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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 19 records
All sourcesNASA NTRSDOE OSTI
DOE OSTI2017

APOBEC3B lysine residues are dispensable for DNA cytosine deamination, HIV-1 restriction, and nuclear localization

The APOBEC3 DNA cytosine deaminase family comprises a fundamental arm of the innate immune response and is best known for retrovirus restriction. Several APOBEC3 enzymes restrict HIV-1 and related retroviruses by deaminating viral cDNA cytosines to uracils compromising viral genomes. Human APOBEC3B (A3B) shows strong virus restriction activities in a variety of experimental systems, and is subjected to tight post-translational regulation evidenced by cell-specific HIV-1 restriction activity and active nuclear import. Here we ask whether lysines and/or lysine post-translational modifications are required for these A3B activities. A lysine-free derivative of human A3B was constructed and shown to be indistinguishable from the wild-type enzyme in DNA cytosine deamination, HIV-1 restriction, and nuclear localization activities. However, lysine loss did render the protein resistant to degradation by SIV Vif. Taken together, we conclude that lysine side chains and modifications thereof are unlikely to be central to A3B function or regulation in human cells. - Highlights: • APOBEC3B is an antiviral DNA cytosine deaminase with nuclear localization. • Lysines are dispensable for DNA cytosine deaminase activity of APOBEC3B. • Lysines are dispensable for HIV restriction activity of APOBEC3B. • Lysines are dispensable for nuclear localization of APOBEC3B. • Lysines are required for SIV Vif-mediated degradation.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2026

Organo-catalyzed deamination of polystyrene sulfonamide for diverse post-polymerization modification of styrenic polymers

Post-polymerization modification allows for the incorporation of functional groups that would otherwise be incompatible with polymerization conditions, enhancing synthetic efficiency and facilitating the creation of complex polymer architectures for specialized applications, such as biomedical devices, electronics, and advanced coatings. Herein, we report a method inspired by late-stage functionalization of small molecules for the post-polymerization modification of aromatic polymers, specifically polystyrene (PS), under mild reaction conditions. First, PS was converted to polystyrene sulfonamide (PSSNH 2 ) with an 85% yield using established procedures. PSSNH 2 was subsequently transformed into a reactive sulfinate by deamination using an N-heterocyclic carbene (NHC) catalyst and benzaldehyde. The catalytic process was optimized by varying catalysts, solvents, bases, temperatures, and reaction times. The highest degree of deamination was 88% with a bicyclic NHC and K 2 CO 3 base in DMSO for 18 hours at 120 °C. The reactive sulfinate was then treated with various functional reagents, resulting in a library of aromatic polymers with different substituents with high degrees of functionalization ranging from 74% to 98%. Similarly, we modified expanded PS waste with trifluorobutyl iodide with a degree of functionalization of 72%, highlighting a new avenue for plastic upgrading. This approach could be used to rapidly generate functionalized polymers from PS with potential applications, including antibacterial properties and flame retardancy.

Shrestha, Tulaja [Univ. of Delaware, Newark, DE (U↗
DOE OSTI2023

Mechanistic insights to drive catalytic hydrogenation of formamide intermediates to methanol via deaminative hydrogenation

Amine-promoted hydrogenation of CO 2 to methanol typically proceeds via a formamide intermediate when amines are used as additives or if the hydrogenation is performed in carbon capture solvents. The catalysts used for the hydrogenation of the formamide intermediate dictate the selectivity of the products formed: 1) Deoxygenative hydrogenation (C–O bond cleavage) resulting in N-methylation of amine and deactivation of the solvent, 2) Deaminative hydrogenation (C–N bond cleavage) resulting in formation of methanol and regeneration of the solvent. To date, catalytic reductions of CO 2 with amine promoters suffer from poor selectively for methanol which we attribute to the limiting formamide intermediate, though to date, the conditions that favor C–N cleavage have yet to be fully understood. To better understand the reactivity of the formamide intermediates, a range of heterogenous catalysts were used to study the hydrogenation of formamide. Well-known gas phase CO 2 hydrogenation catalysts catalyze the hydrogenation of formamide to N-methyl product via C–O bond cleavage. However, the selectivity can be readily shifted to selective C–N bond cleavage by addition of an additive with sufficient basicity for both homogenous and heterogeneous catalytic systems. The base additive shifts the selectivity by deprotonating a hemiaminal intermediate formed in situ during the formamide hydrogenation. This prevents dehydration process leading to N-methylated product, which is a key capture solvent deactivation pathway that hinders amine use in carbon capture, utilization, and storage (CCUS). The findings from this study provide a roadmap on how to improve the selectivity of known heterogenous catalysts, enabling catalytic reduction of captured CO 2 to methanol.

