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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 271 records · Page 15
All sourcesNASA NTRSDOE OSTI
DOE OSTI2022

Bioinspired oxidation of benzyl alcohol: The role of environment and nuclearity of the catalyst evaluated by multivariate analysis

Inspired by copper-containing enzymes such as galactose oxidase and catechol oxidase, in which distinct coordination environments and nuclearities lead to specific catalytic activities, we summarize here the catalytic properties of dinuclear and mononuclear copper species towards benzyl alcohol oxidation using a multivariate statistical approach. Here, the new dinuclear [Cu 2 (μ-L 1 )(μ-pz)] 2+ (1) is compared against the mononuclear [CuL 2 Cl] (2), where (L 1 ) - and (L 2 ) - are the respective deprotonated forms of 2,6-bis((bis(pyridin-2-ylmethyl)amino)methyl)-4-methylphenol, and 3-((bis(pyridin-2-ylmethyl)amino)methyl)-2-hydroxy-5-methylbenzaldehyde and (pz) - is a pyrazolato bridge. Copper(II) perchlorate (CP) is used as control. The catalytic oxidation of benzyl alcohol is pursued, aiming to assess the role of the ligand environment and nuclearity. The multivariate statistical approach allows for the search of optimal catalytic conditions, considering variables such as catalyst load, hydrogen peroxide load, and time. Species 1, 2 and CP promoted selective production of benzaldehyde at different yields, with only negligible amounts of benzoic acid. Under normalized conditions, 2 showed superior catalytic activity. This species is 3.5-fold more active than the monometallic control CP, and points out to the need for an efficient ligand framework. Species 2 is 6-fold more active than the dinuclear 1, and indicates the favored nuclearity for the conversion of alcohols into aldehydes.

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

Mechanistic insights explain the transforming potential of the T507K substitution in the protein-tyrosine phosphatase SHP2

The protein-tyrosine phosphatase SHP2 is an allosteric enzyme critical for cellular events downstream of growth factor receptors. Mutations in the SHP2 gene have been linked to many different types of human diseases, including developmental disorders, leukemia, and solid tumors. Unlike most SHP2-activating mutations, the T507K substitution in SHP2 is unique in that it exhibits oncogenic Ras-like transforming activity. However, the biochemical basis of how the SHP2/T507K variant elicits transformation remains unclear. By combining kinetic and biophysical methods, X-ray crystallography, and molecular modeling, as well as using cell biology approaches, in this work we uncovered that the T507K substitution alters both SHP2 substrate specificity and its allosteric regulatory mechanism. We found that although SHP2/T507K exists in the closed, autoinhibited conformation similar to the WT enzyme, the interactions between its N-SH2 and protein-tyrosine phosphatase domains are weakened such that SHP2/T507K possesses a higher affinity for the scaffolding protein Grb2-associated binding protein 1 (Gab1). We also discovered that the T507K substitution alters the structure of the SHP2 active site, resulting in a change in SHP2 substrate preference for Sprouty1, a known negative regulator of Ras signaling and a potential tumor suppressor. Our results suggest that SHP2/T507K's shift in substrate specificity coupled with its preferential association of SHP2/T507K with Gab1 enable the mutant SHP2 to more efficiently dephosphorylate Sprouty1 at pTyr-53. This dephosphorylation hyperactivates Ras signaling, which is likely responsible for SHP2/T507K's Ras-like transforming activity.

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

Comparison of human poly-N-acetyl-lactosamine synthase structure with GT-A fold glycosyltransferases supports a modular assembly of catalytic subsites

