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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 253 records · Page 14
All sourcesNASA NTRSDOE OSTI
DOE OSTI2023

The vacuolar iron transporter mediates iron detoxification in Toxoplasma gondii

Iron is essential to cells as a cofactor in enzymes of respiration and replication, however without correct storage, iron leads to the formation of dangerous oxygen radicals. In yeast and plants, iron is transported into a membrane-bound vacuole by the vacuolar iron transporter (VIT). This transporter is conserved in the apicomplexan family of obligate intracellular parasites, including in Toxoplasma gondii . Here, we assess the role of VIT and iron storage in T. gondii . By deleting VIT, we find a slight growth defect in vitro, and iron hypersensitivity, confirming its essential role in parasite iron detoxification, which can be rescued by scavenging of oxygen radicals. We show VIT expression is regulated by iron at transcript and protein levels, and by altering VIT localization. In the absence of VIT, T. gondii responds by altering expression of iron metabolism genes and by increasing antioxidant protein catalase activity. We also show that iron detoxification has an important role both in parasite survival within macrophages and in virulence in a mouse model. Together, by demonstrating a critical role for VIT during iron detoxification in T. gondii , we reveal the importance of iron storage in the parasite and provide the first insight into the machinery involved.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2023

Mechanistic manifold in a hemoprotein-catalyzed cyclopropanation reaction with diazoketone

Abstract Hemoproteins have recently emerged as promising biocatalysts for new-to-nature carbene transfer reactions. However, mechanistic understanding of the interplay between productive and unproductive pathways in these processes is limited. Using spectroscopic, structural, and computational methods, we investigate the mechanism of a myoglobin-catalyzed cyclopropanation reaction with diazoketones. These studies shed light on the nature and kinetics of key catalytic steps in this reaction, including the formation of an early heme-bound diazo complex intermediate, the rate-determining nature of carbene formation, and the cyclopropanation mechanism. Our analyses further reveal the existence of a complex mechanistic manifold for this reaction that includes a competing pathway resulting in the formation of an N-bound carbene adduct of the heme cofactor, which was isolated and characterized by X-ray crystallography, UV-Vis, and Mössbauer spectroscopy. This species can regenerate the active biocatalyst, constituting a non-productive, yet non-destructive detour from the main catalytic cycle. These findings offer a valuable framework for both mechanistic analysis and design of hemoprotein-catalyzed carbene transfer reactions.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2024

A hemoprotein with a zinc-mirror heme site ties heme availability to carbon metabolism in cyanobacteria

Abstract Heme has a critical role in the chemical framework of the cell as an essential protein cofactor and signaling molecule that controls diverse processes and molecular interactions. Using a phylogenomics-based approach and complementary structural techniques, we identify a family of dimeric hemoproteins comprising a domain of unknown function DUF2470. The heme iron is axially coordinated by two zinc-bound histidine residues, forming a distinct two-fold symmetric zinc-histidine-iron-histidine-zinc site. Together with structure-guided in vitro and in vivo experiments, we further demonstrate the existence of a functional link between heme binding by Dri1 (Domain related to iron 1, formerly ssr1698) and post-translational regulation of succinate dehydrogenase in the cyanobacterium Synechocystis , suggesting an iron-dependent regulatory link between photosynthesis and respiration. Given the ubiquity of proteins containing homologous domains and connections to heme metabolism across eukaryotes and prokaryotes, we propose that DRI (Domain Related to Iron; formerly DUF2470) functions at the molecular level as a heme-dependent regulatory domain.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2024

Drosophila Evi5 is a critical regulator of intracellular iron transport via transferrin and ferritin interactions

Abstract Vesicular transport is essential for delivering cargo to intracellular destinations. Evi5 is a Rab11-GTPase-activating protein involved in endosome recycling. In humans, Evi5 is a high-risk locus for multiple sclerosis, a debilitating disease that also presents with excess iron in the CNS. In insects, the prothoracic gland (PG) requires entry of extracellular iron to synthesize steroidogenic enzyme cofactors. The mechanism of peripheral iron uptake in insect cells remains controversial. We show that Evi5-depletion in the Drosophila PG affected vesicle morphology and density, blocked endosome recycling and impaired trafficking of transferrin-1, thus disrupting heme synthesis due to reduced cellular iron concentrations. We show that ferritin delivers iron to the PG as well, and interacts physically with Evi5. Further, ferritin-injection rescued developmental delays associated with Evi5-depletion. To summarize, our findings show that Evi5 is critical for intracellular iron trafficking via transferrin-1 and ferritin, and implicate altered iron homeostasis in the etiology of multiple sclerosis.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2024

