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Search indexed NASA NTRS and DOE OSTI research on propulsion, heat transfer, battery materials and energy systems. Follow report and document links to the original sources.

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At least 307 records · Page 17
All sourcesNASA NTRSDOE OSTI
DOE OSTI2026

Phycocyanobilin biosynthesis in Galdieria sulphuraria requires isomerization of phycoerythrobilin synthesized by bilin reductases

Phycobiliproteins are essential components of the light-harvesting antennae in cyanobacteria and red algae, requiring covalently bound open-chain tetrapyrrole chromophores (bilins) for proper function. In the red alga Galdieria sulphuraria , the primary chromophore is phycocyanobilin (PCB), despite the apparent presence of only biosynthetic genes for phycoerythrobilin (PEB) biosynthesis (PEBA and PEBB). This observation suggests the presence of an alternative, atypical biosynthetic pathway for PCB. In this study, we confirmed the presence of PEB:PCB isomerase activity in an enriched protein fraction from G. sulphuraria . To further investigate this unusual pathway, we combined in silico analyses with biochemical assays. Phylogenetic analyses confirmed the placement of the G. sulphuraria ferredoxin-dependent bilin reductases within the PEBA and PEBB lineages, typically associated with PEB synthesis, whereas the related red alga Cyanidioschyzon merolae was found to contain only PCYA. This gene distribution presents a functional paradox. G. sulphuraria harbors PEB biosynthesis genes but no detectable PEB chromophores and lacks known PCB-synthesizing enzymes despite containing PCB. Functional characterization of recombinant GsPEBA (G. sulphuraria PEBA) and GsPEBB ( G. sulphuraria PEBB) confirmed their roles in PEB synthesis, demonstrating that these enzymes have not evolved to synthesize PCB or act as isomerases despite their phylogenetic placement. In contrast, Cm PCYA ( C. merolae PCYA) catalyzed direct PCB formation from biliverdin. Together, these findings reveal an atypical isomerase-based pathway for PCB biosynthesis in G. sulphuraria , expanding our understanding of bilin metabolism and providing new insight into the evolutionary flexibility of photosynthetic pigment biosynthesis in Rhodophyta.

14 SOLAR ENERGY↗
NASA NTRS2001

The Effects of Long-Term Feeding of Rodent Food Bars on Lipid Peroxidation And Antioxidant Enzyme Levels In Fisher Rats

A specialized rodent food bar diet has been developed and utilized successfully for short-duration shuttle missions. Recent tests conducted in preparation for experiments aboard the International Space Station (ISS) indicated that long-term food bar feeding for three months induced hyperlipidemia in rats. This study examined oxidative stress status in livers of these same animals. Spectrophotometric analysis of 79 Fischer rat livers (40 female and 39 male) for lipid peroxidation (LPO) and superoxide dismutase (SOD) was conducted using Bioxytech LPO-587(TM) assay kit and SOD-525(Tm) assay kit, respectively. The treatment groups consisted of 20 male CHOW and 19 male FOOD BAR rats and 20 female CHOW and 20 female FOOD BAR rats. Statistical analysis to compare differences between groups was performed by standard analysis of variance procedures. The male FOOD BAR group LPO mean (3.6 +/- 0.2 mmol/g) was significantly (p less than or equal to 0.05) greater than that of the male CHOW group (2.1 +/-0.1 mmol/g). Moreover the female FOOD BAR group LPO mean (2.9 +/-0.1 mmol/g) was also significantly greater than the female CHOW group mean (2.2 +/-0.1 mmol/g). The mean values for SOD in both male and female groups showed no significant differences between CHOW and FOOD BAR groups. These results show that LPO levels were significantly higher in both the male and female FOOD BAR groups compared to CHOW groups and that there was no concomitant increase in SOD levels across the group. In addition, males showed a greater difference than females in terms of LPO levels. These findings suggest a need for further investigation into the use of the current food bar formulation for long-term experiments such as those planned for the ISS.