Poly-N-acetyl-lactosamine (poly-LacNAc) structures are composed of repeating [-Galβ(1,4)-GlcNAcβ(1,3)-]n glycan extensions. They are found on both N- and O-glycoproteins and glycolipids and play an important role in development, immune function, and human disease. The majority of mammalian poly-LacNAc is synthesized by the alternating iterative action of β1,3-N-acetylglucosaminyltransferase 2 (B3GNT2) and β1,4-galactosyltransferases. B3GNT2 is in the largest mammalian glycosyltransferase family, GT31, but little is known about the structure, substrate recognition, or catalysis by family members. Here we report the structures of human B3GNT2 in complex with UDP:Mg 2+ and in complex with both UDP:Mg 2+ and a glycan acceptor, lacto-N-neotetraose. The B3GNT2 structure conserves the GT-A fold and the DxD motif that coordinates a Mg 2+ ion for binding the UDP-GlcNAc sugar donor. The acceptor complex shows interactions with only the terminal Galβ(1,4)-GlcNAcβ(1,3)- disaccharide unit, which likely explains the specificity for both N- and O-glycan acceptors. Modeling of the UDP-GlcNAc donor supports a direct displacement inverting catalytic mechanism. Comparative structural analysis indicates that nucleotide sugar donors for GT-A fold glycosyltransferases bind in similar positions and conformations without conserving interacting residues, even for enzymes that use the same donor substrate. In contrast, the B3GNT2 acceptor binding site is consistent with prior models suggesting that the evolution of acceptor specificity involves loops inserted into the stable GT-A fold. These observations support the hypothesis that GT-A fold glycosyltransferases employ coevolving donor, acceptor, and catalytic subsite modules as templates to achieve the complex diversity of glycan linkages in biological systems.

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

Insight into CAZymes of Alicyclobacillus mali FL18: Characterization of a New Multifunctional GH9 Enzyme

In the bio-based era, cellulolytic and hemicellulolytic enzymes are biocatalysts used in many industrial processes, playing a key role in the conversion of recalcitrant lignocellulosic waste biomasses. In this context, many thermophilic microorganisms are considered as convenient sources of carbohydrate-active enzymes (CAZymes). In this work, a functional genomic annotation of Alicyclobacillus mali FL18, a recently discovered thermo-acidophilic microorganism, showed a wide reservoir of putative CAZymes. Among them, a novel enzyme belonging to the family 9 of glycosyl hydrolases (GHs), named AmCel9, was identified; in-depth in silico analyses highlighted that AmCel9 shares general features with other GH9 members. The synthetic gene was expressed in Escherichia coli and the recombinant protein was purified and characterized. The monomeric enzyme has an optimal catalytic activity at pH 6.0 and has comparable activity at temperatures ranging from 40 °C to 70 °C. It also has a broad substrate specificity, a typical behavior of multifunctional cellulases; the best activity is displayed on β-1,4 linked glucans. Very interestingly, AmCel9 also hydrolyses filter paper and microcrystalline cellulose. This work gives new insights into the properties of a new thermophilic multifunctional GH9 enzyme, that looks a promising biocatalyst for the deconstruction of lignocellulose.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2024

Phosphorus recycling by mineral-catalyzed ribonucleotide cleavage on iron and manganese oxides

Abstract Phosphorus is an essential element influencing both food security via plant fertilization, and water pollution through excessive phosphorus use, yet the phosphorus cycle in ecosystems is poorly known. In particular, beyond adsorption, the role of iron and manganese oxides in catalyzing the abiotic dephosphorylation of biomolecules is debated. Here, we studied the reactions of ribonucleotides, containing different phosphate bonding, with goethite, hematite, and birnessite. We employed both high-resolution mass spectrometry of solution species and molecular modeling simulations of ribonucleotide-mineral complexes. Results disclose an up to fivefold preferential hydrolytic cleavage of a phosphoanhydride bond over a phosphoester bond, indicating that mineral-catalyzed reactions reflect the hierarchy reported for the activity of phosphatase enzymes. The fourfold higher catalytic reactivity of goethite and birnessite versus hematite is explained by mineral-specific binding rather than surface area differences. Corresponding simulated adsorbate conformations at the water–mineral interfaces are proposed. Overall, our findings provide new insights on the catalytic recycling of organic phosphorus species by mineral oxides.

58 GEOSCIENCES↗
DOE OSTI2025

Divergent viral phosphodiesterases for immune signaling evasion

Cyclic dinucleotides (CDNs) and other short oligonucleotides play fundamental roles in immune system activation in organisms ranging from bacteria to humans. In response, viruses use phosphodiesterase (PDE)-mediated oligonucleotide cleavage for immune evasion, a strategy whose diversity has not yet been explored. Here, we use a canonical 2H PDE (2H PDE) structure-based search of prokaryotic and eukaryotic viral sequences to identify an exceptional diversity of 2H PDEs across the virome, including enzymes not detectable with sequence search methods alone. Despite active site conservation, biochemical experiments reveal remarkable substrate specificity of these PDEs that corresponds to variations in the core 2H fold. This nuanced specificity allows 2H PDEs to selectively degrade oligonucleotide messengers to avoid interfering with host nucleotide signaling. Together, these findings nominate viral 2H PDEs as key regulators of CDN signaling across the tree of life.