Functional anatomy of zinc finger antiviral protein complexes

Abstract ZAP is an antiviral protein that binds to and depletes viral RNA, which is often distinguished from vertebrate host RNA by its elevated CpG content. Two ZAP cofactors, TRIM25 and KHNYN, have activities that are poorly understood. Here, we show that functional interactions between ZAP, TRIM25 and KHNYN involve multiple domains of each protein, and that the ability of TRIM25 to multimerize via its RING domain augments ZAP activity and specificity. We show that KHNYN is an active nuclease that acts in a partly redundant manner with its homolog N4BP1. The ZAP N-terminal RNA binding domain constitutes a minimal core that is essential for antiviral complex activity, and we present a crystal structure of this domain that reveals contacts with the functionally required KHNYN C-terminal domain. These contacts are remote from the ZAP CpG binding site and would not interfere with RNA binding. Based on our dissection of ZAP, TRIM25 and KHNYN functional anatomy, we could design artificial chimeric antiviral proteins that reconstitute the antiviral function of the intact authentic proteins, but in the absence of protein domains that are otherwise required for activity. Together, these results suggest a model for the RNA recognition and action of ZAP-containing antiviral protein complexes.

Science & Technology - Other Topics↗
DOE OSTI2025

Small molecule BLVRB redox inhibitor promotes megakaryocytopoiesis and stress thrombopoiesis in vivo

Biliverdin IXβ reductase (BLVRB) is an NADPH-dependent enzyme previously implicated in a redox-regulated mechanism of thrombopoiesis distinct from the thrombopoietin (TPO)/c-MPL axis. Here, we apply computational modeling to inform molecule design, followed by de novo syntheses and screening of unique small molecules retaining the capacity for selective BLVRB inhibition as a novel platelet-enhancing strategy. Two distinct classes of molecules are identified, and NMR spectroscopy and co-crystallization studies confirm binding modes within the BLVRB active site and ring stacking between the nicotinamide moiety of the NADP+ cofactor. A diazabicyclo derivative displaying minimal off-target promiscuity and excellent bioavailability characteristics promotes megakaryocyte speciation in biphenotypic (erythro/megakaryocyte) cellular models and synergizes with TPO-dependent megakaryocyte formation in hematopoietic stem cells. Upon oral delivery into mice, this inhibitor expands platelet recovery in stress thrombopoietic models with no adverse effects. In this work, we identify and validate a cellular redox inhibitor retaining the potential to selectively promote megakaryocytopoiesis and enhance stress-associated platelet formation in vivo distinct from TPO receptor agonists.

36 MATERIALS SCIENCE↗
DOE OSTI2026

Structural basis of the promiscuity of the unusual Fe(II) and 2-oxoglutarate dependent human aspartate/asparagine-β-hydroxylase

Protein-hydroxylation catalysed by Fe(II) and 2-oxoglutarate (2OG) dependent oxygenases is an important regulatory mechanism in human biology. Such oxygenases typically coordinate their Fe(II) cofactor via a conserved triad of an aspartate- or glutamate- and two histidine-residues. By contrast, aspartate/asparagine β-hydroxylase (AspH), which catalyses asparagine/aspartate-residue oxidation in epidermal growth factor-like domains (EGFDs), has only two histidine-residues (H679, H725), with a water occupying the site normally occupied by an aspartate- or glutamate-residue. We describe mechanistic studies with catalytically active AspH crystals. Turnover studies with single crystals under cryogenic conditions give (3 R)-hydroxylated EGFDs with the product alcohol coordinating Fe(II) trans to H725. Time-resolved serial crystallography of microcrystals using an acoustic droplet ejection system, coupled to X-ray emission analyses, demonstrate turnover within 1.5 s, giving a product complex in which Fe(II) is regenerated. Solution and crystallographic studies with the O 2 surrogate nitric oxide imply O 2 binds to Fe(II) trans to H725. The additional Fe-chelating water is maintained throughout AspH catalysis and is not directly involved in substrate hydroxylation, because O 2 is the sole oxygen source in alcohol products, as shown by 18 O labelling studies. The results reveal how AspH accommodates both aspartate- and asparagine-substrates and will assist in efforts targeting AspH for cancer treatment.