Ramirez, Joel↗
DOE OSTI2024

Characterization of a Unique Pair of Ferredoxin and Ferredoxin NADP+ Reductase Isoforms That Operates in Non-Photosynthetic Glandular Trichomes

Our recent investigations indicated that isoforms of ferredoxin (Fd) and ferredoxin NADP+ reductase (FNR) play essential roles for the reductive steps of the 2C-methyl-D-erythritol 4-phosphate (MEP) pathway of terpenoid biosynthesis in peppermint glandular trichomes (GTs). Based on an analysis of several transcriptome data sets, we demonstrated the presence of transcripts for a leaf-type FNR (L-FNR), a leaf-type Fd (Fd I), a root-type FNR (R-FNR), and two root-type Fds (Fd II and Fd III) in several members of the mint family (Lamiaceae). The present study reports on the biochemical characterization of all Fd and FNR isoforms of peppermint (Mentha × piperita L.). The redox potentials of Fd and FNR isoforms were determined using photoreduction methods. Based on a diaphorase assay, peppermint R-FNR had a substantially higher specificity constant (kcat/Km) for NADPH than L-FNR. Similar results were obtained with ferricyanide as an electron acceptor. When assayed for NADPH–cytochrome c reductase activity, the specificity constant with the Fd II and Fd III isoforms (when compared to Fd I) was slightly higher for L-FNR and substantially higher for R-FNR. Based on real-time quantitative PCR assays with samples representing various peppermint organs and cell types, the Fd II gene was expressed very highly in metabolically active GTs (but also present at lower levels in roots), whereas Fd III was expressed at low levels in both roots and GTs. Our data provide evidence that high transcript levels of Fd II, and not differences in the biochemical properties of the encoded enzyme when compared to those of Fd III, are likely to support the formation of copious amounts of monoterpene via the MEP pathway in peppermint GTs. This work has laid the foundation for follow-up studies to further investigate the roles of a unique R-FNR–Fd II pair in non-photosynthetic GTs of the Lamiaceae.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2022

Acoustic force spectroscopy reveals subtle differences in cellulose unbinding behavior of carbohydrate-binding modules

Protein adsorption to solid carbohydrate interfaces is critical to many biological processes, particularly in biomass deconstruction. To engineer more-efficient enzymes for biomass deconstruction into sugars, it is necessary to characterize the complex protein–carbohydrate interfacial interactions. A carbohydrate-binding module (CBM) is often associated with microbial surface-tethered cellulosomes or secreted cellulase enzymes to enhance substrate accessibility. However, it is not well known how CBMs recognize, bind, and dissociate from polysaccharides to facilitate efficient cellulolytic activity, due to the lack of mechanistic understanding and a suitable toolkit to study CBM–substrate interactions. Our work outlines a general approach to study the unbinding behavior of CBMs from polysaccharide surfaces using a highly multiplexed single-molecule force spectroscopy assay. Here, we apply acoustic force spectroscopy (AFS) to probe a Clostridium thermocellum cellulosomal scaffoldin protein (CBM3a) and measure its dissociation from nanocellulose surfaces at physiologically relevant, low force loading rates. An automated microfluidic setup and method for uniform deposition of insoluble polysaccharides on the AFS chip surfaces are demonstrated. The rupture forces of wild-type CBM3a, and its Y67A mutant, unbinding from nanocellulose surfaces suggests distinct multimodal CBM binding conformations, with structural mechanisms further explored using molecular dynamics simulations. Applying classical dynamic force spectroscopy theory, the single-molecule unbinding rate at zero force is extrapolated and found to agree with bulk equilibrium unbinding rates estimated independently using quartz crystal microbalance with dissipation monitoring. However, our results also highlight critical limitations of applying classical theory to explain the highly multivalent binding interactions for cellulose–CBM bond rupture forces exceeding 15 pN.

