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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 OSTI2022

Imaging active site chemistry and protonation states: NMR crystallography of the tryptophan synthase α-aminoacrylate intermediate

NMR-assisted crystallography-the integrated application of solid-state NMR, X-ray crystallography, and first-principles computational chemistry-holds significant promise for mechanistic enzymology: by providing atomic-resolution characterization of stable intermediates in enzyme active sites, including hydrogen atom locations and tautomeric equilibria, NMR crystallography offers insight into both structure and chemical dynamics. Here, this integrated approach is used to characterize the tryptophan synthase α-aminoacrylate intermediate, a defining species for pyridoxal-5'-phosphate-dependent enzymes that catalyze β-elimination and replacement reactions. For this intermediate, NMR-assisted crystallography is able to identify the protonation states of the ionizable sites on the cofactor, substrate, and catalytic side chains as well as the location and orientation of crystallographic waters within the active site. Most notable is the water molecule immediately adjacent to the substrate β-carbon, which serves as a hydrogen bond donor to the ε-amino group of the acid-base catalytic residue βLys87. From this analysis, a detailed three-dimensional picture of structure and reactivity emerges, highlighting the fate of the L-serine hydroxyl leaving group and the reaction pathway back to the preceding transition state. Reaction of the α-aminoacrylate intermediate with benzimidazole, an isostere of the natural substrate indole, shows benzimidazole bound in the active site and poised for, but unable to initiate, the subsequent bond formation step. When modeled into the benzimidazole position, indole is positioned with C3 in contact with the α-aminoacrylate C $\bar{β}$ and aligned for nucleophilic attack. Here, the chemically detailed, three-dimensional structure from NMR-assisted crystallography is key to understanding why benzimidazole does not react, while indole does.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗
DOE OSTI2022

Toward a glycyl radical enzyme containing synthetic bacterial microcompartment to produce pyruvate from formate and acetate

Formate has great potential to function as a feedstock for biorefineries because it can be sustainably produced by a variety of processes that don’t compete with agricultural production. However, naturally formatotrophic organisms are unsuitable for large-scale cultivation, difficult to engineer, or have inefficient native formate assimilation pathways. Thus, metabolic engineering needs to be developed for model industrial organisms to enable efficient formatotrophic growth. Here, we build a prototype synthetic formate utilizing bacterial microcompartment (sFUT) encapsulating the oxygen-sensitive glycyl radical enzyme pyruvate formate lyase and a phosphate acyltransferase to convert formate and acetyl-phosphate into the central biosynthetic intermediate pyruvate. This metabolic module offers a defined environment with a private cofactor coenzyme A that can cycle efficiently between the encapsulated enzymes. To facilitate initial design-build-test-refine cycles to construct an active metabolic core, we used a “wiffleball” architecture, defined as an icosahedral bacterial microcompartment (BMC) shell with unoccupied pentameric vertices to freely permit substrate and product exchange. The resulting sFUT prototype wiffleball is an active multi enzyme synthetic BMC functioning as platform technology.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2022

Cryo-EM structure of RNA-induced tau fibrils reveals a small C-terminal core that may nucleate fibril formation

In neurodegenerative diseases including Alzheimer’s and amyotrophic lateral sclerosis, proteins that bind RNA are found in aggregated forms in autopsied brains. Evidence suggests that RNA aids nucleation of these pathological aggregates; however, the mechanism has not been investigated at the level of atomic structure. Here, we present the 3.4-Å resolution structure of fibrils of full-length recombinant tau protein in the presence of RNA, determined by electron cryomicroscopy (cryo-EM). The structure reveals the familiar in-register cross-β amyloid scaffold but with a small fibril core spanning residues Glu391 to Ala426, a region disordered in the fuzzy coat in all previously studied tau polymorphs. RNA is bound on the fibril surface to the positively charged residues Arg406 and His407 and runs parallel to the fibril axis. The fibrils dissolve when RNase is added, showing that RNA is necessary for fibril integrity. While this structure cannot exist simultaneously with the tau fibril structures extracted from patients’ brains, it could conceivably account for the nucleating effects of RNA cofactors followed by remodeling as fibrils mature.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2022

