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At least 145 records · Page 8

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DOE OSTI2020

Triose phosphate utilization limitation: an unnecessary complexity in terrestrial biosphere model representation of photosynthesis

Triose phosphates are the principal product of photosynthesis. They are used within the chloroplast for starch synthesis, or translocated to the cytosol where they are used to fuel sucrose synthesis. Use of triose phosphate releases inorganic phosphate, and is under strict metabolic control that matches the supply of triose phosphate from the Calvin-Benson cycle to the demand for carbon by sinks (Heldt & Heldt, 2011; McClain & Sharkey, 2019). However, a low rate of triose phosphate utilization (TPU) can deplete the phosphate pool, restrict ATP synthesis and reduce the availability of ATP to power the Calvin-Benson cycle, thereby limiting photosynthesis (Sharkey, 1985).

54 ENVIRONMENTAL SCIENCES↗

DOE OSTI2022

Structural insights into the assembly and the function of the plant oxidative phosphorylation system

One of the key functions of mitochondria is the production of ATP to support cellular metabolism and growth. The last step of mitochondrial ATP synthesis is performed by the oxidative phosphorylation (OXPHOS) system, an ensemble of protein complexes embedded in the inner mitochondrial membrane. In the last 25 yr, many structures of OXPHOS complexes and supercomplexes have been resolved in yeast, mammals, and bacteria. However, structures of plant OXPHOS enzymes only became available very recently. In this review, we highlight the plant-specific features revealed by the recent structures and discuss how they advance our understanding of the function and assembly of plant OXPHOS complexes. Here we also propose new hypotheses to be tested and discuss older findings to be re-evaluated. Further biochemical and structural work on the plant OXPHOS system will lead to a deeper understanding of plant respiration and its regulation, with significant agricultural, environmental, and societal implications.

59 BASIC BIOLOGICAL SCIENCES↗

DOE OSTI2022

Increasing the Thermodynamic Driving Force of the Phosphofructokinase Reaction in Clostridium thermocellum

Glycolysis is an ancient, widespread, and highly conserved metabolic pathway that converts glucose into pyruvate. In the canonical pathway, the phosphofructokinase (PFK) reaction plays an important role in controlling flux through the pathway. Clostridium thermocellum has an atypical glycolysis and uses pyrophosphate (PP i ) instead of ATP as the phosphate donor for the PFK reaction. The reduced thermodynamic driving force of the PP i -PFK reaction shifts the entire pathway closer to thermodynamic equilibrium, which has been predicted to limit product titers. Here, in this study, we replace the PP i -PFK reaction with an ATP-PFK reaction. We demonstrate that the local changes are consistent with thermodynamic predictions: the ratio of fructose 1,6-bisphosphate to fructose-6-phosphate increases, and the reverse flux through the reaction (determined by 13 C labeling) decreases. The final titer and distribution of fermentation products, however, do not change, demonstrating that the thermodynamic constraints of the PP i -PFK reaction are not the sole factor limiting product titer.

59 BASIC BIOLOGICAL SCIENCES↗

DOE OSTI2020

Metabolic Fluxes of Nitrogen and Pyrophosphate in Chemostat Cultures of Clostridium thermocellum and Thermoanaerobacterium saccharolyticum

This study discusses the fate of pyrophosphate in the metabolism of two thermophilic anaerobes that lack a soluble irreversible pyrophosphatase as present in Escherichia coli but instead use a reversible membrane-bound proton-pumping enzyme. In such organisms, the charging of tRNA with amino acids may become more reversible. This may contribute to the observed excretion of amino acids during sugar fermentation by Clostridium thermocellum and Thermoanaerobacterium saccharolyticum . Calculation of the energetic advantage of reversible pyrophosphate-dependent glycolysis, as occurs in Clostridium thermocellum , could not be properly evaluated, as currently available genome-scale models neglect the anabolic generation of pyrophosphate in, for example, polymerization of amino acids to protein. This anabolic pyrophosphate replaces ATP and thus saves energy. Its amount is, however, too small to cover the pyrophosphate requirement of sugar catabolism in glycolysis. Consequently, pyrophosphate for catabolism is generated according to ATP + P i → ADP + PP i .