09 BIOMASS FUELS↗
DOE OSTI2021

CAZymes from the thermophilic fungus Thermoascus aurantiacus are induced by C5 and C6 sugars

Abstract Background Filamentous fungi are excellent lignocellulose degraders, which they achieve through producing carbohydrate active enzymes (CAZymes). CAZyme production is highly orchestrated and gene expression analysis has greatly expanded understanding of this important biotechnological process. The thermophilic fungus Thermoascus aurantiacus secretes highly active thermostable enzymes that enable saccharifications at higher temperatures; however, the genome-wide measurements of gene expression in response to CAZyme induction are not understood. Results A fed-batch system with plant biomass-derived sugars d -xylose, l -arabinose and cellobiose established that these sugars induce CAZyme expression in T. aurantiacus . The C5 sugars induced both cellulases and hemicellulases, while cellobiose specifically induced cellulases. A minimal medium formulation was developed to enable gene expression studies of T. aurantiacus with these inducers. It was found that d-xylose and l -arabinose strongly induced a wide variety of CAZymes, auxiliary activity (AA) enzymes and carbohydrate esterases (CEs), while cellobiose facilitated lower expression of mostly cellulase genes. Furthermore, putative orthologues of different unfolded protein response genes were up-regulated during the C5 sugar feeding together with genes in the C5 sugar assimilation pathways. Conclusion This work has identified two additional CAZyme inducers for T. aurantiacus , l -arabinose and cellobiose, along with d -xylose. A combination of biochemical assays and RNA-seq measurements established that C5 sugars induce a suite of cellulases and hemicellulases, providing paths to produce broad spectrum thermotolerant enzymatic mixtures.

09 BIOMASS FUELS↗
DOE OSTI2023

Allosteric activation or inhibition of PI3Kγ mediated through conformational changes in the p110γ helical domain

PI3Kγ is a critical immune signaling enzyme activated downstream of diverse cell surface molecules, including Ras, PKCβ activated by the IgE receptor, and Gβγ subunits released from activated GPCRs. PI3Kγ can form two distinct complexes, with the p110γ catalytic subunit binding to either a p101 or p84 regulatory subunit, with these complexes being differentially activated by upstream stimuli. Here, using a combination of cryo electron microscopy, HDX-MS, and biochemical assays, we have identified novel roles of the helical domain of p110γ in regulating lipid kinase activity of distinct PI3Kγ complexes. We defined the molecular basis for how an allosteric inhibitory nanobody potently inhibits kinase activity through rigidifying the helical domain and regulatory motif of the kinase domain. The nanobody did not block either p110γ membrane recruitment or Ras/Gβγ binding, but instead decreased ATP turnover. We also identified that p110γ can be activated by dual PKCβ helical domain phosphorylation leading to partial unfolding of an N-terminal region of the helical domain. PKCβ phosphorylation is selective for p110γ-p84 compared to p110γ-p101, driven by differential dynamics of the helical domain of these different complexes. Nanobody binding prevented PKCβ-mediated phosphorylation. Overall, this work shows an unexpected allosteric regulatory role of the helical domain of p110γ that is distinct between p110γ-p84 and p110γ-p101 and reveals how this can be modulated by either phosphorylation or allosteric inhibitory binding partners. This opens possibilities of future allosteric inhibitor development for therapeutic intervention.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2020

Cofactor Complexes of DesD, a Model Enzyme in the Virulence-related NIS Synthetase Family

The understudied nonribosomal-peptide-synthetase-independent siderophore (NIS) synthetase family has been increasingly associated with virulence in bacterial species due to its key role in the synthesis of hydroxamate and carboxylate "stealth" siderophores. We have identified a model family member, DesD, from Streptomyces coelicolor , to structurally characterize using a combination of a wild-type and a Arg306Gln variant in apo , cofactor product AMP-bound, and cofactor reactant ATP-bound complexes. The kinetics in the family has been limited by solubility and reporter assays, so we have developed a label-free kinetics assay utilizing a single-injection isothermal-titration-calorimetry-based method. We report second-order rate constants that are 50 times higher than the previous estimations for DesD. Our Arg306Gln DesD variant was also tested under identical buffer and substrate conditions, and its undetectable activity was confirmed. These are the first reported structures for DesD, and they describe the critical cofactor coordination. This is also the first label-free assay to unambiguously determine the kinetics for an NIS synthetase.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗
DOE OSTI2023

tRNA shape is an identity element for an archaeal pyrrolysyl-tRNA synthetase from the human gut

