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Results for “Enzyme Reactivity”

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 91 records · Page 5
All sourcesNASA NTRSDOE OSTI
DOE OSTI2023

Expanding the substrate scope of pyrrolysyl-transfer RNA synthetase enzymes to include non-$α$-amino acids in vitro and in vivo

The absence of orthogonal aminoacyl-transfer RNA (tRNA) synthetases that accept non-L-α-amino acids is a primary bottleneck hindering the in vivo translation of sequence-defined hetero-oligomers and biomaterials. Here we report that pyrrolysyl-tRNA synthetase (PylRS) and certain PylRS variants accept α-hydroxy, α-thio and N-formyl-L-α-amino acids, as well as α-carboxy acid monomers that are precursors to polyketide natural products. These monomers are accommodated and accepted by the translation apparatus in vitro; those with reactive nucleophiles are incorporated into proteins in vivo. High-resolution structural analysis of the complex formed between one PylRS enzyme and a m-substituted 2-benzylmalonic acid derivative revealed an active site that discriminates prochiral carboxylates and accommodates the large size and distinct electrostatics of an α-carboxy substituent. This work emphasizes the potential of PylRS-derived enzymes for acylating tRNA with monomers whose α-substituent diverges substantially from the α-amine of proteinogenic amino acids. These enzymes or derivatives thereof could synergize with natural or evolved ribosomes and/or translation factors to generate diverse sequence-defined non-protein heteropolymers.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2023

Carbonic Anhydrase Enhanced UV-Crosslinked PEG-DA/PEO Extruded Hydrogel Flexible Filaments and Durable Grids for CO 2 Capture

In this study, poly (ethylene glycol) diacrylate/poly (ethylene oxide) (PEG-DA/PEO) interpenetrating polymer network hydrogels (IPNH) were extruded into 1D filaments and 2D grids. The suitability of this system for enzyme immobilization and CO 2 capture application was validated. IPNH chemical composition was verified spectroscopically using FTIR. The extruded filament had an average tensile strength of 6.5 MPa and elongation at break of 80%. IPNH filament can be twisted and bent and therefore is suitable for further processing using conventional textile fabrication methods. Initial activity recovery of the entrapped carbonic anhydrase (CA) calculated from esterase activity, showed a decrease with an increase in enzyme dose, while activity retention of high enzyme dose samples was over 87% after 150 days of repeated washing and testing. IPNH 2D grids that were assembled into spiral roll structured packings exhibited increased CO 2 capture efficiency with increasing enzyme dose. Long-term CO 2 capture performance of the CA immobilized IPNH structured packing was tested in a continuous solvent recirculation experiment for 1032 h, where 52% of the initial CO 2 capture performance and 34% of the enzyme contribution were retained. These results demonstrate the feasibility of using rapid UV-crosslinking to form enzyme-immobilized hydrogels by a geometrically-controllable extrusion process that uses analogous linear polymers for both viscosity enhancement and chain entanglement purposes, and achieves high activity retention and performance stability of the immobilized CA. Potential uses for this system extend to 3D printing inks and enzyme immobilization matrices for such diverse applications as biocatalytic reactors and biosensor fabrication.

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

Advances in supramolecular host-mediated reactivity

Since the trailblazing discoveries of Lehn, Cram and Pedersen, supramolecular chemistry has established itself as a cornerstone of organic chemistry. Supramolecular hosts offer defined microenvironments that mimic the active sites of enzymes, utilizing specific host–guest interactions to enable remarkable rate enhancements and product selectivity. The development of a diverse array of self-assembled hosts, coupled with the increased demand for shorter and greener synthetic routes, have spurred significant progress in the field of supramolecular catalysis. This Review Article covers recent advances in the field, ranging from novel organic reactivity aided by supramolecular hosts to catalytic cooperation between hosts and organometallic compounds or metal nanoparticles. Furthermore, strides have also been made in the synthetic application of these hosts in site-selective substrate modifications and challenging photochemical reactions. These efforts have enabled the incorporation of non-covalent macromolecular catalysis in natural product syntheses, evidencing their unique advantages as a synthetic tool, and their powerful potential for practical applications.

