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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 289 records · Page 16
All sourcesNASA NTRSDOE OSTI
DOE OSTI2016

Negative Cooperativity in the Nitrogenase Fe Protein Electron Delivery Cycle

Nitrogenase catalyzes the ATP-dependent reduction of dinitrogen (N2) to two ammonia (NH3) molecules. Catalysis involves the participation of two protein components designated the MoFe and Fe proteins. Electron transfer (ET) from the Fe protein to the catalytic MoFe protein involves a series of synchronized events requiring the transient association of one Fe protein with each aß half of the MoFe protein. This process is referred to as the Fe protein cycle and includes: binding of two ATP to an Fe protein, association of an Fe protein with the MoFe protein, ET from the Fe protein to the MoFe protein, hydrolysis of two ATP to two ADP and two Pi for each ET, Pi release, and dissociation of oxidized Fe protein-(ADP)2 from the MoFe protein. Because the MoFe protein tetramer has two separate aß active units, it has the capacity to participate in two independent Fe protein cycles. The present work examines whether the two catalytic halves of an individual MoFe protein function independently or, instead, communicate with each other during catalysis. Kinetic analyses of ET, ATP hydrolysis, and Pi release during the pre-steady state phase indicate that the two halves of the complex in fact cooperate. A global fit of the data is consistent with a two-branch negative-cooperativity kinetic model. A possible mechanism for communication between the two halves of nitrogenase is suggested by normal-mode calculations suggesting correlated and anti-correlated motions between the two nitrogenase halves.

Danyal, Karamatullah↗
DOE OSTI2025

An argument for using anaerobes as microbial cell factories to advance synthetic biology and biomanufacturing

Anaerobes thrive in the absence of oxygen and are an untapped reservoir of biotechnological potential. Therefore, bioprospecting efforts focused on anaerobic microbial diversity could rapidly uncover new enzymes, pathways, and chassis organisms to drive biotechnology innovation. Despite their potential utility, anaerobic fermenters are viewed as inefficient from a biochemical perspective because their metabolisms produce fewer ATP (~2) per molecule of glucose processed than heterotrophic respirers (~32–38 ATP). While aerobes excel at ATP generation, they are often less efficient than anaerobes at processes that compete with ATP generation for cellular resources. This perspective highlights how anaerobic adaptations are advantageous for synthetic biology and biomanufacturing applications through the engineering of microbial cell factories. We further highlight emerging applications of anaerobic bioprocessing, including the use of anaerobic metabolisms for lignocellulosic bioprocessing, human and environmental health, and value-added bioproduction.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2019

Partitioning of Recombinant Pseudomonas putida POS-F84 Proline Dehydrogenase in Aqueous Two-Phase Systems: Optimization Using Response Surface Methodology

Empirical modeling the partition behavior and recovery of a recombinant Pseudomonas putida POS-F84 proline dehydrogenase (ProDH) in aqueous two-phase systems (ATPS) was carried out by response surface methodology (RSM). Polyethylene glycol 1000 (PEG-1000) concentration, sodium carbonate concentration, and pH, which were the most important factors, were chosen for modeling the partition feature of enzyme. The adequacy of the models was investigated by means of variance analysis. Also, to confirm the efficiency of the ATPS in partition and purification of recombinant ProDH, purity and enzymatic activity was studied. After numerical optimization, an optimal ATPS composed of 14.33% PEG-1000 and 11.79% sodium carbonate at pH 7.48 was achieved. Yield, purification factor, and recovery were 81.41%, 60.82, and 270.82%, respectively. Purified recombinant ProDH was found as a single protein band into the upper PEG-rich phase and the specific activity was calculated to be 46.23 ± 2.1 U/mg. Collectively, our data showed that the RSM could be an appropriate and powerful tool to define the best ATPS system for recovery and purification of P. putida ProDH.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2023

On the co-nucleation of adjoining twin pairs at grain boundaries in hexagonal close-packed materials