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

Structural basis of sequence-specific cytosine deamination by double-stranded DNA deaminase toxin DddA

The interbacterial deaminase toxin DddA catalyzes cytosine-to-uracil conversion in double-stranded (ds) DNA and enables CRISPR-free mitochondrial base editing, but the molecular mechanisms underlying its unique substrate selectivity have remained elusive. Here, we report crystal structures of DddA bound to a dsDNA substrate containing the 5'-TC target motif. These structures show that DddA binds to the minor groove of a sharply bent dsDNA and engages the target cytosine extruded from the double helix. DddA Phe1375 intercalates in dsDNA and displaces the 5' (-1) thymine, which in turn replaces the target (0) cytosine and forms a noncanonical T–G base pair with the juxtaposed guanine. This tandem displacement mechanism allows DddA to locate a target cytosine without flipping it into the active site. Biochemical experiments demonstrate that DNA base mismatches enhance the DddA deaminase activity and relax its sequence selectivity. On the basis of the structural information, we further identified DddA mutants that exhibit attenuated activity or altered substrate preference. Our studies may help design new tools useful in genome editing or other applications.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2017

Uracil Accumulation and Mutagenesis Dominated by Cytosine Deamination in CpG Dinucleotides in Mice Lacking UNG and SMUG1

Both a DNA lesion and an intermediate for antibody maturation, uracil is primarily processed by base excision repair (BER), either initiated by uracil-DNA glycosylase (UNG) or by single-strand selective monofunctional uracil DNA glycosylase (SMUG1). The relative in vivo contributions of each glycosylase remain elusive. To assess the impact of SMUG1 deficiency, we measured uracil and 5-hydroxymethyluracil, another SMUG1 substrate, in Smug1 - / - mice. Here, we found that 5-hydroxymethyluracil accumulated in Smug1 - / - tissues and correlated with 5-hydroxymethylcytosine levels. The highest increase was found in brain, which contained about 26-fold higher genomic 5-hydroxymethyluracil levels than the wild type. Smug1 - / - mice did not accumulate uracil in their genome and Ung - / - mice showed slightly elevated uracil levels. Contrastingly, Ung - / - Smug1 - / - mice showed a synergistic increase in uracil levels with up to 25-fold higher uracil levels than wild type. Whole genome sequencing of UNG/SMUG1-deficient tumours revealed that combined UNG and SMUG1 deficiency leads to the accumulation of mutations, primarily C to T transitions within CpG sequences. This unexpected sequence bias suggests that CpG dinucleotides are intrinsically more mutation prone. In conclusion, we showed that SMUG1 efficiently prevent genomic uracil accumulation, even in the presence of UNG, and identified mutational signatures associated with combined UNG and SMUG1 deficiency.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2020

Understanding the structural basis of HIV-1 restriction by the full length double-domain APOBEC3G

APOBEC3G, a member of the double-domain cytidine deaminase (CD) APOBEC, binds RNA to package into virions and restrict HIV-1 through deamination-dependent or deamination-independent inhibition. Mainly due to lack of a full-length double-domain APOBEC structure, it is unknown how CD1/CD2 domains connect and how dimerization/multimerization is linked to RNA binding and virion packaging for HIV-1 restriction. We report rhesus macaque A3G structures that show different inter-domain packing through a short linker and refolding of CD2. The A3G dimer structure has a hydrophobic dimer-interface matching with that of the previously reported CD1 structure. A3G dimerization generates a surface with intensified positive electrostatic potentials (PEP) for RNA binding and dimer stabilization. Unexpectedly, mutating the PEP surface and the hydrophobic interface of A3G does not abolish virion packaging and HIV-1 restriction. The data support a model in which only one RNA-binding mode is critical for virion packaging and restriction of HIV-1 by A3G.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2018

Insights into DNA substrate selection by APOBEC3G from structural, biochemical, and functional studies