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

Advancing Our Understanding of Pyranopterin-Dithiolene Contributions to Moco Enzyme Catalysis

The pyranopterin dithiolene ligand is remarkable in terms of its geometric and electronic structure and is uniquely found in mononuclear molybdenum and tungsten enzymes. The pyranopterin dithiolene is found coordinated to the metal ion, deeply buried within the protein, and non-covalently attached to the protein via an extensive hydrogen bonding network that is enzyme-specific. However, the function of pyranopterin dithiolene in enzymatic catalysis has been difficult to determine. This focused account aims to provide an overview of what has been learned from the study of pyranopterin dithiolene model complexes of molybdenum and how these results relate to the enzyme systems. This work begins with a summary of what is known about the pyranopterin dithiolene ligand in the enzymes. We then introduce the development of inorganic small molecule complexes that model aspects of a coordinated pyranopterin dithiolene and discuss the results of detailed physical studies of the models by electronic absorption, resonance Raman, X-ray absorption and NMR spectroscopies, cyclic voltammetry, X-ray crystallography, and chemical reactivity.

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

Deciphering the molecular components of the quorum sensing system in the fungus Ophiostoma piceae

Quorum sensing (QS) is a complex cell-cell communication mechanism that coordinates population-level behaviors in microbes. In eukaryotes, this phenomenon has been extensively described in the dimorphic yeast Candida albicans as its main QS molecule, the sesquiterpene alcohol farnesol, is responsible for various phenotypic (i.e., inhibition of yeast-to-hyphae transition, biofilm formation, and, hence, pathogenesis) and metabolic (i.e., induction of oxidative stress and apoptosis) changes. Ophiostoma piceae CECT 20416 is a dimorphic saprotrophic ascomycete with biotechnological interest that also produces farnesol as QS molecule, but in this case, the alcohol promotes the morphological transition to the mycelial form, biofilm formation, enzyme secretion, and melanin production. Here, we characterized the physiological response of Ophiostoma piceae to farnesol, the molecular components of the QS system of this fungus have been investigated using a “multiomics” approach that involved genomic, transcriptomic, and proteomic analyses. Some genes identified in this work are proposed as key factors in farnesol transport and signaling. We have also cataloged the genes undergoing major transcriptional changes triggered by the presence of the autoinducer, such as cell-wall remodeling, reactive oxygen species (ROS) protection, and melanin biosynthesis, using self-organizing maps. This analysis could be useful for applications in the forestry industry, for enzymes production, and for the valorization of residues. Furthermore, it might as well help to investigate the QS mechanisms of clinically relevant fungi phylogenetically related to Ophiostoma.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2024

Photoenzymatic Asymmetric Hydroamination for Chiral Alkyl Amine Synthesis

Chiral alkyl amines are common structural motifs in pharmaceuticals, natural products, synthetic intermediates, and bioactive molecules. An attractive method to prepare these molecules is the asymmetric radical hydroamination; however, this approach has not been explored with dialkyl amine-derive nitrogen-centered radicals since designing a catalytic system to generate the aminium radical cation, to suppress deleterious side reactions such as α-deprotonation and H atom abstraction, and to facilitate enantioselective hydrogen atom transfer is a formidable task. Herein, we describe the application of photoenzymatic catalysis to generate and harness the aminium radical cation for asymmetric intermolecular hydroamination. In this reaction, the flavin-dependent ene-reductase photocatalytically generates the aminium radical cation from the corresponding hydroxylamine and catalyzes the asymmetric intermolecular hydroamination to furnish the enantioenriched tertiary amine, whereby enantioinduction occurs through enzyme-mediated hydrogen atom transfer. Furthermore, this work highlights the use of photoenzymatic catalysis to generate and control highly reactive radical intermediates for asymmetric synthesis, addressing a long-standing challenge in chemical synthesis.

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

Tungsten enzymes play a role in detoxifying food and antimicrobial aldehydes in the human gut microbiome

Significance The diverse microorganisms contained within the human gut are known to have significant effects on human health. Herein, we show that genes encoding members of the tungsten oxidoreductase (WOR) family of enzymes and a tungstate-specific transporter are prevalent in the human gut microbiome and metagenome. We demonstrate that two model gut microbes assimilate tungsten into multiple WOR enzymes and that some of these enzymes catalyze the conversion of gut aldehydes to the corresponding acid, likely as a detoxification strategy to remove these reactive compounds.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2025

Genome-wide identification of novel flagellar motility genes in Pseudomonas syringae pv. tomato DC3000