Here this work addresses the formation of adjoining twin pairs (ATPs) at grain boundaries (GBs) in hexagonal close-packed (hcp) metals from the perspective of the co-nucleation (CN) of pairs of deformation twins. A continuum defect mechanics model is proposed to investigate the energetic feasibility of the CN of ATPs resulting from the dissociation of GB dislocations. Application of the model to a set of GBs in Mg reveals that CN is largely preferred over the nucleation of a single twin variant for low misorientation angle GBs, which is consistent with previous experimental analyses. The CN events are subsequently analyzed considering the GB character, and the twin system alignment ($m$') and Schmid factors to obtain better insights into the conditions governing the formation of ATPs via CN. The likelihood of CN even at low $m$' values reveals that CN events could be responsible for the formation of ATP pairs in these scenarios.

36 MATERIALS SCIENCE↗
DOE OSTI2017

Periplasmic Binding Protein Dimer Has a Second Allosteric Event Tied to Ligand Binding

Here, the ligand-induced conformational changes of periplasmic binding proteins (PBP) play a key role in the acquisition of metabolites in ATP binding cassette (ABC) transport systems. This conformational change allows for differential recognition of the ligand occupancy of the PBP by the ABC transporter. This minimizes futile ATP hydrolysis in the transporter, a phenomenon in which ATP hydrolysis is not coupled to metabolite transport. In many systems, the PBP conformational change is insufficient at eliminating futile ATP hydrolysis. Here we identify an additional state of the PBP that is also allosterically regulated by the ligand. Ligand binding to the homodimeric apo PBP leads to a tightening of the interface alpha-helices so that the hydrogen bonding pattern shifts to that of a 3 10 helix, in-turn altering the contacts and the dynamics of the protein interface so that the monomer exists in the presence of ligand.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗
DOE OSTI2014

Mechanistic determinants of the directionality and energetics of active export by a heterodimeric ABC transporter

The ATP-binding cassette (ABC) transporter associated with antigen processing (TAP) participates in immune surveillance by moving proteasomal products into the endoplasmic reticulum (ER) lumen for major histocompatibility complex class I loading and cell surface presentation to cytotoxic T cells. Here we delineate the mechanistic basis for antigen translocation. Notably, TAP works as a molecular diode, translocating peptide substrates against the gradient in a strict unidirectional way. We reveal the importance of the D-loop at the dimer interface of the two nucleotide-binding domains (NBDs) in coupling substrate translocation with ATP hydrolysis and defining transport vectoriality. Substitution of the converved aspartate, which coordinates the ATP-binding site, decreases NBD dimerization affinity and turns the unidirectional primary active pump into a passive bidirectional nucleotide-gated facilitator. Thus, ATP hydrolysis is not required for translocation per se , but is essential for both active and unidirectional transport. As a result, our data provide detailed mechanistic insight into how heterodimeric ABC exporters operate.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2019

Characterizing the role of adjoining twins at grain boundaries in hexagonal close packed materials

Abstract Hexagonal close packed (HCP) Mg and Zr are being used in transportation and nuclear industries, respectively. The ductility and formability of these materials is significantly limited by the activation of prevalent deformation twinning. Twins in HCP polycrystals usually nucleate at grain boundaries (GBs), propagate into the grain, and they either terminate at opposing GBs (isolated-twins) or transmit into a neighboring grain (adjoining-twin-pairs: ATPs). Because twin interfaces provide a path for crack propagation, twin transmission is relevant to material ductility. This study combines electron backscatter diffraction (EBSD) based statistical analysis of twinning microstructures and crystal plasticity modeling, to characterize twin thickening processes away from and near GBs. Analysis of deformed Mg and Zr microstructures reveals that local twin thicknesses at GBs are statistically larger for ATPs compared to isolated-twins. Further, thicknesses are found to decrease with increasing GB misorientation angle. Full-field Fast-Fourier-Transform micromechanics modeling shows that shear-transformation induced backstress are locally relaxed at GBs for ATPs, but not for isolated-twins. As a consequence, ATPs can thicken locally at GBs and the preferential site for twin thickening shifts from the middle of the twin to common GB.

