shows the strategic positioning of the E53Q side chain in close proximity to the external aldimine and THF and reinforces the proposed role for E53 as the driver of proton transfer events along the reaction pathway.

Drago, Victoria N. [Oak Ridge National Laboratory ↗

NASA NTRS1975

Effect of fluorocarbons on acetylcholinesterase activity and some counter measures

An isolated vagal sympathetic heart system has been successfully used for the study of the effect of fluorocarbons (FCs) on cardiac performance and in situ enzyme activity. Dichlorodifluoromethane sensitizes this preparation to sympathetic stimulation and to exogenous epinephrine challenge. Partial and complete A-V block and even cardiac arrest have been induced by epinephrine challenge in the FC sensitized heart. Potassium chloride alone restores the rhythmicity but not the normal contractility of the heart in such a situation. Addition of glucose will, however, completely restore the normal function of the heart which is sensitized by dichlorodifluoromethane. The ED 50 values of acetylcholinesterase activity which are used as a measure of relative effectiveness of fluorocarbons are compared with the maximum permissible concentration. Kinetic studies indicate that all the fluorocarbons tested so far are noncompetitive.

Young, W.↗

DOE OSTI2020

DNA sequence-dependent activity and base flipping mechanisms of DNMT1 regulate genome-wide DNA methylation

DNA methylation maintenance by DNMT1 is an essential process in mammals but molecular mechanisms connecting DNA methylation patterns and enzyme activity remain elusive. Here, we systematically analyzed the specificity of DNMT1, revealing a pronounced influence of the DNA sequences flanking the target CpG site on DNMT1 activity. We determined DNMT1 structures in complex with preferred DNA substrates revealing that DNMT1 employs flanking sequence-dependent base flipping mechanisms, with large structural rearrangements of the DNA correlating with low catalytic activity. Moreover, flanking sequences influence the conformational dynamics of the active site and cofactor binding pocket. Importantly, we show that the flanking sequence preferences of DNMT1 highly correlate with genomic methylation in human and mouse cells, and 5-azacytidine triggered DNA demethylation is more pronounced at CpG sites with flanks disfavored by DNMT1. Overall, our findings uncover the intricate interplay between CpG-flanking sequence, DNMT1-mediated base flipping and the dynamic landscape of DNA methylation.

59 BASIC BIOLOGICAL SCIENCES↗

DOE OSTI2025

Structure of human MUTYH and functional profiling of cancer-associated variants reveal an allosteric network between its [4Fe-4S] cluster cofactor and active site required for DNA repair

Abstract MUTYH is a clinically important DNA glycosylase that thwarts mutations by initiating base-excision repair at 8-oxoguanine (OG):A lesions. The roles for its [4Fe-4S] cofactor in DNA repair remain enigmatic. Functional profiling of cancer-associated variants near the [4Fe-4S] cofactor reveals that most variations abrogate both retention of the cofactor and enzyme activity. Surprisingly, R241Q and N238S retained the metal cluster and bound substrate DNA tightly, but were completely inactive. We determine the crystal structure of human MUTYH bound to a transition state mimic and this shows that Arg241 and Asn238 build an H-bond network connecting the [4Fe-4S] cluster to the catalytic Asp236 that mediates base excision. The structure of the bacterial MutY variant R149Q, along with molecular dynamics simulations of the human enzyme, support a model in which the cofactor functions to position and activate the catalytic Asp. These results suggest that allosteric cross-talk between the DNA binding [4Fe-4S] cofactor and the base excision site of MUTYH regulate its DNA repair function.

Science & Technology - Other Topics↗

DOE OSTI2023

Rapid Biocatalytic Synthesis of Aromatic Acid CoA Thioesters by Using Microbial Aromatic Acid CoA Ligases

Abstract Chemically labile ester linkages can be introduced into lignin by incorporation of monolignol conjugates, which are synthesized in planta by acyltransferases that use a coenzyme A (CoA) thioester donor and a nucleophilic monolignol alcohol acceptor. The presence of these esters facilitates processing and aids in the valorization of renewable biomass feedstocks. However, the effectiveness of this strategy is potentially limited by the low steady‐state levels of aromatic acid thioester donors in plants. As part of an effort to overcome this, aromatic acid CoA ligases involved in microbial aromatic degradation were identified and screened against a broad panel of substituted cinnamic and benzoic acids involved in plant lignification. Functional fingerprinting of this ligase library identified four robust, highly active enzymes capable of facile, rapid, and high‐yield synthesis of aromatic acid CoA thioesters under mild aqueous reaction conditions mimicking in planta activity.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗

DOE OSTI2023

Insights into the Ecological Diversification of the Hymenochaetales based on Comparative Genomics and Phylogenomics With an Emphasis on Coltricia

To elucidate the genomic traits of ecological diversification in the Hymenochaetales, we sequenced 15 new genomes, with attention to ectomycorrhizal (EcM) Coltricia species. Together with published data, 32 genomes, including 31 Hymenochaetales and one outgroup, were comparatively analyzed in total. Compared with those of parasitic and saprophytic members, EcM species have significantly reduced number of plant cell wall degrading enzyme genes, and expanded transposable elements, genome sizes, small secreted proteins, and secreted proteases. EcM species still retain some of secreted carbohydrate-active enzymes (CAZymes) and have lost the key secreted CAZymes to degrade lignin and cellulose, while possess a strong capacity to degrade a microbial cell wall containing chitin and peptidoglycan. There were no significant differences in secreted CAZymes between fungi growing on gymnosperms and angiosperms, suggesting that the secreted CAZymes in the Hymenochaetales evolved before differentiation of host trees into gymnosperms and angiosperms. Nevertheless, parasitic and saprophytic species of the Hymenochaetales are very similar in many genome features, which reflect their close phylogenetic relationships both being white rot fungi. Phylogenomic and molecular clock analyses showed that the EcM genus Coltricia formed a clade located at the base of the Hymenochaetaceae and divergence time later than saprophytic species. And Coltricia remains one to two genes of AA2 family. These indicate that the ancestors of Coltricia appear to have originated from saprophytic ancestor with the ability to cause a white rot. This study provides new genomic data for EcM species and insights into the ecological diversification within the Hymenochaetales based on comparative genomics and phylogenomics analyses.

59 BASIC BIOLOGICAL SCIENCES↗