Pseudomonas syringaepv.tomatoDC3000 (PstDC3000) is a plant pathogenic bacterium that possesses complicated motility regulation pathways including a typical chemotaxis system. A significant portion of our understanding about the genes functioning inPstDC3000 motility is based on comparison to other bacteria. This leaves uncertainty about whether gene functions are conserved, especially since specific regulatory modules can have opposite functions in sets ofPseudomonas. In this study, we used a competitive selection to enrich for mutants with altered swimming motility and used random barcode transposon-site sequencing (RB-TnSeq) to identify genes with significant roles in swimming motility. Besides many of the known or predicted chemotaxis and motility genes, our method identified PSPTO_0406 (dipA), PSPTO_1042 (chrR) and PSPTO_4229 (hypothetical protein) as novel motility regulators. PSPTO_0406 is a homolog ofdipA, a known cyclic di-GMP degrading enzyme inP. aeruginosa. PSPTO_1042 is part of an extracytoplasmic sensing system that controls gene expression in response to reactive oxygen species, suggesting that PSPTO_1042 may function as part of a mechanism that enablesPstDC3000 to alter motility when encountering oxidative stressors. PSPTO_4229 encodes a protein containing an HD-related output domain (HDOD), but with no previously identified functions. We found that deletion and overexpression of PSPTO_4229 both reduce swimming motility, suggesting that its function is sensitive to expression level. We used the overexpression phenotype to screen for nonsense and missense mutants of PSPTO_4229 that no longer reduce swimming motility and found a pair of conserved arginine residues that are necessary for motility suppression. Together these results provide a global perspective on regulatory and structural genes controlling flagellar motility inPstDC3000.

Microbiology↗
DOE OSTI2023

Reactive iron, not fungal community, drives organic carbon oxidation potential in floodplain soils

Wetlands host ∼20% of terrestrial organic carbon and serve as a major sink for atmospheric carbon. Anoxic soils and sediments accrue soil organic carbon (SOC) partly by hampering the activity of extracellular oxidative enzymes that break down phenolic polymers. Upon aeration, fungal-driven oxidative enzymatic depolymerization and microbial respiration of released monomers ensue. Redox-active metals can simultaneously catalyze abiotic nonspecific oxidation of SOC, notable examples including Mn(III) or Fe(II) through Fenton-like, hydrogen peroxide-catalyzed oxidative radical production. However, the extent of reactive metal contributions to biotic and abiotic SOC degradation is not understood in the context of natural environments with diverse redox chemistry. We tested the relative contributions of fungi, Mn(III) and Fe(II) to phenolic substrate (L-DOPA) oxidation in floodplain soils representing a range of transient redox conditions driven by permanent vs. periodic flooding. Phenol oxidative potential was highest in permanently flooded soils with fewer fungal taxa known for observed (per)oxidase activity and instead correlated with HCl-extractable Fe(II), Fe(total) and Fe(II)/Fe(total), suggesting a specific role of Fe(II). Fe(II) additions enhanced phenol oxidative potential in sterilized and non-sterilized soils in the presence of hydrogen peroxide, indicating abiotic Fe-mediated radical chemistry could significantly enhance wetland SOC oxidative depolymerization throughout redox-active floodplain soils. Fungal community composition did not correlate to phenol oxidative potential overall and only more oxic soils adjacent to the river with diverse fungal communities showed declining oxidative potential after sterilization. Mn(III) addition did not significantly enhance phenol oxidative potential across all soils, although it appeared to drive fungal-mediated oxidative potential in the most aerated floodplain soils. Understanding how metals mediate SOC depolymerization as abiotic oxidants or microbially-harnessed enzyme cofactors and substrates in soils under variable hydrologic controls will improve our ability to represent depolymerization in terrestrial carbon models in wetland and other frequently saturated soils.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2021

Biocatalytic Yarn for Peroxide Decomposition with Controlled Liquid Transport

In this study, a robust biocatalytic yarn with controllable liquid transport properties is created by coating thin layers of chitosan containing catalase onto a cellulosic yarn. The resulting material integrates enzyme catalytic functionality with protective coating properties of chitosan and structural functionality of the textile. Mild immobilization conditions and good affinity between the two polysaccharides minimize enzyme inactivation during the preparation steps and prevent enzyme from leaching during peroxide decomposition testing and washing, providing a novel and versatile enzyme immobilization strategy. The catalytic efficiency of enzymes in a reaction containing solid, liquid, and gas phases is facilitated when dissolved enzyme substrate is transported by liquid flowing through the coated textile structure. The flow-through configuration decomposes at least two times more peroxide in a twenty-times smaller reaction zone volume compared to a stirred tank configuration. Liquid transport through the yarn and liquid spatial distribution within the yarn are investigated by in situ neutron radiography and neutron computed tomography, revealing a constrained wicking mechanism that benefits biocatalytic yarn performance. This new class of sustainable and flexible biocatalytic textile matrices has beneficial multifunctional properties, not previously described, that are applicable for numerous small- and large-scale applications including controlled flow reactors and reactive filtration.