36 MATERIALS SCIENCE↗
DOE OSTI2017

Systems-level understanding of ethanol-induced stresses and adaptation in E. coli

Understanding ethanol-induced stresses and responses in biofuel-producing bacteria at systems level has significant implications in engineering more efficient biofuel producers. We present a computational study of transcriptomic and genomic data of both ethanol-stressed and ethanol-adapted E. coli cells with computationally predicated ethanol-binding proteins and experimentally identified ethanol tolerance genes. Our analysis suggests: (1) ethanol damages cell wall and membrane integrity, causing increased stresses, particularly reactive oxygen species, which damages DNA and reduces the O 2 level; (2) decreased cross-membrane proton gradient from membrane damage, coupled with hypoxia, leads to reduced ATP production by aerobic respiration, driving cells to rely more on fatty acid oxidation, anaerobic respiration and fermentation for ATP production; (3) the reduced ATP generation results in substantially decreased synthesis of macromolecules; (4) ethanol can directly bind 213 proteins including transcription factors, altering their functions; (5) all these changes together induce multiple stress responses, reduced biosynthesis, cell viability and growth; and (6) ethanol-adapted E. coli cells restore the majority of these reduced activities through selection of specific genomic mutations and alteration of stress responses, ultimately restoring normal ATP production, macromolecule biosynthesis, and growth. These new insights into the energy and mass balance will inform design of more ethanol-tolerant strains.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2019

Statistical properties of autonomous flows in 2D active nematics

We study the dynamics of a tunable 2D active nematic liquid crystal composed of microtubules and kinesin motors confined to an oil–water interface. Kinesin motors continuously inject mechanical energy into the system through ATP hydrolysis, powering the relative microscopic sliding of adjacent microtubules, which in turn generates macroscale autonomous flows and chaotic dynamics. We use particle image velocimetry to quantify two-dimensional flows of active nematics and extract their statistical properties. In agreement with the hydrodynamic theory, we find that the vortex areas comprising the chaotic flows are exponentially distributed, which allows us to extract the characteristic system length scale. We probe the dependence of this length scale on the ATP concentration, which is the experimental knob that tunes the magnitude of the active stress. In conclusion, our data suggest a possible mapping between the ATP concentration and the active stress that is based on the Michaelis–Menten kinetics that governs the motion of individual kinesin motors.

99 GENERAL AND MISCELLANEOUS↗
DOE OSTI2021

Probing remdesivir nucleotide analogue insertion to SARS-CoV-2 RNA dependent RNA polymerase in viral replication

Remdesivir (RDV) prodrug can be metabolized into a triphosphate form nucleotide analogue (RDV-TP) to bind and insert into the active site of viral RNA dependent RNA polymerase (RdRp) to further interfere with viral genome replication. Here in this work, we computationally studied how RDV-TP binds and inserts to the SARS-CoV-2 RdRp active site, in comparison with natural nucleotide substrate adenosine triphosphate (ATP). To do that, we first constructed atomic structural models of an initial binding complex (active site open) and a substrate insertion complex (active site closed), based on high-resolution cryo-EM structures determined recently for SARS-CoV-2 RdRp or non-structural protein (nsp) 12, in complex with accessory protein factors nsp7 and nsp8. By conducting all-atom molecular dynamics simulation with umbrella sampling strategies on the nucleotide insertion between the open and closed state RdRp complexes, our studies show that RDV-TP can initially bind in a comparatively stabilized state to the viral RdRp active site, as it primarily forms base stacking with the template uracil nucleotide (nt +1), which under freely fluctuations supports a low free energy barrier of the RDV-TP insertion (~1.5 kcal mol -1 ). In comparison, the corresponding natural substrate ATP binds initially to the RdRp active site in Watson–Crick base pairing with the template nt, and inserts into the active site with a medium low free energy barrier (~2.6 kcal mol -1 ), when the fluctuations of the template nt are well quenched. The simulations also show that the initial base stacking of RDV-TP with the template can be specifically stabilized by motif C-S759, S682 (near motif B) with the base, and motif G-K500 with the template backbone. Although the RDV-TP insertion can be hindered by motif F-R555/R553 interaction with the triphosphate, the ATP insertion seems to be facilitated by such interactions. The inserted RDV-TP and ATP can be further distinguished by specific sugar interaction with motif B-T687 and motif A-D623, respectively.