de Munnik, Mariska↗
DOE OSTI2025

Model of metabolism and gene expression predicts proteome allocation in Pseudomonas putida

Abstract The genome-scale model of metabolism and gene expression (ME-model) forPseudomonas putidaKT2440,iPpu1676-ME, provides a comprehensive representation of biosynthetic costs and proteome allocation. Compared to a metabolic-only model,iPpu1676-ME significantly expands on gene expression, macromolecular assembly, and cofactor utilization, enabling accurate growth predictions without additional constraints. Multi-omics analysis using RNA sequencing and ribosomal profiling data revealed translational prioritization inP. putida, with core pathways, such as nicotinamide biosynthesis and queuosine metabolism, exhibiting higher translational efficiency, while secondary pathways displayed lower priority. Notably, the ME-model significantly outperformed the M-model in alignment with multi-omics data, thereby validating its predictive capacity. Thus,iPpu1676-ME offers valuable insights intoP. putida’s proteome allocation and presents a powerful tool for understanding resource allocation in this industrially relevant microorganism.

Mathematical & Computational Biology↗
DOE OSTI2024

Stereodivergent photobiocatalytic radical cyclization through the repurposing and directed evolution of fatty acid photodecarboxylases

Despite their intriguing photophysical and photochemical activities, naturally occurring photoenzymes have not yet been repurposed for new-to-nature activities. Here we engineered fatty acid photodecarboxylases (FAPs) to catalyse unnatural photoredox radical C–C bond formation by leveraging strongly oxidizing excited-state flavoquinone cofactor. Through genome mining, rational engineering and directed evolution, we developed a panel of radical photocyclases (RAPs) to facilitate decarboxylative radical cyclisation with excellent chemo-, enantio-, and diastereoselectivities. Our high-throughput experimental workflow allowed for the directed evolution of FAPs. An orthogonal set of RAPs was engineered to access all four possible stereoisomers of stereochemical dyad, affording fully diastereo- and enantiodivergent biotransformations in asymmetric radical biocatalysis. Molecular dynamics simulations show that our evolved RAPs allow near-attack conformations to be easily accessed, enabling chemoselective radical cyclisation. The development of stereoselective RAPs provides unnatural C–C bond forming activities in natural photoenzyme families, which can be used to tame the stereochemistry of free radical-mediated reactions.

Ju, Shuyun (ORCID:000000020322104X)↗
DOE OSTI2020

Ultrafast structural changes within a photosynthetic reaction centre

Photosynthetic reaction centres harvest the energy content of sunlight by transporting electrons across an energy-transducing biological membrane. In this study we use time-resolved serial femtosecond crystallography using an X-ray free-electron laser to observe light-induced structural changes in the photosynthetic reaction centre of Blastochloris viridis on a timescale of picoseconds. Structural perturbations first occur at the special pair of chlorophyll molecules of the photosynthetic reaction centre that are photo-oxidized by light. Electron transfer to the menaquinone acceptor on the opposite side of the membrane induces a movement of this cofactor together with lower amplitude protein rearrangements. These observations reveal how proteins use conformational dynamics to stabilize the charge-separation steps of electron-transfer reactions.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2025

Random heteropolymers as enzyme mimics

Despite successes in replicating the primary–secondary–tertiary structure hierarchy of protein, it remains elusive to synthetically materialize protein functions that are deeply rooted in their chemical, structural and dynamic heterogeneities. We propose that for polymers with backbone chemistries different from that of proteins, programming spatial and temporal projections of sidechains at the segmental level can be effective in replicating protein behaviours; and leveraging the rotational freedom of polymer can mitigate deficiencies in monomeric sequence specificity and achieve behaviour uniformity at the ensemble level. Here, guided by the active site analysis of about 1,300 metalloproteins, we design random heteropolymers (RHPs) as enzyme mimics based on one-pot synthesis. We introduce key monomers as the equivalents of the functional residues of protein and statistically modulate the chemical characteristics of key monomer-containing segments, such as segmental hydrophobicity. The resultant RHPs form pseudo-active sites that provide key monomers with protein-like microenvironments, co-localize substrates with catalytic or cofactor-binding sidechains and catalyse reactions such as oxidation and cyclization of citronellal with isopulegol/menthoglycol selectivity. This RHP design led to enzyme-like materials that can retain catalytic activity under non-biological conditions, are compatible with scalable processing and have expanded substrate scope, including environmentally long-lasting antibiotic tetracycline.