36 MATERIALS SCIENCE↗
DOE OSTI2023

Characterization of a monoclonal antibody specific to California serogroup orthobunyaviruses and development as a chimeric immunoglobulin M-positive control in human diagnostics

ABSTRACT California serogroup viruses (CSGVs) of medical importance in the United States include La Crosse virus, Jamestown Canyon virus (JCV), California encephalitis virus, and snowshoe hare virus. Current diagnosis of CSGVs relies heavily on serologic techniques for detecting immunoglobulin M (IgM), an indication of a recent CSGV infection. However, human-positive control sera reactive to viruses in the serogroup are scarce because detection of recent infections is rare. Here, we describe the development of new murine monoclonal antibodies (MAbs) reactive to CSGVs and the engineering of a human-murine chimeric antibody by combining the variable regions of the broadly CSGV cross-reactive murine MAb, 3-3B6/2-3B2 and the constant region of the human IgM. MAb 3-3B6/2-3B2 recognizes a tertiary epitope on the Gn/Gc heterodimer, and epitopes important in JCV neutralization were mapped to the Gc glycoprotein. This engineered human IgM constitutively expressed in a HEK-293 stable cell line can replace human-positive control sera in diagnostic serological techniques such as IgM antibody capture enzyme-linked immunosorbent assay (MAC-ELISA). Compared to the parent murine MAbs, the human-chimeric IgM antibody had identical serological activity to CSGVs in ELISA and demonstrated equivalent reactivity compared to human immune sera in the MAC-ELISA. Importance Orthobunyaviruses in the California serogroup cause severe neurological disease in children and adults. While these viruses are known to circulate widely in North America, their occurrence is rare. Serological testing for CSGVs is hindered by the limited availability and volumes of human-positive specimens needed as controls in serologic assays. Here, we described the development of a murine monoclonal antibody cross-reactive to CSGVs engineered to contain the variable regions of the murine antibody on the backbone of human IgM. The chimeric IgM produced from the stably expressing HEK293 cell line was evaluated for use as a surrogate human-positive control in a serologic diagnostic test.

Microbiology↗
DOE OSTI2021

Calcium‐Ion Binding Mediates the Reversible Interconversion of Cis and Trans Peroxido Dicopper Cores

Abstract Coupled dinuclear copper oxygen cores (Cu 2 O 2 ) featured in type III copper proteins (hemocyanin, tyrosinase, catechol oxidase) are vital for O 2 transport and substrate oxidation in many organisms. μ ‐1,2‐ cis peroxido dicopper cores ( C P ) have been proposed as key structures in the early stages of O 2 binding in these proteins; their reversible isomerization to other Cu 2 O 2 cores are directly relevant to enzyme function. Despite the relevance of such species to type III copper proteins and the broader interest in the properties and reactivity of bimetallic C P cores in biological and synthetic systems, the properties and reactivity of C P Cu 2 O 2 species remain largely unexplored. Herein, we report the reversible interconversion of μ ‐1,2‐ trans peroxido ( T P ) and C P dicopper cores. Ca II mediates this process by reversible binding at the Cu 2 O 2 core, highlighting the unique capability for metal‐ion binding events to stabilize novel reactive fragments and control O 2 activation in biomimetic systems.

Vargo, Natasha P.↗
DOE OSTI2020

Activity–Based Protein Profiling of Bile Salt Hydrolysis in the Human Gut Microbiome with Beta–Lactam or Acrylamide–Based Probes

Microbial bile salt hydrolases (BSHs) found in the intestine catalyze the deconjugation of taurine– and glycine–linked bile salts produced in the liver. The resulting bile salts are biological detergents and are critical in aiding lipophilic nutrient digestion. Therefore, the activity of BSHs in the gut microbiome is directly linked to human metabolism and overall health. Bile salt metabolism has also been associated with disease phenotypes such as liver and colorectal cancer. In order to reshape the gut microbiome to optimize bile salt metabolism, tools to characterize and quantify these processes must exist to enable a much–improved understanding of how metabolism goes awry in the face of disease, and how it can be improved through an altered lifestyle and environment. Furthermore, it is necessary to attribute metabolic activity to specific members and BSHs within the microbiome. To this end, we have developed activity–based probes with two different reactive groups to target bile salt hydrolases. Finally, these probes bind similarly to the authentic bile salt substrates, and we demonstrate enzyme labeling of active bile salt hydrolases by using purified protein, cell lysates, and in human stool.