36 MATERIALS SCIENCE↗
DOE OSTI2015

Mycobacterial nicotinate mononucleotide adenylyltransferase: Structure, mechanism, and implications for drug discovery

Nicotinate mononucleotide adenylyltransferase NadD is an essential enzyme in the biosynthesis of the NAD cofactor, which has been implicated as a target for developing new antimycobacterial therapies. Here we report the crystal structure of Mycobacterium tuberculosis NadD ( Mt NadD) at a resolution of 2.4 Å. A remarkable new feature of the MtNadD structure, compared with other members of this enzyme family, is a 310 helix that locks the active site in an over-closed conformation. As a result, Mt NadD is rendered inactive as it is topologically incompatible with substrate binding and catalysis. Directed mutagenesis was also used to further dissect the structural elements that contribute to the interactions of the two Mt NadD substrates, i.e. ATP and nicotinic acid mononucleotide (NaMN). For inhibitory profiling of partially active mutants and wild type Mt NadD, we used a small molecule inhibitor of MtNadD with moderate affinity ( K i ~ 25 μM) and antimycobacterial activity (MIC 80 ) ~ 40-80 μM). This analysis revealed interferences with some of the residues in the NaMN binding subsite consistent with the competitive inhibition observed for the NaMN substrate (but not ATP). A detailed steady-state kinetic analysis of Mt NadD suggests that ATP must first bind to allow efficient NaMN binding and catalysis. This sequential mechanism is consistent with the requirement of transition to catalytically competent (open) conformation hypothesized from structural modeling. A possible physiological significance of this mechanism is to enable the down-regulation of NAD synthesis under ATP-limiting dormancy conditions. Lastly, these findings point to a possible new strategy for designing inhibitors that lock the enzyme in the inactive over-closed conformation.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2019

Structures of maintenance of carboxysome distribution Walker-box McdA and McdB adaptor homologs

Carboxysomes, protein-coated organelles in cyanobacteria, are important in global carbon fixation. However, these organelles are present at low copy in each cell and hence must be segregated to ensure transmission from one generation to the next. Recent studies revealed that a DNA partitionlike ParA–ParB system mediates carboxysome maintenance, called McdA-McdB. Here, we describe the first McdA and McdB homolog structures. McdA is similar to partition ParA Walker-box proteins, but lacks the P-loop signature lysine involved in ATP binding. Strikingly, a McdA-ATP structure shows that a lysine distant from the P-loop and conserved in McdA homologs, enables ATP-dependent nucleotide sandwich dimer formation. Similar to partition ParA proteins this ATP-bound form binds nonspecific-DNA. McdB, which we show directly binds McdA, harbors a unique fold and appears to form higher-order oligomers like partition ParB proteins. Thus, our data reveal a new signature motif that enables McdA dimer formation and indicates that, similar to DNA segregation, carboxysome maintenance systems employ Walker-box proteins as DNA-binding motors while McdB proteins form higher order oligomers, which could function as adaptors to link carboxysomes and provide for stable transport by the McdA proteins.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2015

Structure of AadA from Salmonella enterica: a monomeric aminoglycoside (3′′)(9) adenyltransferase