Proton-coupled electron transfer of macrocyclic ring hydrogenation: The chlorinphlorin

Significance The chemical reduction of unsaturated bonds occurs by hydrogenation with H 2 as the reductant. Conversely, in biology, the unavailability of H 2 engenders the typical reduction of unsaturated bonds with electrons and protons from different cofactors, requiring olefin hydrogenation to occur by proton-coupled electron transfer (PCET). Moreover, the redox noninnocence of tetrapyrrole macrocycles furnishes unusual PCET intermediates, including the phlorin, which is an intermediate in tetrapyrrole ring reductions. Whereas the phlorin of a porphyrin is well established, the phlorin of a chlorin is enigmatic. By controlling the PCET reactivity of a chlorin, including the use of a hangman functionality to manage the proton transfer, the formation of a chlorinphlorin by PCET is realized, and the mechanism for its formation is defined.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗
DOE OSTI2022

Root angle is controlled by EGT1 in cereal crops employing an antigravitropic mechanism

Root angle in crops represents a key trait for efficient capture of soil resources. Root angle is determined by competing gravitropic versus antigravitropic offset (AGO) mechanisms. Here we report a root angle regulatory gene termed ENHANCED GRAVITROPISM1 ( EGT1 ) that encodes a putative AGO component, whose loss-of-function enhances root gravitropism. Mutations in barley and wheat EGT1 genes confer a striking root phenotype, where every root class adopts a steeper growth angle. EGT1 encodes an F-box and Tubby domain-containing protein that is highly conserved across plant species. Haplotype analysis found that natural allelic variation at the barley EGT1 locus impacts root angle. Gravitropic assays indicated that Hvegt1 roots bend more rapidly than wild-type. Transcript profiling revealed Hvegt1 roots deregulate reactive oxygen species (ROS) homeostasis and cell wall-loosening enzymes and cofactors. ROS imaging shows that Hvegt1 root basal meristem and elongation zone tissues have reduced levels. Atomic force microscopy measurements detected elongating Hvegt1 root cortical cell walls are significantly less stiff than wild-type. In situ analysis identified HvEGT1 is expressed in elongating cortical and stele tissues, which are distinct from known root gravitropic perception and response tissues in the columella and epidermis, respectively. We propose that EGT1 controls root angle by regulating cell wall stiffness in elongating root cortical tissue, counteracting the gravitropic machinery’s known ability to bend the root via its outermost tissues. We conclude that root angle is controlled by EGT1 in cereal crops employing an antigravitropic mechanism.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2022

The pathway for coenzyme M biosynthesis in bacteria

Mercaptoethane sulfonate or coenzyme M (CoM) is the smallest known organic cofactor and is most commonly associated with the methane-forming step in all methanogenic archaea but is also associated with the anaerobic oxidation of methane to CO 2 in anaerobic methanotrophic archaea and the oxidation of short-chain alkanes in Syntrophoarchaeum species. It has also been found in a small number of bacteria capable of the metabolism of small organics. Although many of the steps for CoM biosynthesis in methanogenic archaea have been elucidated, a complete pathway for the biosynthesis of CoM in archaea or bacteria has not been reported. Here, we present the complete CoM biosynthesis pathway in bacteria, revealing distinct chemical steps relative to CoM biosynthesis in methanogenic archaea. The existence of different pathways represents a profound instance of convergent evolution. The five-step pathway involves the addition of sulfite, the elimination of phosphate, decarboxylation, thiolation, and the reduction to affect the sequential conversion of phosphoenolpyruvate to CoM. The salient features of the pathway demonstrate reactivities for members of large aspartase/fumarase and pyridoxal 5'-phosphate–dependent enzyme families.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2024

Perturbative diffraction methods resolve a conformational switch that facilitates a two-step enzymatic mechanism