Human apolipoprotein B mRNA-editing enzyme-catalytic polypeptide-like 3 (A3) proteins are a family of cytidine deaminases that catalyze the conversion of deoxycytidine (dC) to deoxyuridine (dU) in single-stranded DNA (ssDNA). A3 proteins act in the innate immune response to viral infection by mutating the viral ssDNA. One of the most well-studied human A3 family members is A3G, which is a potent inhibitor of HIV-1. Each A3 protein prefers a specific substrate sequence for catalysis—for example, A3G deaminates the third dC in the CCCA sequence motif. However, the interaction between A3G and ssDNA is difficult to characterize due to poor solution behavior of the full-length protein and loss of DNA affinity of the truncated protein. Here, we present a novel DNA-anchoring fusion strategy using the protection of telomeres protein 1 (Pot1) which has nanomolar affinity for ssDNA, with which we captured an A3G-ssDNA interaction. We crystallized a non-preferred adenine in the -1 nucleotide-binding pocket of A3G. The structure reveals a unique conformation of the catalytic site loops that sheds light onto how the enzyme scans substrate in the -1 pocket. Furthermore, our biochemistry and virology studies provide evidence that the nucleotide-binding pockets on A3G influence each other in selecting the preferred DNA substrate. Together, the results provide insights into the mechanism by which A3G selects and deaminates its preferred substrates and help define how A3 proteins are tailored to recognize specific DNA sequences. This knowledge contributes to a better understanding of the mechanism of DNA substrate selection by A3G, as well as A3G antiviral activity against HIV-1.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2017

Protection against herbivores

The present invention relates to genes, proteins and methods comprising molecules that alter amino acid levels. In one embodiment, the present invention relates to altering guanidino substrate hydrolysis activities in plants, arthropods and microorganisms using molecules within the arginase family and other molecules that alter an amino acid levels. In ones embodiment, the present invention relates to altering threonine substrate deamination and dehydration activities in plants, arthropods and microorganisms using molecules within the threonine deaminase family and other molecules that alter amino acid levels. In one embodiment, the present invention relates to using genes, proteins and methods comprising arginase or threonine deaminase for altering the pathophysiology of plants, arthropods and microorganisms. In a preferred embodiment, the present invention relates to altering guanidino substrate hydrolysis activity in plants, arthropods, and microorganisms using arginase. In another preferred embodiment, the invention relates to altering threonine substrated deamination and dehydration activity in plants, arthropods, and microorganisms using threonine deaminase. In some embodiments, the invention related to overexpression and increased activity of arginase, threonine deaminase and a proteinase inhibitor.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2017

Allelopathic effects of exogenous phenylalanine: a comparison of four monocot species

Isotopic labeling of biomolecules through biosynthesis from deuterated precursors has successfully been employed for both structural studies and metabolic analysis. Phenylalanine is the precursor of many products synthesized by plants, including the monolignols used for synthesis of lignin. Possible allelochemical effects of phenylalanine have not been reported, although its deamination product cinnamic acid is known to have deleterious effects on root elongation and growth of several plant species. In this work, the effects of phenylalanine and its deuterated analog phenylalanine-d8 added to growth media were studied for annual ryegrass (Lolium multiflorum), winter grain rye (Secale cereale), and switchgrass (Panicum virgatum) cultivated under hydroponic conditions. Growth of annual ryegrass was inhibited by phenylalanine while switchgrass and rye were not significantly affected. Growth was less affected by deuterated phenylalanine-d8 than by its protiated counterpart, which may be a typical deuterium kinetic isotope effect resulting in slower enzymatic reaction rates. Deuterium incorporation levels of 2-3% were achieved in biomass of switchgrass and annual ryegrass. Both protiated and deuterated phenylalanine were moderately toxic (IC25 values 0.6 and 0.8 mM, respectively) to duckweed (Lemna minor) grown using a 12 h diurnal cycle under photoautotrophic conditions. A significant increase in toxicity, greater for the deuterated form, was noted when duckweed was grown under higher intensity, full spectrum illumination with a metal halide lamp compared to fluorescent plant growth lamps emitting in the blue and red spectral regions. Lastly, supplementation with glucose increased toxicity of phenylalanine consistent with synergy between hexose and amino acid uptake that has been reported for duckweed.

09 BIOMASS FUELS↗
DOE OSTI2018

Studies of lysine cyclodeaminase from Streptomyces pristinaespiralis: Insights into the complex transition NAD+ state

Highlights: • LCD and its three structure complexes are determined for the first time. • A NAD{sup +} gate function was revealed in LCD by combining molecular simulation methods. • A substrate tunnel towards NAD{sup +} was observed in LCD via alignment of structures. • This study expands our understanding of catalytic NAD{sup +} usage in various enzymes. Lysine cyclodeaminase (LCD) catalyzes the piperidine ring formation in macrolide-pipecolate natural products metabolic pathways from a lysine substrate through a combination of cyclization and deamination. This enzyme belongs to a unique enzyme class, which uses NAD{sup +} as the catalytic prosthetic group instead of as the co-substrate. To understand the molecular details of NAD{sup +} functions in lysine cyclodeaminase, we have determined four ternary crystal structure complexes of LCD-NAD{sup +} with pipecolic acid (LCD-PA), lysine (LCD-LYS), and an intermediate (LCD-INT) as ligands at 2.26-, 2.00-, 2.17- and 1.80 Å resolutions, respectively. By combining computational studies, a NAD{sup +}-mediated “gate keeper” function involving NAD{sup +}/NADH and Arg49 that control the binding and entry of the ligand lysine was revealed, confirming the critical roles of NAD{sup +} in the substrate access process. Further, in the gate opening form, a substrate delivery tunnel between ε-carboxyl moiety of Glu264 and the α-carboxyl moiety of Asp236 was observed through a comparison of four structure complexes. The LCD structure details including NAD{sup +}-mediated “gate keeper” and substrate tunnel may assist in the exploration the NAD{sup +} function in this unique enzyme class, and in regulation of macrolide-pipecolate natural product synthesis.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2018