59 BASIC BIOLOGICAL SCIENCES↗

DOE OSTI2023

RecJ3/4-aRNase J form a Ubl-associated nuclease complex functioning in survival against DNA damage in Haloferax volcanii

Nucleases are strictly regulated and often localized in the cell to avoid the uncontrolled degradation of DNA and RNA. Here, a new type of nuclease complex, composed of RecJ3, RecJ4, and aRNase J, was identified through its ATP-dependent association with the ubiquitin-like SAMP1 and AAA-ATPase Cdc48a. The complex was discovered in Haloferax volcanii , an archaeon lacking an RNA exosome. Genetic analysis revealed aRNase J to be essential and RecJ3, RecJ4, and Cdc48a to function in the recovery from DNA damage including genotoxic agents that generate double-strand breaks. The RecJ3:RecJ4:aRNase J complex (isolated in 2:2:1 stoichiometry) functioned primarily as a 3′−5′ exonuclease in hydrolyzing RNA and ssDNA, with the mechanism non-processive for ssDNA. aRNase J could also be purified as a homodimer that catalyzed endoribonuclease activity and, thus, was not restricted to the 5′−3′ exonuclease activity typical of aRNase J homologs. Moreover, RecJ3 and RecJ4 could be purified as a 560-kDa subcomplex in equimolar subunit ratio with nuclease activities mirroring the full RecJ3/4-aRNase J complex. These findings prompted reconstitution assays that suggested RecJ3/4 could suppress, alter, and/or outcompete the nuclease activities of aRNase J. Based on the phenotypic results, this control mechanism of aRNase J by RecJ3/4 is not necessary for cell growth but instead appears important for DNA repair. IMPORTANCE Nucleases are critical for various cellular processes including DNA replication and repair. Here, a dynamic type of nuclease complex is newly identifiedidentifiedidentifiedin the archaeon Haloferax volcanii , which is missing the canonical RNA exosome. The complex, composed of RecJ3, RecJ4, and aRNase J, functions primarily as a 3′−5′ exonuclease and was discovered through its ATP-dependent association with the ubiquitin-like SAMP1 and Cdc48a. aRNase J alone forms a homodimer that has endonuclease function and, thus, is not restricted to 5′−3′ exonuclease activity typical of other aRNase J enzymes. RecJ3/4 appears to suppress, alter, and/or outcompete the nuclease activities of aRNase J. While aRNase J is essential for growth, RecJ3/4, Cdc48a, and SAMPs are important for recovery against DNA damage. These biological distinctions may correlate with the regulated nuclease activity of aRNase J in the RecJ3/4-aRNaseJ complex.

59 BASIC BIOLOGICAL SCIENCES↗

DOE OSTI2021

Metabolic Model of the Nitrogen-Fixing Obligate Aerobe Azotobacter vinelandii Predicts Its Adaptation to Oxygen Concentration and Metal Availability

There is considerable interest in promoting biological nitrogen fixation (BNF) as a mechanism to reduce the inputs of nitrogenous fertilizers in agriculture, but considerable fundamental knowledge gaps still need to be addressed. BNF is catalyzed by nitrogenase, which requires a large input of energy in the form of ATP and low potential electrons. Diazotrophs that respire aerobically have an advantage in meeting the ATP demands of BNF but face challenges in protecting nitrogenase from inactivation by oxygen. Here, we constructed a genome-scale metabolic model of the nitrogen-fixing bacterium Azotobacter vinelandii, which uses a complex respiratory protection mechanism to consume oxygen at a high rate to keep intracellular conditions microaerobic. Our model accurately predicts growth rate under high oxygen and substrate concentrations, consistent with a large electron flux directed to the respiratory protection mechanism. While a partially decoupled electron transport chain compensates for some of the energy imbalance under high-oxygen conditions, it does not account for all substrate intake, leading to increased maintenance rates. Interestingly, the respiratory protection mechanism is required for accurate predictions even when ammonia is supplemented during growth, suggesting that the respiratory protection mechanism might be a core principle of metabolism and not just used for nitrogenase protection. We have also shown that rearrangement of flux through the electron transport system allows A. vinelandii to adapt to different oxygen concentrations, metal availability, and genetic disruption, which cause an ammonia excretion phenotype. Accurately determining the energy balance in an aerobic nitrogen-fixing metabolic model is required for future engineering approaches.