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

Reactive transport modeling of organic carbon degradation in marine methane hydrate systems

Abstract Natural methane hydrate has often been observed in sand layers that contain no particulate organic carbon (POC), but are surrounded by organic-rich, fine-grained marine muds. In this paper, we develop a reactive transport model (RTM) of a microbially-mediated set of POC degradation reactions, including hydrolysis of POC driven by extracellular enzymes, fermentation of the resulting high-molecular weight dissolved organic carbon (HMW-DOC), and methanogenesis that consumes low-molecular weight dissolved organic carbon (LMW-DOC). These processes are mediated by two groups of microbes, fermenters and methanogens that are heterogeneously distributed in different lithologies, with the largest numbers of microbes in the large pores of coarse-grained layers. We find that the RTM can reproduce methane hydrate occurrences observed in two different geological environments, at Walker Ridge Site 313-H (Gulf of Mexico) and IODP Site U1325 (Cascadia Margin). We also find that microbes can degrade POC even if they are physically separated, as extracellular enzymes and DOC can diffuse away from where they are produced by microbes. Microbial activity is highest at relatively early times after burial at shallow depths and near lithological boundaries, where concentration gradients transport solutes to intervals that contain the most microbes.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗
DOE OSTI2025

Reversible enzyme-catalysed NAD + /NADH electrochemistry

The formate dehydrogenase subcomplex FdsBG from Cupriavidus necator has been electrochemically activated to reversibly interconvert NAD + /NADH and provide unique insight to its kinetics and reactivity.

Giang, Peter D. [School of Chemistry and Molecular↗
DOE OSTI2021

Calcium-Ion Binding Mediates the Reversible Interconversion of Cis and Trans Peroxido Dicopper Cores

Coupled dinuclear copper oxygen cores (Cu 2 O 2 ) featured in type III copper proteins (hemocyanin, tyrosinase, catechol oxidase) are vital for O 2 transport and substrate oxidation in many organisms. μ-1,2-cis peroxido dicopper cores ( C P) have been proposed as key structures in the early stages of O 2 binding in these proteins; their reversible isomerization to other Cu 2 O 2 cores are directly relevant to enzyme function. Furthermore, despite the relevance of such species to type III copper proteins and the broader interest in the properties and reactivity of bimetallic C P cores in biological and synthetic systems, the properties and reactivity of C P Cu 2 O 2 species remain largely unexplored. Herein, we report the reversible interconversion of μ-1,2-trans peroxido ( T P) and C P dicopper cores. Ca II mediates this process by reversible binding at the Cu 2 O 2 core, highlighting the unique capability for metal-ion binding events to stabilize novel reactive fragments and control O 2 activation in biomimetic systems.

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

Chlorophyllase from Arabidopsis thaliana Reveals an Emerging Model for Controlling Chlorophyll Hydrolysis

Chlorophyll (Chl) is one of Nature’s most complex pigments to biosynthesize and derivatize. This pigment is vital for survival and also paradoxically toxic if overproduced or released from a protective protein scaffold. Therefore, along with the mass production of Chl, organisms also invest in mechanisms to control its degradation and recycling. One important enzyme that is involved in these latter processes is chlorophyllase. This enzyme is employed by numerous photosynthetic organisms to hydrolyze the phytol tail of Chl. Although traditionally thought to catalyze the first step of Chl degradation, recent work suggests that chlorophyllase is instead employed during times of abiotic stress or conditions that produce reactive oxygen species. However, the molecular details regarding how chlorophyllases are regulated to function under such conditions remain enigmatic. Here, we investigate the Arabidopsis thaliana chlorophyllase isoform AtCLH2 using site-directed mutagenesis, mass spectrometry, dynamic light scattering, size-exclusion multiangle light scattering, and both steady-state enzyme kinetic and thermal stability measurements. Through these experiments, we show that AtCLH2 exists as a monomer in solution and contains two disulfide bonds. One disulfide bond putatively maps to the active site, whereas the other links two N-terminal Cys residues together. These disulfide bonds are cleaved by chemical or chemical and protein-based reductants, respectively, and are integral to maintaining the activity, stability, and substrate scope of the enzyme. This work suggests that Cys residue oxidation in chlorophyllases is an emerging regulatory strategy for controlling the hydrolysis of Chl pigments.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2022

Tyrosinase-Mediated Synthesis of Nanobody–Cell Conjugates

A convenient enzymatic strategy is reported for the modification of cell surfaces. Using a tyrosinase enzyme isolated from Agaricus bisporus, unique tyrosine residues introduced at the C-termini of nanobodies can be site-selectively oxidized to reactive o-quinones. These reactive intermediates undergo rapid modification with nucleophilic thiol, amine, and imidazole residues present on cell surfaces, producing novel nanobody–cell conjugates that display targeted antigen binding. We extend this approach toward the synthesis of nanobody–NK cell conjugates for targeted immunotherapy applications. The resulting NK cell conjugates exhibit targeted cell binding and elicit targeted cell death.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