Protein translation is orchestrated through tRNA aminoacylation and ribosomal elongation. Among the highly conserved structure of tRNAs, they have distinguishing features which promote interaction with their cognate aminoacyl tRNA synthetase (aaRS). These key features are referred to as identity elements. In our study, we investigated the tRNA:aaRS pair that installs the 22nd amino acid, pyrrolysine (tRNAPyl:PylRS). Pyrrolysyl-tRNA synthetases (PylRSs) are naturally encoded in some archaeal and bacterial genomes to acylate tRNAPyl with pyrrolysine. Their large amino acid binding pocket and poor recognition of the tRNA anticodon have been instrumental in incorporating > 200 noncanonical amino acids. PylRS enzymes can be divided into three classes based on their genomic structure. Two classes contain both an N-terminal and C-terminal domain, however the third class (ΔpylSn) lacks the N-terminal domain. In this study we explored the tRNA identity elements for a ΔpylSn tRNAPyl from Candidatus Methanomethylophilus alvus which drives the orthogonality seen with its cognate PylRS (MaPylRS). From aminoacylation and translation assays we identified five key elements in ΔpylSn tRNAPyl necessary for MaPylRS activity. The absence of a base (position 8) and a G–U wobble pair (G28:U42) were found to affect the high-resolution structure of the tRNA, while molecular dynamic simulations led us to acknowledge the rigidity imparted from the G–C base pairs (G3:C70 and G5:C68).

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2021

USP18 promotes tumor metastasis in esophageal squamous cell carcinomas via deubiquitinating ZEB1

Highlights: • High USP18 expression is positively correlated with the malignant phenotype and poor survival in ESCC patients. • Knockdown of USP18 suppressed the metastasis ability of ESCC cells in vitro and in vivo. • USP18 contributes to ESCC cell metastasis by inducing ZEB1 mediated EMT process. • USP18 acts as a deubiquitinase, and stabilises ZEB1 by reducing its poly-ubiquitination. The dysregulation of deubiquitinating enzymes (DUBs), which regulate the stability of most cellular proteins, have been implicated in many human diseases, including cancers. Ubiquitin-specific protease 18 (USP18), a member of the DUBs family, functions as a potential tumour promoter in various cancers. However, the biological function and clinical significance of USP18 in esophageal squamous cell carcinomas (ESCC) are still unclear. Here, we found that ESCC tumors had higher USP18 expression compared with that of normal esophageal epithelial tissues, and high USP18 level was significantly correlated with malignant phenotype and shorter survival in patients with ESCC. In functional experiments, USP18 knockdown significantly inhibited ESCC invasion and metastasis in vitro. Consistently, a xenograft assay showed that knockdown of USP18 in ESCC cell suppressed their dissemination to lung tissue in vivo. Furthermore, we showed that USP18 promoted ESCC cell metastasis by inducing ZEB1 mediated epithelial-mesenchymal transition (EMT). Importantly, our results demonstrated that the oncogenic effect of USP18 in ESCC is partially dependent on ZEB1 enhancement. Mechanistic investigations revealed that USP18 directly bound ZEB1 and decreased its ubiquitination to enhance the protein stability of ZEB1 in ESCC cells. Overall, our data highlighted an essential role of USP18 in ESCC metastasis, suggesting that it could be a potential diagnostic and therapeutic target for ESCC.