59 BASIC BIOLOGICAL SCIENCES↗

DOE OSTI2024

Insights into posttranslational regulation of skeletal muscle contractile function by the acetyltransferases, p300 and CBP

Here, mice with skeletal muscle-specific and inducible double knockout of the lysine acetyltransferases, p300 (E1A binding protein p300) and CBP (cAMP-response element-binding protein binding protein), referred to as i-mPCKO, demonstrate a dramatic loss of contractile function in skeletal muscle and ultimately die within 7 days. Given that many proteins involved in ATP generation and cross-bridge cycling are acetylated, we investigated whether these processes are dysregulated in skeletal muscle from i-mPCKO mice and, thus, whether they could underlie the rapid loss of muscle contractile function. Just 4–5 days after inducing knockout of p300 and CBP in skeletal muscle from adult i-mPCKO mice, there was ~90% reduction in ex vivo contractile function in the extensor digitorum longus (EDL) and a ~65% reduction in in vivo ankle dorsiflexion torque, as compared with wild type (WT; i.e., Cre negative) littermates. Despite this profound loss of contractile force in i-mPCKO mice, there were no genotype-driven differences in fatigability during repeated contractions, nor were there genotype differences in mitochondrial-specific pathway enrichment of the proteome, intermyofibrillar mitochondrial volume, or mitochondrial respiratory function. As it relates to cross-bridge cycling, remarkably, the overt loss of contractile function in i-mPCKO muscle was reversed in permeabilized fibers supplied with exogenous Ca 2+ and ATP, with active tension being similar between i-mPCKO and WT mice, regardless of Ca 2+ concentration. Actin-myosin motility was also similar in skeletal muscle from i-mPCKO and WT mice. In conclusion, neither mitochondrial abundance/function, nor actomyosin cross-bridge cycling, are the underlying driver of contractile dysfunction in i-mPCKO mice.

59 BASIC BIOLOGICAL SCIENCES↗

DOE OSTI2024

yihuishen/metabolic_flux_regulation

Metabolic efficiency profoundly influences organismal fitness. Heterotrophs, from yeast to mammals, derive usable energy primarily through glycolysis and respiration. While respiration is more energy-efficient, some cells favor glycolysis even when oxygen is available (aerobic glycolysis, Warburg effect). A leading explanation is that glycolysis is more efficient in terms of ATP production per unit mass of protein (i.e. faster). Through quantitative flux analysis and proteomics, we find however that mitochondrial respiration is actually more proteome-efficient than aerobic glycolysis. This is shown across yeasts, T cells, cancer cells, and tissues and tumors in vivo. Instead of aerobic glycolysis being valuable for fast ATP production, it correlates with high glycolytic protein expression, which is valuable for hypoxic growth. Aerobic glycolytic yeasts do not excel at aerobic growth, but outgrow respiratory cells in oxygen limitation. Thus, aerobic glycolysis emerges from cells maintaining a proteome conducive to both aerobic and hypoxic growth.

Shen, Yihui↗

DOE OSTI2024

yihuishen/T_cell_MFA

Shen, Yihui↗

DOE OSTI2021

Altered Cardiac Energetics and Mitochondrial Dysfunction in Hypertrophic Cardiomyopathy