Enzymes catalyze biochemical reactions through precise positioning of substrates, cofactors, and amino acids to modulate the transition-state free energy. However, the role of conformational dynamics remains poorly understood due to poor experimental access. This shortcoming is evident with Escherichia coli dihydrofolate reductase (DHFR), a model system for the role of protein dynamics in catalysis, for which it is unknown how the enzyme regulates the different active site environments required to facilitate proton and hydride transfer. Here, we describe ligand-, temperature-, and electric-field-based perturbations during X-ray diffraction experiments to map the conformational dynamics of the Michaelis complex of DHFR. We resolve coupled global and local motions and find that these motions are engaged by the protonated substrate to promote efficient catalysis. This result suggests a fundamental design principle for multistep enzymes in which pre-existing dynamics enable intermediates to drive rapid electrostatic reorganization to facilitate subsequent chemical steps.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2024

Identification and characterization of a small-molecule metallophore involved in lanthanide metabolism

Many bacteria secrete metallophores, low-molecular-weight organic compounds that bind ions with high selectivity and affinity, in order to access essential metals from the environment. Previous work has elucidated the structures and biosynthetic machinery of metallophores specific for iron, zinc, nickel, molybdenum, and copper. No physiologically relevant lanthanide-binding metallophore has been discovered despite the knowledge that lanthanide metals (Ln) have been revealed to be essential cofactors for certain alcohol dehydrogenases across a diverse range of phyla. Here, we report the biosynthetic machinery, the structure, and the physiological relevance of a lanthanophore, methylolanthanin. The structure of methylolanthanin exhibits a unique 4-hydroxybenzoate moiety which has not previously been described in other metallophores. We find that production of methylolanthanin is required for normal levels of Ln accumulation in the methylotrophic bacterium Methylobacterium extorquens AM1, while overexpression of the molecule greatly increases bioaccumulation and adsorption. Our results provide a clearer understanding of how Ln-utilizing bacteria sense, scavenge, and store Ln; essential processes in the environment where Ln are poorly bioavailable. More broadly, the identification of this lanthanophore opens doors for study of how biosynthetic gene clusters are repurposed for additional functions and the complex relationship between metal homeostasis and fitness.

Science & Technology - Other Topics↗
DOE OSTI2024

An RNA aptamer exploits exosite-dependent allostery to achieve specific inhibition of coagulation factor IXa

Hemostasis relies on a reaction network of serine proteases and their cofactors to form a blood clot. Coagulation factor IXa (protease) plays an essential role in hemostasis as evident from the bleeding disease associated with its absence. RNA aptamers specifically targeting individual coagulation factors have potential as anticoagulants and as probes of the relationship between structure and function. Here, we report X-ray structures of human factor IXa without a ligand bound to the active site either in the apo-form or in complex with an inhibitory aptamer specific for factor IXa. The aptamer binds to an exosite in the catalytic domain and allosterically distorts the active site. Our studies reveal a conformational ensemble of IXa states, wherein large movements of Trp 215 near the active site drive functional transitions between the closed (aptamer-bound), latent (apo), and open (substrate-bound) states. The latent state of the apo-enzyme may bear on the uniquely poor catalytic activity of IXa compared to other coagulation proteases. The exosite, to which the aptamer binds, has been implicated in binding VIIIa and heparin, both of which regulate IXa function. Our findings reveal the importance of exosite-driven allosteric modulation of IXa function and new strategies to rebalance hemostasis for therapeutic gain.

Science & Technology - Other Topics↗
DOE OSTI2024

Molecular basis of hemoglobin binding and heme removal in Corynebacterium diphtheriae

To successfully mount infections, nearly all bacterial pathogens must acquire iron, a key metal cofactor that primarily resides within human hemoglobin. Corynebacterium diphtheriae causes the life-threatening respiratory disease diphtheria and captures hemoglobin for iron scavenging using the surface-displayed receptor HbpA. Here, we show using X-ray crystallography, NMR, and in situ binding measurements that C. diphtheriae selectively captures iron-loaded hemoglobin by partially ensconcing the heme molecules of its α subunits. Quantitative growth and heme release measurements are compatible with C. diphtheriae acquiring heme passively released from hemoglobin’s β subunits. We propose a model in which HbpA and heme-binding receptors collectively function on the C. diphtheriae surface to capture hemoglobin and its spontaneously released heme. Acquisition mechanisms that exploit the propensity of hemoglobin’s β subunit to release heme likely represent a common strategy used by bacterial pathogens to obtain iron during infections.