60 APPLIED LIFE SCIENCES↗
NASA NTRS2017

BioWires: Conductive DNA Nanowires in a Computationally-Optimized, Synthetic Biological Platform for Nanoelectronic Fabrication

DNA is an ideal template for a biological nanowire-it has a linear structure several atoms thick; it possesses addressable nucleobase geometry that can be precisely defined; and it is massively scalable into branched networks. Until now, the drawback of DNA as a conducting nanowire been, simply put, its low conductance. To address this deficiency, we extensively characterize a chemical variant of canonical DNA that exploits the affinity of natural cytosine bases for silver ions. We successfully construct chains of single silver ions inside double-stranded DNA, confirm the basic dC-Ag+-dC bond geometry and kinetics, and show length-tunability dependent on mismatch distribution, ion availability and enzyme activity. An analysis of the absorbance spectra of natural DNA and silver-binding, poly-cytosine DNA demonstrates the heightened thermostability of the ion chain and its resistance to aqueous stresses such as precipitation, dialysis and forced reduction. These chemically critical traits lend themselves to an increase in electrical conductivity of over an order of magnitude for 11-base silver-paired duplexes over natural strands when assayed by STM break junction. We further construct and implement a genetic pathway in the E. coli bacterium for the biosynthesis of highly ionizable DNA sequences. Toward future circuits, we construct a model of transcription network architectures to determine the most efficient and robust connectivity for cell-based fabrication, and we perform sequence optimization with a genetic algorithm to identify oligonucleotides robust to changes in the base-pairing energy landscape. We propose that this system will serve as a synthetic biological fabrication platform for more complex DNA nanotechnology and nanoelectronics with applications to deep space and low resource environments.

DNA↗
DOE OSTI2021

LOXG473A induces the formation of osteoclasts in RAW264.7 cells via IL-6/JAK2/STAT3 signaling

Formation of osteoclasts is known to be closely associated with osteoporosis progression. LOX is a key enzyme that catalyzes the synthesis of collagen, which is the new mediator in osteoclast formation. However, the effect of LOXG473A on of osteoclast formation needs to be explored. Thereby, we sought to explore the effect of LOX{sub G473A} on formation of osteoclasts and its underlying mechanism. To investigate the function of LOX{sub G473A} in osteoclast formation, Raw264.7 cells were stably transfected with LOX-WT or LOX-MUT (LOX{sub G473A}). Real-time PCR and western blotting were used to detect the relative levels of osteoclast formation related genes and proteins. TRAP staining and immunofluorescence staining were used to test the ability of Raw264.7 cells to form osteoclasts and the ability of cells to form rings, respectively. Bone erosion assay was used to test bone resorptive activity. The data indicated that LOX{sub G473A} significantly enhanced the ability of osteoclasts forming, ring-forming and bone resorpting in Raw264.7 cells. Mechanically, LOX{sub G473A} upregulated the expressions of NFATC1, ACP5, CTSK, IL-6, and the proportion of p-JAK2/JAK2 and p-STAT3/STAT3, thereby promoting the formation of osteoclasts. In conclusion, we have verified that LOX{sub G473A} induces the proliferation of osteoclasts in Raw264.7 cells via IL-6/JAK2/STAT3 signaling, suggesting a novel strategy for studying osteoporosis.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2020

Purification and characterization of a native lytic polysaccharide monooxygenase from Thermoascus aurantiacus

Lytic polysaccharide monooxygenases (LPMOs) have emerged as key proteins for depolymerization of cellulose. These copper-containing enzymes oxidize C-1 and/or C-4 bonds in cellulose, promoting increased hydrolysis of the oxidized cellulose chains. The LPMO from Thermoascus aurantiacus, a thermophilic ascomycete fungus, has been extensively studied and has served as a model LPMO. A method was developed to purify the LPMO from culture filtrates of T. aurantiacus along with its native cellobiohydrolase and endoglucanase. The activity of the purified LPMO was measured with a colorimetric assay that established the T opt of the native LPMO at 60 °C. Purification of the components of the T. aurantiacus cellulase mixture also enabled quantification of the amounts of cellobiohydrolase, endoglucanase and LPMO present in the T. aurantiacus culture filtrate, establishing that the LPMO was the most abundant protein in the culture supernatants. Here, the importance of the LPMO to activity of the mixture was demonstrated by saccharifications with Avicel and acid-pretreated corn stover.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2023