Background: Hypertrophic cardiomyopathy (HCM) is a complex disease partly explained by the effects of individual gene variants on sarcomeric protein biomechanics. At the cellular level, HCM mutations most commonly enhance force production, leading to higher energy demands. Despite significant advances in elucidating sarcomeric structure–function relationships, there is still much to be learned about the mechanisms that link altered cardiac energetics to HCM phenotypes. Here, we test the hypothesis that changes in cardiac energetics represent a common pathophysiologic pathway in HCM. Methods: We performed a comprehensive multiomics profile of the molecular (transcripts, metabolites, and complex lipids), ultrastructural, and functional components of HCM energetics using myocardial samples from 27 HCM patients and 13 normal controls (donor hearts). Results: Integrated omics analysis revealed alterations in a wide array of biochemical pathways with major dysregulation in fatty acid metabolism, reduction of acylcarnitines, and accumulation of free fatty acids. HCM hearts showed evidence of global energetic decompensation manifested by a decrease in high energy phosphate metabolites (ATP, ADP, and phosphocreatine) and a reduction in mitochondrial genes involved in creatine kinase and ATP synthesis. Accompanying these metabolic derangements, electron microscopy showed an increased fraction of severely damaged mitochondria with reduced cristae density, coinciding with reduced citrate synthase activity and mitochondrial oxidative respiration. These mitochondrial abnormalities were associated with elevated reactive oxygen species and reduced antioxidant defenses. However, despite significant mitochondrial injury, HCM hearts failed to upregulate mitophagic clearance. Conclusions: Overall, our findings suggest that perturbed metabolic signaling and mitochondrial dysfunction are common pathogenic mechanisms in patients with HCM. These results highlight potential new drug targets for attenuation of the clinical disease through improving metabolic function and reducing mitochondrial injury.

60 APPLIED LIFE SCIENCES↗

DOE OSTI2026

Data for "Metabolic Engineering Strategies to Produce Medium-Chain Oleochemicals via Acyl-ACP:CoA Transacylase Activity"

Microbial lipid metabolism is an attractive route for producing oleochemicals. The predominant strategy centers on heterologous thioesterases to synthesize desired chain-length fatty acids. To convert acids to oleochemicals (e.g., fatty alcohols, ketones), the narrowed fatty acid pool needs to be reactivated as coenzyme A thioesters at cost of one ATP per reactivation – an expense that could be saved if the acyl-chain was directly transferred from ACP- to CoA-thioester. Here, we demonstrate such an alternative acyl-transferase strategy by heterologous expression of PhaG, an enzyme first identified in Pseudomonads, that transfers 3-hydroxy acyl-chains between acyl-carrier protein and coenzyme A thioester forms for creating polyhydroxyalkanoate monomers. We use it to create a pool of acyl-CoA’s that can be redirected to oleochemical products. Through bioprospecting, mutagenesis, and metabolic engineering, we develop three strains of Escherichia coli capable of producing over 1 g/L of medium-chain free fatty acids, fatty alcohols, and methyl ketones.

Bioproducts↗

DOE OSTI2021

Thermodynamic and Kinetic Modeling of Co-utilization of Glucose and Xylose for 2,3-BDO Production by Zymomonas mobilis

Prior engineering of the ethanologen Zymomonas mobilis has enabled it to metabolize xylose and to produce 2,3-butanediol (2,3-BDO) as a dominant fermentation product. When co-fermenting with xylose, glucose is preferentially utilized, even though xylose metabolism generates ATP more efficiently during 2,3-BDO production on a BDO-mol basis. To gain a deeper understanding of Z. mobilis metabolism, we first estimated the kinetic parameters of the glucose facilitator protein of Z. mobilis by fitting a kinetic uptake model, which shows that the maximum transport capacity of glucose is seven times higher than that of xylose, and glucose is six times more affinitive to the transporter than xylose. With these estimated kinetic parameters, we further compared the thermodynamic driving force and enzyme protein cost of glucose and xylose metabolism. It is found that, although 20% more ATP can be yielded stoichiometrically during xylose utilization, glucose metabolism is thermodynamically more favorable with 6% greater cumulative Gibbs free energy change, more economical with 37% less enzyme cost required at the initial stage and sustains the advantage of the thermodynamic driving force and protein cost through the fermentation process until glucose is exhausted. Glucose-6-phosphate dehydrogenase (g6pdh), glyceraldehyde-3-phosphate dehydrogenase (gapdh) and phosphoglycerate mutase (pgm) are identified as thermodynamic bottlenecks in glucose utilization pathway, as well as two more enzymes of xylose isomerase and ribulose-5-phosphate epimerase in xylose metabolism. Acetolactate synthase is found as potential engineering target for optimized protein cost supporting unit metabolic flux. Pathway analysis was then extended to the core stoichiometric matrix of Z. mobilis metabolism. Growth was simulated by dynamic flux balance analysis and the model was validated showing good agreement with experimental data. Dynamic FBA simulations suggest that a high agitation is preferable to increase 2,3-BDO productivity while a moderate agitation will benefit the 2,3-BDO titer. Taken together, this work provides thermodynamic and kinetic insights of Z. mobilis metabolism on dual substrates, and guidance of bioengineering efforts to increase hydrocarbon fuel production.