Crystal structure of the nicotinamidase/pyrazinamidase PncA from Bacillus subtilis

Highlights: • Crystal structure of B. Subtilis PncA consists of an α/β domain and a subdomain. • The metal-binding site of BsPncA is located at the bottom of a large cavity between the core and the subdomain. • Zinc ion is pentahedrally coordinated within several highly conserved amino acid residues and one water molecule. • Both dimers and tetramers were observed in BsPncA protein crystals, but only dimers were observed in solution. The nicotinamidase/pyrazinamidase PncA is a member of a large family of hydrolase enzymes that catalyze the deamination of nicotinamide to nicotinic acid. PncA also functions as a pyrazinamidase in a wide variety of eubacteria and is an essential coenzyme in many cellular redox reactions in living systems. We report the crystal structure of substrate-free PncA from Bacillus subtilis (BsPncA) at 2.0 Å resolution to improve our understanding of the PncA family. The structure of BsPncA consists of an α/β domain and a subdomain. The subdomain of BsPncA has a different conformation than that of PncA enzymes from other organisms. The B-factor analysis revealed a rigid structure of the α/β domain, while the subdomain is highly flexible. Both dimers and tetramers were observed in BsPncA protein crystals, but only dimers were observed in solution. Our results provide useful information that will further enhance our understanding of the molecular functions of PncA family members.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2020

Ring-opening and hydrodenitrogenation of indole under hydrothermal conditions over Ni, Pt, Ru, and Ni-Ru bimetallic catalysts

Here, the activity and selectivity of activated-carbon-supported Ni, Pt, Ru, and Ni-Ru bimetallic catalysts was examined for hydrothermal denitrogenation of indole. The molar yield of pyrrole ring-opening compounds, without an added hydrogen source, are in the order: Ni < Pt < Ni 90 Ru 10 < Ni 75 Ru 25 < Ni 50 Ru 50 ~ Ni 25 Ru 75 ~ Ru. Ru-containing catalysts facilitated production of hydrocarbons (hydrodenitrogenation (HDN) products) when used with added formic acid (hydrogen source). We elucidated catalytic hydrothermal HDN pathways for indole based on the product distributions and the variation of their yields with time. Hydrogenation of indole to indoline is the primary pathway and ring-opening of indoline to form alkyl anilines is faster than forming HDN products (alkyl benzenes). DFT calculations confirmed experimental activity trends, showing Ru is more active than Ni for indole ring opening and for o-toluidine deamination. If no hydrogen source is present, directly breaking the N-C bond in the pyrrole ring is more favorable than breaking the C-N bond with an aromatic carbon. If a H source is provided, the pyrrole ring hydrogenates first, forming indoline, followed by cleavage of the C-N bond.

42 ENGINEERING↗
DOE OSTI2017

AID Recognizes Structured DNA for Class Switch Recombination

We discuss how activation-induced cytidine deaminase (AID) initiates both class switch recombination (CSR) and somatic hypermutation (SHM) in antibody diversification. Mechanisms of AID targeting and catalysis remain elusive despite its critical immunological roles and off-target effects in tumorigenesis. Here, we produced active human AID and revealed its preferred recognition and deamination of structured substrates. G-quadruplex (G4)-containing substrates mimicking the mammalian immunoglobulin switch regions are particularly good AID substrates in vitro. By solving crystal structures of maltose binding protein (MBP)-fused AID alone and in complex with deoxycytidine monophosphate, we surprisingly identify a bifurcated substrate-binding surface that explains structured substrate recognition by capturing two adjacent single-stranded overhangs simultaneously. Moreover, G4 substrates induce cooperative AID oligomerization. Structure-based mutations that disrupt bifurcated substrate recognition or oligomerization both compromise CSR in splenic B cells. Collectively, our data implicate intrinsic preference of AID for structured substrates and uncover the importance of G4 recognition and oligomerization of AID in CSR.

59 BASIC BIOLOGICAL SCIENCES↗