CBASS↗
DOE OSTI2022

Effects of experimental nitrogen deposition on soil organic carbon storage in Southern California drylands

Atmospheric nitrogen (N) deposition is enriching soils with N across biomes. Soil N enrichment can increase plant productivity and affect microbial activity, thereby increasing soil organic carbon (SOC), but such responses vary across biomes. Drylands cover ~45% of Earth's land area and store ~33% of global SOC contained in the top 1 m of soil. Nitrogen fertilization could, therefore, disproportionately impact carbon (C) cycling, yet whether dryland SOC storage increases with N remains unclear. To understand how N enrichment may change SOC storage, we separated SOC into plant–derived, particulate organic C (POC), and largely microbially derived, mineral–associated organic C (MAOC) at four N deposition experimental sites in Southern California. Theory suggests that N enrichment increases the efficiency by which microbes build MAOC (C stabilization efficiency) if soil pH stays constant. But if soils acidify, a common response to N enrichment, then microbial biomass and enzymatic organic matter decay may decrease, increasing POC but not MAOC. We found that N enrichment had no effect on C fractions except for a decrease in MAOC at one site. Specifically, despite reported increases in plant biomass in three sites and decreases in microbial biomass and extracellular enzyme activities in two sites that acidified, POC did not increase. Furthermore, microbial C use and stabilization efficiency increased in a non–acidified site, but without increasing MAOC. Instead, MAOC decreased by 16% at one of the sites that acidified, likely because it lost 47% of the exchangeable calcium (Ca) relative to controls. Indeed, MAOC was strongly and positively affected by Ca, which directly and, through its positive effect on microbial biomass, explained 58% of variation in MAOC. Finally, long–term effects of N fertilization on dryland SOC storage appear abiotic in nature, such that drylands where Ca–stabilization of SOC is prevalent and soils acidify, are most at risk for significant C loss.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2021

Inhibition of Pyruvate Kinase From Thermoanaerobacterium saccharolyticum by IMP Is Independent of the Extra-C Domain

The pyruvate kinase (PYK) isozyme from Thermoanaerobacterium saccharolyticum (TsPYK) has previously been used in metabolic engineering for improved ethanol production. This isozyme belongs to a subclass of PYK isozymes that include an extra C-domain. Like other isozymes that include this extra C-domain, we found that TsPYK is activated by AMP and ribose-5-phosphate (R5P). Our use of sugar-phosphate analogs generated a surprising result in that IMP and GMP are allosteric inhibitors (rather than activators) of TsPYK. We believe this to be the first report of any PYK isozyme being inhibited by IMP and GMP. A truncated protein that lacks the extra C-domain is also inhibited by IMP. A screen of several other bacterial PYK enzymes (include several that have the extra-C domain) indicates that the inhibition by IMP is specific to only a subset of those isozymes.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2020

Smart Microbial Cells Couple Catalysis and Sensing to Provide High-Throughput Selection of an Organophosphate Hydrolase

Enzyme engineering for gain of function requires navigating a large combinatorial sequence space efficiently. Typically, many mutations are needed to get significant improvements, while a single “bad” mutation can inactivate the enzyme. To establish high-throughput screening and achieve enhanced resolution between two variants, genetic libraries of the organophosphate hydrolase enzyme paraoxonase 1 (PON 1 ) were rapidly screened via an engineered positive-feedback circuit: a p-nitrophenol (PNP)-specific transcription factor (TF) regulated expression of PON 1 , which catalyzed paraoxon breakdown and PNP production. Rare active mutant colonies, picked by simple visual fluorescence of a PON 1 –green fluorescent protein (GFP) fusion, were characterized. Furthermore, in a single screening round, high (library-scale) throughput enabled the discovery of enhanced paraoxon degradation activity in PON 1 , including structurally unexpected mutations.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2022

Characterization of the Biomass Degrading Enzyme GuxA from Acidothermus cellulolyticus