The crystal structure of the aminoglycoside-adenylating enzyme AadA is reported together with functional experiments providing insights into its oligomeric state, ligand binding and catalysis. Aminoglycoside resistance is commonly conferred by enzymatic modification of drugs by aminoglycoside-modifying enzymes such as aminoglycoside nucleotidyltransferases (ANTs). Here, the first crystal structure of an ANT(3′′)(9) adenyltransferase, AadA from Salmonella enterica, is presented. AadA catalyses the magnesium-dependent transfer of adenosine monophosphate from ATP to the two chemically dissimilar drugs streptomycin and spectinomycin. The structure was solved using selenium SAD phasing and refined to 2.5 Å resolution. AadA consists of a nucleotidyltransferase domain and an α-helical bundle domain. AadA crystallizes as a monomer and is a monomer in solution as confirmed by small-angle X-ray scattering, in contrast to structurally similar homodimeric adenylating enzymes such as kanamycin nucleotidyltransferase. Isothermal titration calorimetry experiments show that ATP binding has to occur before binding of the aminoglycoside substrate, and structure analysis suggests that ATP binding repositions the two domains for aminoglycoside binding in the interdomain cleft. Candidate residues for ligand binding and catalysis were subjected to site-directed mutagenesis. In vivo resistance and in vitro binding assays support the role of Glu87 as the catalytic base in adenylation, while Arg192 and Lys205 are shown to be critical for ATP binding.

75 CONDENSED MATTER PHYSICS, SUPERCONDUCTIVITY AND↗
DOE OSTI2016

Mechanistic insight into protein modification and sulfur mobilization activities of noncanonical E1 and associated ubiquitin-like proteins of Archaea

Here we provide the first detailed biochemical study of a noncanonical E1-like enzyme with broad specificity for cognate ubiquitin-like (Ubl) proteins that mediates Ubl protein modification and sulfur mobilization to form molybdopterin and thiolated tRNA. Isothermal titration calorimetry and in vivo analyses proved useful in discovering that environmental conditions, ATP binding and Ubl type controlled the mechanism of association of the Ubl protein with its cognate E1-like enzyme (SAMP and UbaA of the archaeon Haloferax volcanii, respectively). Further analysis revealed ATP hydrolysis triggered the formation of thioester and peptide bonds within the Ubl:E1-like complex. Importantly, the thioester was an apparent precursor to Ubl protein modification but not sulfur mobilization. Comparative modeling to MoeB/ThiF guided the discovery of key residues within the adenylation domain of UbaA that were needed to bind ATP as well as residues that were specifically needed to catalyze the downstream reactions of sulfur mobilization and/or Ubl protein modification. UbaA was also found to be Ubl-automodified at lysine residues required for early (ATP binding) and late (sulfur mobilization) stages of enzyme activity revealing multiple layers of auto-regulation. Cysteine residues, distinct from the canonical E1 ‘active site’ cysteine, were found important in UbaA function supporting a model that this non-canonical E1 is structurally flexible in its active site to allow Ubl~adenylate, Ubl~E1-like thioester and cysteine persulfide(s) intermediates to form.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2025

Biochemical properties of glycerol kinase from the hypersaline-adapted archaeon Haloferax volcanii