NMR↗
DOE OSTI2025

An electron-bifurcating “plug” to a protein nanowire in tungsten-dependent aldehyde detoxification

Members of the tungsten-containing oxidoreductase (WOR) family, which contain a tungstopyranopterin (Tuco) cofactor, are typically either monomeric (WorL) or heterodimeric (WorLS). These enzymes oxidize aldehydes to the corresponding acids while reducing the redox protein ferredoxin. They have been structurally characterized mainly using WORs from hyperthermophilic archaea. The WORs of some bacteria contain three additional subunits of the BfuABC family and these chimeric WorABCSL enzymes catalyze an electron-bifurcating reaction in which aldehyde oxidation is coupled to the simultaneous reduction of ferredoxin and nicotinamide adenine dinucleotide. In human gut microbes, electron bifurcation by WorABSL is proposed to enable the detoxification of aldehydes generated from cooked foods and in the tungstocentric production of beneficial short chain fatty acids from lactate, potentially impacting health. Herein we present the high-resolution cryogenic electron microscopy (cryo-EM) structure of the WorABCSL purified from the bacteriumAcetomicrobium mobile.The structure reveals a surprising 1:3 stoichiometry between WorABC and WorSL, with the WorSL units forming a nanowire-like architecture leading from three Tuco-containing catalytic sites in WorL via strings of multiple iron-sulfur clusters in WorS to a single bifurcating WorABC core. Our structure uncovers a distinct domain arrangement that links three Tuco-dependent aldehyde oxidation sites with the bifurcation process and potentially facilitates environmental aldehyde oxidation.

Science & Technology - Other Topics↗
DOE OSTI2025

An unusual Co–S bond links B 12 chaperones in an interprotein complex

Chaperones in trafficking pathways ensure specificity of transition metal loading and protection against adventitious side reactions. In B 12 , an essential cofactor for humans, a cobalt ion is coordinated to a >1.3 kDa tetrapyrrolic scaffold, posing logistical challenges for its translocation. In this study, we report the 3.4 Å crystal structure of the human MMACHC and MMADHC B 12 chaperones, tethered via a rarely seen covalent cobalt–sulfur bond. B 12 is bound in the base-off state to MMACHC, with Cys-261 on MMADHC serving as the upper axial ligand; the lower-axial position is vacant. The propensity of thiolato-cobalamin derivatives bound to MMACHC to undergo spontaneous decomposition via general acid catalysis or reduction/oxidation chemistry is averted in the interprotein complex with MMADHC. An exposed face in the complex suggests an exit route for B 12 . No known clinical variants localize to the interprotein interface, consistent with the cobalt–sulfur bond being key to forming the high-affinity complex.

Science & Technology - Other Topics↗
DOE OSTI2026

Photoenzymatic Csp 3 –Csp 3 bond formation via enzyme-templated radical–radical coupling

Cross-couplings are essential reactions in modern chemical synthesis, enabling the rapid construction of complex molecules from simple precursors. Transition metal catalysts are prized for these transformations because their reactivity and selectivity can be tuned via judicious selection of the metal and ligand. Although enzymes offer analogous opportunities for tuning via protein engineering, their application to cross-coupling remains limited, as nature relies on alternative paradigms for building molecular complexity. Here, we report the cross-coupling of alkyl halides and benzylic carboxylic acids using an engineered flavin-dependent lactate monooxygenase—a photoenzyme. The enzyme achieves this feat by exploiting the redox versatility of the flavin cofactor. Stoichiometric experiments, ultrafast spectroscopy, and computational studies support a mechanism in which photoexcited flavin quinone initiates the reaction via oxidative decarboxylation to generate a benzylic radical. The resulting flavin semiquinone can reduce the alkyl halide to form a second organic radical within the protein active site, which rapidly engages in C(sp 3 )–C(sp 3 ) bond formation. A variant was engineered to control the stereochemical outcome of this radical–radical coupling event, highlighting the ability of the protein to alter the energetic barrier for a mechanistic step that is traditionally understood to be near barrierless. This work demonstrates that the scope for nonnative reaction mechanisms in biocatalysis far exceeds previously established bounds and has potential to solve a variety of reactivity challenges in cross-coupling chemistry.