Microbial conversion of biomass relies on a complex combination of enzyme systems promoting synergy to overcome biomass recalcitrance. Some thermophilic bacteria have been shown to exhibit particularly high levels of cellulolytic activity, making them of particular interest for biomass conversion. These bacteria use varying combinations of CAZymes that vary in complexity from a single catalytic domain to large multi-modular and multi-functional architectures to deconstruct biomass. Since the discovery of CelA from Caldicellulosiruptor bescii which was identified as one of the most active cellulase so far identified, the search for efficient multi-modular and multi-functional CAZymes has intensified. One of these candidates, GuxA (previously Acel_0615), was recently shown to exhibit synergy with other CAZymes in C. bescii, leading to a dramatic increase in growth on biomass when expressed in this host. GuxA is a multi-modular and multi-functional enzyme from Acidothermus cellulolyticus whose catalytic domains include a xylanase/endoglucanase GH12 and an exoglucanase GH6, representing a unique combination of these two glycoside hydrolase families in a single CAZyme. These attributes make GuxA of particular interest as a potential candidate for thermophilic industrial enzyme preparations. Here, we present a more complete characterization of GuxA to understand the mechanism of its activity and substrate specificity. In addition, we demonstrate that GuxA exhibits high levels of synergism with E1, a companion endoglucanase from A. cellulolyticus. We also present a crystal structure of one of the GuxA domains and dissect the structural features that might contribute to its thermotolerance.

09 BIOMASS FUELS↗
DOE OSTI2025

Cell-free synthetic biology for natural product biosynthesis and discovery

Natural products have applications as biopharmaceuticals, agrochemicals, and other high-value chemicals. However, there are challenges in isolating natural products from their native producers (e.g. bacteria, fungi, plants). In many cases, synthetic chemistry or heterologous expression must be used to access these important molecules. The biosynthetic machinery to generate these compounds is found within biosynthetic gene clusters, primarily consisting of the enzymes that biosynthesise a range of natural product classes (including, but not limited to ribosomal and nonribosomal peptides, polyketides, and terpenoids). Cell-free synthetic biology has emerged in recent years as a bottom-up technology applied towards both prototyping pathways and producing molecules. Recently, it has been applied to natural products, both to characterise biosynthetic pathways and produce new metabolites. This review discusses the core biochemistry of cell-free synthetic biology applied to metabolite production and critiques its advantages and disadvantages compared to whole cell and/or chemical production routes. Specifically, we review the advances in cell-free biosynthesis of ribosomal peptides, analyse the rapid prototyping of natural product biosynthetic enzymes and pathways, highlight advances in novel antimicrobial discovery, and discuss the rising use of cell-free technologies in industrial biotechnology and synthetic biology.

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

Structure of the poxvirus decapping enzyme D9 reveals its mechanism of cap recognition and catalysis

Poxviruses encode decapping enzymes that remove the protective 5' cap from both host and viral mRNAs to commit transcripts for decay by the cellular exonuclease Xrn1. Decapping by these enzymes is critical for poxvirus pathogenicity by means of simultaneously suppressing host protein synthesis and limiting the accumulation of viral double-stranded RNA (dsRNA), a trigger for antiviral responses. Here we present a high-resolution structural view of the vaccinia virus decapping enzyme D9. This Nudix enzyme contains a domain organization different from other decapping enzymes in which a three-helix bundle is inserted into the catalytic Nudix domain. The 5' mRNA cap is positioned in a bipartite active site at the interface of the two domains. Specificity for the methylated guanosine cap is achieved by stacking between conserved aromatic residues in a manner similar to that observed in canonical cap-binding proteins VP39, eIF4E, and CBP20, and distinct from eukaryotic decapping enzyme Dcp2.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2021

Electronic control of redox reactions inside Escherichia coli using a genetic module

Microorganisms regulate the redox state of different biomolecules to precisely control biological processes. These processes can be modulated by electrochemically coupling intracellular biomolecules to an external electrode, but current approaches afford only limited control and specificity. Here we describe specific electrochemical control of the reduction of intracellular biomolecules in Escherichia coli through introduction of a heterologous electron transfer pathway. E . coli expressing cymAmtrCAB from Shewanella oneidensis MR-1 consumed electrons directly from a cathode when fumarate or nitrate, both intracellular electron acceptors, were present. The fumarate-triggered current consumption occurred only when fumarate reductase was present, indicating all the electrons passed through this enzyme. Moreover, CymAMtrCAB-expressing E . coli used current to stoichiometrically reduce nitrate. Thus, our work introduces a modular genetic tool to reduce a specific intracellular redox molecule with an electrode, opening the possibility of electronically controlling biological processes such as biosynthesis and growth in any microorganism.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2024