Inhibitor-3 inhibits Protein Phosphatase 1 via a metal binding dynamic protein–protein interaction

To achieve substrate specificity, protein phosphate 1 (PP1) forms holoenzymes with hundreds of regulatory and inhibitory proteins. Inhibitor-3 (I3) is an ancient inhibitor of PP1 with putative roles in PP1 maturation and the regulation of PP1 activity. Here, we show that I3 residues 27–68 are necessary and sufficient for PP1 binding and inhibition. In addition to a canonical RVxF motif, which is shared by nearly all PP1 regulators and inhibitors, and a non-canonical SILK motif, I3 also binds PP1 via multiple basic residues that bind directly in the PP1 acidic substrate binding groove, an interaction that provides a blueprint for how substrates bind this groove for dephosphorylation. Unexpectedly, this interaction positions a CCC (cys-cys-cys) motif to bind directly across the PP1 active site. Using biophysical and inhibition assays, we show that the I3 CCC motif binds and inhibits PP1 in an unexpected dynamic, fuzzy manner, via transient engagement of the PP1 active site metals. Together, these data not only provide fundamental insights into the mechanisms by which IDP protein regulators of PP1 achieve inhibition, but also shows that fuzzy interactions between IDPs and their folded binding partners, in addition to enhancing binding affinity, can also directly regulate enzyme activity.

59 BASIC BIOLOGICAL SCIENCES↗
NASA NTRS1998

Analysis of proteins involved in biodegradation of crop biomass

The biodegradation of crop biomass for re-use in crop production is part of the bioregenerative life support concept proposed by the National Aeronautics and Space Administration (NASA) for long duration, manned space exploration. The current research was conducted in the laboratory to evaluate the use of electrophoretic analysis as a means of rapidly assaying for constitutive and induced proteins associated with the bacterial degradation of crop residue. The proteins involved in crop biomass biodegradation are either constitutive or induced. As a result, effluent and cultures were examined to investigate the potential of using electrophoretic techniques as a means of monitoring the biodegradation process. Protein concentration for optimum banding patterns was determined using the Bio-Rad Protein Assay kit. Four bacterial soil isolates were obtained from the G.W. Carver research Farm at Tuskegee University and used in the decomposition of components of plant biomass. The culture, WDSt3A was inoculated into 500 mL of either Tryptic Soy Broth or Nutrient Broth. Incubation, with shaking of each flask was for 96 hours at 30 C. The cultures consistently gave unique banding patterns under denaturing protein electrophoresis conditions, The associated extracellular enzymes also yielded characteristic banding patterns over a 14-day period, when native electrophoresis techniques were used to examine effluent from batch culture bioreactors. The current study evaluated sample preparation and staining protocols to determine the ease of use, reproducibility and reliability, as well as the potential for automation.

Crawford, Kamau↗
DOE OSTI2026

Rethinking Suicide Thi4 Thiazole Synthases: Comparative Genomic Insights and Pilot Functional Evidence

Suicide thiazole synthases (Thi4) are mononuclear metal enzymes that form the thiazole moiety of thiamin from NAD + , glycine, and a sulfur atom that is stripped from an active-site cysteine residue, causing enzyme inactivation. Comparative genomic analysis shows that prokaryotic Thi4 genes often cluster on the chromosomal regions encoding ThiS, ThiF, and other proteins that can produce, relay, or use persulfide or thiocarboxylate sulfur. These recurring genomic associations raise the possibility that, in some microorganisms, suicide Thi4s may interact with sulfur-relay systems, i.e., they can possibly operate in a nonsuicide mode. This proof-of-concept study explores this possibility via complementation assays using Escherichia coli as a heterologous platform. A representative bacterial Thi4 that clustered with thiS and thiF complemented an E. coli ΔthiG (thiazole auxotroph) single mutant better than a ΔthiG ΔthiF ΔthiS triple mutant. Although (in)direct sulfur transfer could not be assessed in the scope of our investigation, the initial results suggest a dependence on host sulfur relay components, consistent with predicted interactions with the host sulfide transfer chain. Collectively, this new perspective provides a useful guide for future biochemical studies on alternative modes of action for “suicide Thi4s” and accessory proteins.