biocatalysis↗
DOE OSTI2020

Structure and function of a flavin-dependent S-monooxygenase from garlic ( Allium sativum )

Allicin is a component of the characteristic smell and flavor of garlic ( Allium sativum ). A flavin-containing monooxygenase (FMO) produced by A. sativum (AsFMO) was previously proposed to oxidize S-allyl- L -cysteine (SAC) to alliin, an allicin precursor. In this paper, we present a kinetic and structural characterization of AsFMO that suggests a possible contradiction to this proposal. Results of steady-state kinetic analyses revealed that AsFMO exhibited negligible activity with SAC; however, the enzyme was highly active with L -cysteine, N-acetyl- L -cysteine, and allyl mercaptan. We found that allyl mercaptan with NADPH was the preferred substrate-cofactor combination. Rapid-reaction kinetic analyses showed that NADPH binds tightly ($K_D$ of ~2 μM) to AsFMO and that the hydride transfer occurs with pro-R stereospecificity. We detected the formation of a long-wavelength band when AsFMO was reduced by NADPH, probably representing the formation of a charge-transfer complex. In the absence of substrate, the reduced enzyme, in complex with NADP + , reacted with oxygen and formed an intermediate with a spectrum characteristic of C4a-hydroperoxyflavin, which decays several orders of magnitude more slowly than the $k$ cat . The presence of substrate enhanced C4a-hydroperoxyflavin formation and, upon hydroxylation, oxidation occurred with a rate constant similar to the $k$ cat . The structure of AsFMO complexed with FAD at 2.08-Å resolution features two domains for binding of FAD and NADPH, representative of class B flavin monooxygenases. These biochemical and structural results are consistent with AsFMO being an S-monooxygenase involved in allicin biosynthesis through direct formation of sulfenic acid and not SAC oxidation.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗
DOE OSTI2020

Zymogen and activated protein C have similar structural architecture

Activated protein C is a trypsin-like protease with anticoagulant and cytoprotective properties that is generated by thrombin from the zymogen precursor protein C in a reaction greatly accelerated by the cofactor thrombomodulin. The molecular details of this activation remain elusive due to the lack of structural information. We now fill this gap by providing information on the overall structural organization of these proteins using single molecule Förster resonance energy transfer and small angle X-ray scattering. Under physiological conditions, both zymogen and protease adopt a conformation with all domains vertically aligned along an axis 76 Å long and maximal particle size of 120 Å. This conformation is stabilized by binding of Ca 2+ to the Gla domain and is affected minimally by interaction with thrombin. Hence, the zymogen protein C likely interacts with the thrombin-thrombomodulin complex through a rigid body association that produces a protease with essentially the same structural architecture. This scenario stands in contrast to an analogous reaction in the coagulation cascade where conversion of the zymogen prothrombin to the protease meizothrombin by the prothrombinase complex is linked to a large conformational transition of the entire protein. The presence of rigid EGF domains in protein C as opposed to kringles in prothrombin likely accounts for the different conformational plasticity of the two zymogens. The new structural features reported here for protein C have general relevance to vitamin K-dependent clotting factors containing EGF domains, such as factors VII, IX, and X.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗
DOE OSTI2021

Beyond the usual suspects: methanogenic communities in eastern North American peatlands are also influenced by nickel and copper concentrations