23-butanediol↗

DOE OSTI2022

Genome-Wide Association Study Reveals Complex Genetic Architecture of Cadmium and Mercury Accumulation and Tolerance Traits in Medicago truncatula

Heavy metals are an increasing problem due to contamination from human sources that and can enter the food chain by being taken up by plants. Understanding the genetic basis of accumulation and tolerance in plants is important for reducing the uptake of toxic metals in crops and crop relatives, as well as for removing heavy metals from soils by means of phytoremediation. Following exposure of Medicago truncatula seedlings to cadmium (Cd) and mercury (Hg), we conducted a genome-wide association study using relative root growth (RRG) and leaf accumulation measurements. Cd and Hg accumulation and RRG had heritability ranging 0.44 – 0.72 indicating high genetic diversity for these traits. The Cd and Hg trait associations were broadly distributed throughout the genome, indicated the traits are polygenic and involve several quantitative loci. For all traits, candidate genes included several membrane associated ATP-binding cassette transporters, P-type ATPase transporters, oxidative stress response genes, and stress related UDP-glycosyltransferases. The P-type ATPase transporters and ATP-binding cassette protein-families have roles in vacuole transport of heavy metals, and our findings support their wide use in physiological plant responses to heavy metals and abiotic stresses. We also found associations between Cd RRG with the genes CAX3 and PDR3, two linked adjacent genes, and leaf accumulation of Hg associated with the genes NRAMP6 and CAX9. When plant genotypes with the most extreme phenotypes were compared, we found significant divergence in genomic regions using population genomics methods that contained metal transport and stress response gene ontologies. Several of these genomic regions show high linkage disequilibrium (LD) among candidate genes suggesting they have evolved together. Minor allele frequency (MAF) and effect size of the most significant SNPs was negatively correlated with large effect alleles being most rare. This is consistent with purifying selection against alleles that increase toxicity and abiotic stress. Conversely, the alleles with large affect that had higher frequencies that were associated with the exclusion of Cd and Hg. Overall, macroevolutionary conservation of heavy metal and stress response genes is important for improvement of forage crops by harnessing wild genetic variants in gene banks such as the Medicago HapMap collection.

59 BASIC BIOLOGICAL SCIENCES↗

DOE OSTI2024

Polyphosphate kinase deletion increases laboratory productivity in cyanobacteria

Identification and manipulation of cellular energy regulation mechanisms may be a strategy to increase productivity in photosynthetic organisms. This work tests the hypothesis that polyphosphate synthesis and degradation play a role in energy management by storing or dissipating energy in the form of ATP. A polyphosphate kinase ( ppk ) knock-out strain unable to synthesize polyphosphate was generated in the cyanobacterium Synechocystis sp. PCC 6803. This mutant strain demonstrated higher ATP levels and faster growth than the wildtype strain in high-carbon conditions and had a growth defect under multiple stress conditions. In a strain that combined ppk deletion with heterologous expression of ethylene-forming enzyme, higher ethylene productivity was observed than in the wildtype background. These results support the role of polyphosphate synthesis and degradation as an energy regulation mechanism and suggest that such mechanisms may be effective targets in biocontainment design.

59 BASIC BIOLOGICAL SCIENCES↗

DOE OSTI2022

Structure and Function of the Zinc Binding Protein ZrgA from Vibrio cholerae

ATP binding cassette (ABC) transporters are the primary means by which bacteria acquire trace elements from the environment. They rely on solute binding proteins (SBPs) to bind the relevant substrate and deliver it to the integral membrane permease for ATP-powered import into the cytoplasm. SBPs of cluster A-I are known to facilitate the transport of essential metals zinc, manganese, and iron, and many have been characterized to date. A group of ABC transporter operons dubbed zinc-regulated genes (zrg) have recently been shown to transport zinc with putative SBPs (zrgA) bearing no homology to the classical cluster A-I family, and a recent crystal structure of a representative protein from Pseudomonas aeruginosa shows no structural similarity to classical SBPs. Thus, the ZrgA proteins appear to represent a newly discovered family of zinc SBPs widespread among Gram-negative bacteria, including human pathogens. Here, we have determined the crystal structure of ZrgA from Vibrio cholerae and characterized its zinc binding in vitro and function in vivo. We also assessed the role of a histidine-rich sequence that appears to be a hallmark of ZrgA proteins that is particularly long in V. cholerae ZrgA. The results show that the zrgA gene is critical to the function of the operon, consistent with a function as an SBP in this system. Further, the His-rich region is not essential to the function of ZrgA, but it does provide additional zinc binding sites in vitro. The structure and zinc binding data for ZrgA reveal interesting differences between it and its homologue from P. aeruginosa, illustrating diversity within this little-studied protein family.