The first intron and promoter of Arabidopsis DIACYLGLYCEROL ACYLTRANSFERASE 1 exert synergistic effects on pollen and embryo lipid accumulation

Summary Accumulation of triacylglycerols (TAGs) is crucial during various stages of plant development. In Arabidopsis , two enzymes share overlapping functions to produce TAGs, namely acyl‐CoA:diacylglycerol acyltransferase 1 (DGAT1) and phospholipid:diacylglycerol acyltransferase 1 (PDAT1). Loss of function of both genes in a dgat1‐1/pdat1‐2 double mutant is gametophyte lethal. However, the key regulatory elements controlling tissue‐specific expression of either gene has not yet been identified. We transformed a dgat1‐1/dgat1‐1//PDAT1/pdat1‐2 parent with transgenic constructs containing the Arabidopsis DGAT1 promoter fused to the AtDGAT1 open reading frame either with or without the first intron. Triple homozygous plants were obtained, however, in the absence of the DGAT1 first intron anthers fail to fill with pollen, seed yield is c . 10% of wild‐type, seed oil content remains reduced (similar to dgat1‐1/dgat1‐1 ), and non‐Mendelian segregation of the PDAT1/pdat1‐2 locus occurs. Whereas plants expressing the AtDGAT1pro:AtDGAT1 transgene containing the first intron mostly recover phenotypes to wild‐type. This study establishes that a combination of the promoter and first intron of AtDGAT1 provides the proper context for temporal and tissue‐specific expression of AtDGAT1 in pollen. Furthermore, we discuss possible mechanisms of intron mediated regulation and how regulatory elements can be used as genetic tools to functionally replace TAG biosynthetic enzymes in Arabidopsis .

McGuire, Sean T.↗
DOE OSTI2024

Directed Evolution of an Adenylation Domain Alters Substrate Specificity and Generates a New Catechol Siderophore in Escherichia coli

Nonribosomal peptide synthetases (NRPS) biosynthesize numerous natural products with therapeutic, agricultural, and industrial significance. Reliably altering substrate selection in these enzymes has been a longstanding goal, as this would enable the production of tailor-made peptides with desired activities. In this study, the NRPS EntF and the associated biosynthesis of the siderophore enterobactin (ENT) were used as a model system to interrogate substrate selection by an adenylation (A) domain. We employed a directed evolution pipeline that harnesses an in vivo genetic selection for siderophore production to alter A domain substrate selection. Surprisingly, this led to the formation of a new, physiologically active catechol siderophore in Escherichia coli. We characterized the enzyme variants in vitro and demonstrated transferability of our findings to the well-studied TycC and GrsB NRPSs. Furthermore, this work identifies critical binding pocket residues that allow for altered substrate selection in our model system and expands upon our understanding of iron acquisition in E. coli.

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 OSTI2025

Multi‐Omics Analyses Reveal Divergent Molecular Mechanisms Underlying Plant Biomass Conversion by Five Fungi

Fungal plant biomass conversion (FPBC) is of great importance to the global carbon cycle and has been increasingly applied for the production of biofuel and biochemicals from lignocellulose. However, the comprehensive understanding of relevant molecular mechanisms in different fungi remains challenging. Here, we comparatively analyzed the transcriptome, proteome and metabolome profile of four ascomycetes and one basidiomycete fungi during their growth on two common agricultural feedstocks (soybean hulls and corn stover). We revealed strong time‐, substrate‐ and species‐specific responses at multi‐omics levels for the tested fungi, highlighting species‐specific carbon utilization approaches and evolutionary adaptation to environmental niches. Notably, a remarkable expressional diversity of lignocellulose degrading enzymes, sugar transporter and metabolic genes, as well as industrially relevant metabolites were identified across different fungi and cultivation conditions. The findings improves our understanding of complex molecular networks underlying FPBC and fungal ecological roles, offering novel insights that can guide future genetic engineering of fungi for valorization of agriculture waste into value‐added bioproducts.

CAZy↗