Peatlands both accumulate carbon and release methane, but their broad range in environmental conditions means that the diversity of microorganisms responsible for carbon cycling is still uncertain. Here, we describe a community analysis of methanogenic archaea responsible for methane production in 17 peatlands from 36 to 53 N latitude across the eastern half of North America, including three metal-contaminated sites. Methanogenic community structure was analysed through Illumina amplicon sequencing of the mcrA gene. Whether metal-contaminated sites were included or not, metal concentrations in peat were a primary driver of methanogenic community composition, particularly nickel, a trace element required in the F 430 cofactor in methyl-coenzyme M reductase that is also toxic at high concentrations. Copper was also a strong predictor, likely due to inhibition at toxic levels and/or to cooccurrence with nickel, since copper enzymes are not known to be present in anaerobic archaea. Here, the methanogenic groups Methanocellales and Methanosarcinales were prevalent in peatlands with low nickel concentrations, while Methanomicrobiales and Methanomassiliicoccales were abundant in peatlands with higher nickel concentrations. Results suggest that peat-associated trace metals are predictors of methanogenic communities in peatlands.

54 ENVIRONMENTAL SCIENCES↗
DOE OSTI2026

From photosynthetic electron flow to gene regulation: redox signal transduction in cyanobacteria

In cyanobacteria, the free-living ancestors of chloroplasts, photosynthesis simultaneously sustains growth and generates reactive oxygen species (ROS) that damage proteins, lipids, and DNA when light capture outpaces carbon fixation. Maintaining redox balance, therefore, requires cells to read photosynthetic electron flow as a signal that continuously tunes gene expression and protein activity. This review traces how these redox signals are transduced to transcription machinery through three routes: membrane-localized sensors, cytoplasmic redox sensors downstream of photosystem I, and ROS generated when electron sinks are saturated. Membrane-bound histidine kinases (two-component systems) relay the redox state of the plastoquinone pool to control photosystem remodeling, pigment biosynthesis, and circadian timing. Cytoplasmic one-component regulators, by contrast, sense redox directly through thiol-disulfide switches, glutathionylation, iron-sulfur clusters, and metal-catalyzed oxidation to control photosystem-cofactor, electron-carrier, and transition-metal homeostasis. Because many of these regulators persist in algal and plant chloroplasts, cyanobacteria illuminate principles of redox control across photosynthetic eukaryotes. Post-transcriptional and translational control further shapes redox-dependent gene expression programs through transcript stability, ribosome assembly, and translation initiation, extending redox regulation beyond transcription to every step of protein synthesis and even activity modulation. Finally, we connect redox regulation to photosynthetic physiology, stress resilience, and the rational engineering of cyanobacteria for sustainable bioproduction.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2024

Phylogenetic distribution and experimental characterization of corrinoid production and dependence in soil bacterial isolates

Abstract Soil microbial communities impact carbon sequestration and release, biogeochemical cycling, and agricultural yields. These global effects rely on metabolic interactions that modulate community composition and function. However, the physicochemical and taxonomic complexity of soil and the scarcity of available isolates for phenotypic testing are significant barriers to studying soil microbial interactions. Corrinoids—the vitamin B12 family of cofactors—are critical for microbial metabolism, yet they are synthesized by only a subset of microbiome members. Here, we evaluated corrinoid production and dependence in soil bacteria as a model to investigate the ecological roles of microorganisms involved in metabolic interactions. We isolated and characterized a taxonomically diverse collection of 161 soil bacteria from a single study site. Most corrinoid-dependent bacteria in the collection prefer B12 over other corrinoids, while all tested producers synthesize B12, indicating metabolic compatibility between producers and dependents in the collection. Furthermore, a subset of producers release B12 at levels sufficient to support dependent isolates in laboratory culture at estimated ratios of up to 1000 dependents per producer. Within our isolate collection, we did not find strong phylogenetic patterns in corrinoid production or dependence. Upon investigating trends in the phylogenetic dispersion of corrinoid metabolism categories across sequenced bacteria from various environments, we found that these traits are conserved in 47 out of 85 genera. Together, these phenotypic and genomic results provide evidence for corrinoid-based metabolic interactions among bacteria and provide a framework for the study of nutrient-sharing ecological interactions in microbial communities.

59 BASIC BIOLOGICAL SCIENCES↗