59 BASIC BIOLOGICAL SCIENCES↗

DOE OSTI2025

Long-Range Allosteric Communication Modulated by Active Site Mn(II) Coordination Drives Catalysis in Xanthobacter autotrophicus Acetone Carboxylase

Acetone carboxylase (AC) from Xanthobacter autotrophicus is a 360 KDa α2β2γ2 heterohexamer that catalyzes the ATP-dependent formation of phosphorylated acetone and bicarbonate intermediates that react at Mn(II) metal active sites to form acetoacetate. Structural models of X. autotrophicus AC (XaAC) with and without nucleotides reveal that the binding and phosphorylation of the two substrates occurs ~40 Å from the Mn(II) active sites where acetoacetate is formed. Based on the crystal structures, a significant conformational change was proposed to open and close a tunnel that facilitates the passage of reaction intermediates between the sites for nucleotide binding and phosphorylation of substrates and Mn(II) sites of acetoacetate formation. We have employed electron paramagnetic resonance (EPR), kinetic assays, and hydrogen/deuterium exchange mass spectrometry (HDX-MS) of poised ligand-bound states and site-specific amino acid variants to complete an in-depth analysis of Mn(II) coordination and allosteric communication throughout the catalytic cycle. In contrast with the established paradigms for carboxylation, our analyses of XaAC suggested a carboxylate shift that couples both local and long-range structural transitions. Shifts in the coordination mode of a single carboxylic acid residue (αE89) mediate both catalysis proximal to a Mn(II) center and communication with an ATP active site in a separate subunit of a 180 kDa α2β2γ2 complex at a distance of 40 Å. This work demonstrates the power of combining structural models from X-ray crystallography with solution-phase spectroscopy and biophysical techniques to elucidate functional aspects of a multi-subunit enzyme.

Biochemistry & Molecular Biology↗

DOE OSTI2022

Selenocyanate derived Se-incorporation into the nitrogenase Fe protein cluster

The nitrogenase Fe protein mediates ATP-dependent electron transfer to the nitrogenase MoFe protein during nitrogen fixation, in addition to catalyzing MoFe protein-independent substrate (CO 2 ) reduction and facilitating MoFe protein metallocluster biosynthesis. The precise role(s) of the Fe protein Fe 4 S 4 cluster in some of these processes remains ill-defined. Herein, we report crystallographic data demonstrating ATP-dependent chalcogenide exchange at the Fe 4 S 4 cluster of the nitrogenase Fe protein when potassium selenocyanate is used as the selenium source, an unexpected result as the Fe protein cluster is not traditionally perceived as a site of substrate binding within nitrogenase. The observed chalcogenide exchange illustrates that this Fe 4 S 4 cluster is capable of core substitution reactions under certain conditions, adding to the Fe protein’s repertoire of unique properties.

59 BASIC BIOLOGICAL SCIENCES↗

DOE OSTI2021

Initial Feasibility and Clinical Implementation of Daily MR-Guided Adaptive Head and Neck Cancer Radiation Therapy on a 1.5T MR-Linac System: Prospective R-IDEAL 2a/2b Systematic Clinical Evaluation of Technical Innovation

This prospective study is, to our knowledge, the first report of daily adaptive radiation therapy (ART) for head and neck cancer (HNC) using a 1.5T magnetic resonance imaging-linear accelerator (MR-linac) with particular focus on safety and feasibility and dosimetric results of an online rigid registration-based adapt to position (ATP) workflow.

62 RADIOLOGY AND NUCLEAR MEDICINE↗