36 MATERIALS SCIENCE↗
DOE OSTI2026

Magnetic resonance control of spin-correlated radical pair dynamics in vivo

Magnetic fields can influence reactions involving spin-correlated radical pairs (SCRPs). This provides a mechanism by which both static and time-varying magnetic fields can affect living systems at the biomolecular level. However, an engineered SCRP system conferring magnetic sensitivity to a non-native biochemical process in a multicellular organism has not yet been demonstrated. Here, in this study, we demonstrate control of SCRP dynamics using magnetic resonance in a live transgenic animal. We show that the emission of various red fluorescent proteins (RFPs), in the presence of a flavin cofactor, can be modified by a combination of static and radiofrequency magnetic fields applied near the electron spin resonance frequency. This effect was measured at room temperature both in vitro and in the nematode Caenorhabditis elegans, genetically modified to express the RFP mScarlet. These observations suggest that the magnetic field effects measured in RFP-flavin systems are due to quantum-correlated radical pairs with a coherence time larger than 4 ns. Our experiments demonstrate that radiofrequency magnetic fields can influence dynamics of reactions involving SCRPs in vivo, potentially enabling new methods for remotely controlling biomolecular processes, such as gene expression, and suggest broader potential for quantum tools in biology.

Burd, Shaun C. [Stanford Univ., CA (United States)↗
DOE OSTI2023

Cofactorless oxygenases guide anthraquinone-fused enediyne biosynthesis

The anthraquinone-fused enediynes (AFEs) combine an anthraquinone moiety and a ten-membered enediyne core capable of generating a cytotoxic diradical species. AFE cyclization is triggered by opening the F-ring epoxide, which is also the site of the most structural diversity. Previous studies of tiancimycin A, a heavily modified AFE, have revealed a cryptic aldehyde blocking installation of the epoxide, and no unassigned oxidases could be predicted within the tnm biosynthetic gene cluster. Here, in this study, we identify two consecutively acting cofactorless oxygenases derived from methyltransferase and α/β-hydrolase protein folds, TnmJ and TnmK2, respectively, that are responsible for F-ring tailoring in tiancimycin biosynthesis by comparative genomics. Further biochemical and structural characterizations reveal that the electron-rich AFE anthraquinone moiety assists in catalyzing deformylation, epoxidation and oxidative ring cleavage without exogenous cofactors. These enzymes therefore fill important knowledge gaps for the biosynthesis of this class of molecules and the underappreciated family of cofactorless oxygenases. Cofactorless oxygenases are rare in nature and natural product biosynthesis. Here the authors describe the biochemical and structural characterization of two such oxygenases catalyzing deformylation, ring cleavage and epoxidation in the biosynthesis of the enediyne natural product tiancimycin A.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗
DOE OSTI2024

Top-down mass spectrometry of native proteoforms and their complexes: a community study

The combination of native electrospray ionization with top-down fragmentation in mass spectrometry (MS) allows simultaneous determination of the stoichiometry of noncovalent complexes and identification of their component proteoforms and cofactors. Although this approach is powerful, both native MS and top-down MS are not yet well standardized, and only a limited number of laboratories regularly carry out this type of research. Further, to address this challenge, the Consortium for Top-Down Proteomics initiated a study to develop and test protocols for native MS combined with top-down fragmentation of proteins and protein complexes across 11 instruments in nine laboratories. Here we report the summary of the outcomes to provide robust benchmarks and a valuable entry point for the scientific community.