Bacteria↗
DOE OSTI2025

Development of an ultrahigh affinity, trimeric ACE2 biologic as a universal SARS-CoV-2 antagonist

Abstract Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), responsible for the COVID-19 pandemic, utilizes membrane-bound, angiotensin-converting enzyme II (ACE2) for internalization and infection. We describe the development of a biologic that takes advantage of the proximity of the N-terminus of bound ACE2 to the three-fold symmetry axis of the spike protein to create an ultrapotent, trivalent ACE2 entry antagonist. Distinct disulfide bonds were added to enhance serum stability and a single point mutation was introduced to eliminate enzymatic activity. Through surface plasmon resonance, pseudovirus neutralization assays, and single-particle cryo-electron microscopy, we show this antagonist binds to and inhibits SARS-CoV-2 variants. We further show the antagonist binds to and inhibits a 2003 SARS-CoV-1 strain. Collectively, structural insight has allowed us to design a universal trivalent antagonist against all variants of SARS-CoV-2 tested, suggesting it will be active against the emergence of future mutants.

Gonzales, Juliet (ORCID:0000000327219566)↗
NASA NTRS2002

Basal glycogenolysis in mouse skeletal muscle: in vitro model predicts in vivo fluxes

A previously published mammalian kinetic model of skeletal muscle glycogenolysis, consisting of literature in vitro parameters, was modified by substituting mouse specific Vmax values. The model demonstrates that glycogen breakdown to lactate is under ATPase control. Our criteria to test whether in vitro parameters could reproduce in vivo dynamics was the ability of the model to fit phosphocreatine (PCr) and inorganic phosphate (Pi) dynamic NMR data from ischemic basal mouse hindlimbs and predict biochemically-assayed lactate concentrations. Fitting was accomplished by optimizing four parameters--the ATPase rate coefficient, fraction of activated glycogen phosphorylase, and the equilibrium constants of creatine kinase and adenylate kinase (due to the absence of pH in the model). The optimized parameter values were physiologically reasonable, the resultant model fit the [PCr] and [Pi] timecourses well, and the model predicted the final measured lactate concentration. This result demonstrates that additional features of in vivo enzyme binding are not necessary for quantitative description of glycogenolytic dynamics.

Non-NASA Center↗
DOE OSTI2026

Hypophosphite Is a Naturally Occurring Selective Inhibitor of Syntrophic Methanogenesis

Microbial methanogenesis is a major contributor to global warming, and methane fluxes represent a loss of energy and electrons from industrial ecosystems. The chemical space of methane control strategies is still underexplored. Most known methanogenesis inhibitors target methanogenic archaeal enzymes. However, interference with the exchange of syntrophic electron carriers (H 2 or formate) in methanogenic systems presents an additional target for methane control. Here, we show that hypophosphite (H 2 PO 2 − ), an inorganic formate analogue, is a potent and selective inhibitor of syntrophic methanogenesis versus primary fermentation in rice field sediments and cattle rumens. Hypophosphite is also generally recognized as safe and relatively nontoxic to plants and animals. Genetic screens and physiological assays in the model methanogen Methanococcus maripaludis S2 implicate formate metabolism as the target of hypophosphite inhibition. Currently, there is no known biological pathway for anaerobic hypophosphite oxidation, and hypophosphite is stable in anoxic sediments for weeks to months. Given its widespread natural occurrence, we propose that hypophosphite may modulate the carbon cycle in natural environments. Taken together, our results suggest that hypophosphite could be used as a safe, inexpensive strategy for methane control in syntrophic methanogenic ecosystems.

chemical inhibitor↗