ABSTRACT Extremophilic microorganisms are promising candidates for industrial and analytical biocatalysis.Haloferax volcanii, a halophilic archaeon that prefers glycerol over glucose, channels this substrate into central metabolism through glycerol kinase (GK). Here, we report the biochemical properties ofH. volcaniiGK and its potential for biotechnological applications. An N-terminal His-tagged GK was functionalin vivoand yielded 3 mg/L culture—4.5 times more enzyme than a C-terminal StrepII-tagged version. Size exclusion chromatography revealed a glycerol-induced oligomeric shift from homodimer to a dimer-dominant state with detectable tetramer. The purified enzyme showed robust activity across broad pH and salinity ranges, with optimal activity at 100 mM NaCl and 50°C–60°C. It retained catalytic activity in 5%–10% dimethyl sulfoxide (DMSO) and crude glycerol containing methanol. His-GK was freeze-thaw stable and thermotolerant in 2 M NaCl buffers. In the absence of ligands, the enzyme’s melting temperature (T m ) was 80°C. Glycerol increased the T m to 85°C, and combinations with MgCl₂ (84°C) or ATP (88°C) provided further stabilization. The highest T m (89°C) occurred with all three ligands, suggesting a cumulative stabilizing effect. Kinetic analyses revealed positive cooperativity for glycerol, ATP, and Mg² + ; Mn² + and Co² + also supported the activity.H. volcaniiGK is the first known GK to exhibit positive cooperativity with glycerol and ATP. Its high stability and substrate flexibility support its use in biodiesel waste valorization,in vitrobiocatalysis, and biosensor development—applications demanding robust, specific, and stable enzymes. IMPORTANCE This study reveals thatH. volcaniiGK exhibits positive cooperativity for glycerol, ATP, and Mg² + , a kinetic feature not previously reported for glycerol kinases. This behavior enables steep, switch-like responses to small substrate changes, offering unique advantages for biosensor design. Importantly,H. volcaniiGK also maintains high activity under extreme salinity, temperature, broad pH, and solvent conditions that typically limit enzyme use in industrial and environmental applications. These traits make this GK an ideal candidate for enzyme-based biosensors, which often suffer from poor tolerance to pH, solvent, and thermal stress. Its robustness supports its use in cross-linked enzyme crystals, an immobilization method that enhances enzyme stability and reusability under harsh conditions. Moreover, GKs are already employed in Mg² + detection kits; however,H. volcaniiGK’s ability to tolerate and respond to diverse divalent cations (e.g., Co² + , Mn² + ) broadens their potential for pollutant detection and environmental monitoring. These features collectively positionH. volcaniiGK as a valuable biocatalyst for biosensing,in vitrodiagnostics, and biotechnological applications requiring both precision and durability.

Biotechnology & Applied Microbiology↗
DOE OSTI2014

Unique Helicase Determinants in the Essential Conjugative TraI Factor from Salmonella enterica Serovar Typhimurium Plasmid pCU1

The widespread development of multidrug-resistant bacteria is a major health emergency. Conjugative DNA plasmids, which harbor a wide range of antibiotic resistance genes, also encode the protein factors necessary to orchestrate the propagation of plasmid DNA between bacterial cells through conjugative transfer. Successful conjugative DNA transfer depends on key catalytic components to nick one strand of the duplex DNA plasmid and separate the DNA strands while cell-to-cell transfer occurs. The TraI protein from the conjugative Salmonella plasmid pCU1 fulfills these key catalytic roles, as it contains both single-stranded DNA-nicking relaxase and ATP-dependent helicase domains within a single, 1,078-residue polypeptide. In this work, we unraveled the helicase determinants of Salmonella pCU1 TraI through DNA binding, ATPase, and DNA strand separation assays. TraI binds DNA substrates with high affinity in a manner influenced by nucleic acid length and the presence of a DNA hairpin structure adjacent to the nick site. TraI selectively hydrolyzes ATP, and mutations in conserved helicase motifs eliminate ATPase activity. Surprisingly, the absence of a relatively short (144-residue) domain at the extreme C terminus of the protein severely diminishes ATP-dependent strand separation. Collectively, these data define the helicase motifs of the conjugative factor TraI from Salmonella pCU1 and reveal a previously uncharacterized C-terminal functional domain that uncouples ATP hydrolysis from strand separation activity.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2017

Cellobiose Consumption Uncouples Extracellular Glucose Sensing and Glucose Metabolism in Saccharomyces cerevisiae

Glycolysis is central to energy metabolism in most organisms and is highly regulated to enable optimal growth. In the yeast Saccharomyces cerevisiae, feedback mechanisms that control flux through glycolysis span transcriptional control to metabolite levels in the cell. Using a cellobiose consumption pathway, we decoupled glucose sensing from carbon utilization, revealing new modular layers of control that induce ATP consumption to drive rapid carbon fermentation. Alterations of the beta subunit of phosphofructokinase-1 (PFK2), H + -plasma membrane ATPase (PMA1), and glucose sensors (SNF3 and RGT2) revealed the importance of coupling extracellular glucose sensing to manage ATP levels in the cell. Controlling the upper bound of cellular ATP levels may be a general mechanism used to regulate energy levels in cells, via a regulatory network that can be uncoupled from ATP concentrations under perceived starvation conditions.

59 BASIC BIOLOGICAL SCIENCES↗