47 OTHER INSTRUMENTATION↗
DOE OSTI2019

Structural analysis of mycobacterial homoserine transacetylases central to methionine biosynthesis reveals druggable active site

Mycobacterium tuberculosis is the cause of the world’s most deadly infectious disease. Efforts are underway to target the methionine biosynthesis pathway, as it is not part of the host metabolism. The homoserine transacetylase MetX converts L-homoserine to O-acetyl-L-homoserine at the committed step of this pathway. In order to facilitate structure-based drug design, we determined the high-resolution crystal structures of three MetX proteins, including M. tuberculosis (MtMetX), Mycolicibacterium abscessus (MaMetX), and Mycolicibacterium hassiacum (MhMetX). A comparison of homoserine transacetylases from other bacterial and fungal species reveals a high degree of structural conservation amongst the enzymes. Utilizing homologous structures with bound cofactors, we analyzed the potential ligandability of MetX. The deep active-site tunnel surrounding the catalytic serine yielded many consensus clusters during mapping, suggesting that MtMetX is highly druggable.

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

Structure determination of the HgcAB complex using metagenome sequence data: insights into microbial mercury methylation

Bacteria and archaea possessing the hgcAB gene pair methylate inorganic mercury (Hg) to form highly toxic methylmercury. HgcA consists of a corrinoid binding domain and a transmembrane domain, and HgcB is a dicluster ferredoxin. However, their detailed structure and function have not been thoroughly characterized. We modeled the HgcAB complex by combining metagenome sequence data mining, coevolution analysis, and Rosetta structure calculations. In addition, we overexpressed HgcA and HgcB in Escherichia coli, confirmed spectroscopically that they bind cobalamin and [4Fe-4S] clusters, respectively, and incorporated these cofactors into the structural model. Surprisingly, the two domains of HgcA do not interact with each other, but HgcB forms extensive contacts with both domains. The model suggests that conserved cysteines in HgcB are involved in shuttling HgII, methylmercury, or both. These findings refine our understanding of the mechanism of Hg methylation and expand the known repertoire of corrinoid methyltransferases in nature.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2023

Revealing protonation states and tracking substrate in serine hydroxymethyltransferase with room-temperature X-ray and neutron crystallography

Pyridoxal 5’-phosphate (PLP)-dependent enzymes utilize a vitamin B 6 -derived cofactor to perform a myriad of chemical transformations on amino acids and other small molecules. Some PLP-dependent enzymes, such as serine hydroxymethyltransferase (SHMT), are promising drug targets for the design of small-molecule antimicrobials and anticancer therapeutics, while others have been used to synthesize pharmaceutical building blocks. Understanding PLP-dependent catalysis and the reaction specificity is crucial to advance structure-assisted drug design and enzyme engineering. Here we report the direct determination of the protonation states in the active site of Thermus thermophilus SHMT (TthSHMT) in the internal aldimine state using room-temperature joint X-ray/neutron crystallography. Conserved active site architecture of the model enzyme TthSHMT and of human mitochondrial SHMT (hSHMT2) were compared by obtaining a room-temperature X-ray structure of hSHMT2, suggesting identical protonation states in the human enzyme. The amino acid substrate serine pathway through the TthSHMT active site cavity was tracked, revealing the peripheral and cationic binding sites that correspond to the pre-Michaelis and pseudo-Michaelis complexes, respectively. At the peripheral binding site, the substrate is bound in the zwitterionic form. By analyzing the observed protonation states, Glu53, but not His residues, is proposed as the general base catalyst, orchestrating the retro-aldol transformation of L-serine into glycine.

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

Fe protein docking transduces conformational changes to MoFe nitrogenase active site in a nucleotide-dependent manner

The reduction of dinitrogen to ammonia catalyzed by nitrogenase involves a complex series of events, including ATP hydrolysis, electron transfer, and activation of metal clusters for N 2 reduction. Early evidence shows that an essential part of the mechanism involves transducing information between the nitrogenase component proteins through conformational dynamics. Here, millisecond time-resolved hydrogen-deuterium exchange mass spectrometry was used to unravel peptide-level protein motion on the time scale of catalysis of Mo-dependent nitrogenase from Azotobacter vinelandii. Normal mode analysis calculations complemented this data, providing insights into the specific signal transduction pathways that relay information across protein interfaces at distances spanning 100 Å. Together, these results show that conformational changes induced by protein docking are rapidly transduced to the active site, suggesting a specific mechanism for activating the metal cofactor in the enzyme active site.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