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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 955 records · Page 53
All sourcesNASA NTRSDOE OSTI
DOE OSTI2022

Fission yeast Duf89 and Duf8901 are cobalt/nickel-dependent phosphatase–pyrophosphatases that act via a covalent aspartyl–phosphate intermediate

Domain of Unknown Function 89 (DUF89) proteins are metal-dependent phosphohydrolases. Exemplary DUF89 enzymes differ in their metal and phosphosubstrate preferences. Here, we interrogated the activities and structures of two DUF89 paralogs from fission yeast—Duf89 and Duf8901. We find that Duf89 and Duf8901 are cobalt/nickel-dependent phosphohydrolases adept at hydrolyzing p-nitrophenylphosphate and PP i . Crystal structures of metal-free Duf89 and Co 2+ -bound Duf8901 disclosed two enzyme conformations that differed with respect to the position of a three-helix module, which is either oriented away from the active site in Duf89 or forms a lid over the active site in Duf8901. Lid closure results in a 16 Å movement of Duf8901 Asp195, vis-à-vis Asp199 in Duf89, that brings Asp195 into contact with an octahedrally coordinated cobalt. Reaction of Duf8901 with BeCl 2 and NaF in the presence of divalent cations Co 2+ , Ni 2+ , or Zn 2+ generated covalent Duf8901-(Asp248)–beryllium trifluoride (BeF 3 )•Co 2+ , Duf8901-(Asp248)–BeF 3 •Ni 2+ , or Duf8901-(Asp248)–BeF 3 •Zn 2+ adducts, the structures of which suggest a two-step catalytic mechanism via formation and hydrolysis of an enzyme-(aspartyl)–phosphate intermediate. Alanine mutations of Duf8901 Asp248, Asn249, Lys401, Asp286, and Asp195 that interact with BeF 3 •Co 2+ squelched p-nitrophenylphosphatase activity. A 1.8 Å structure of a Duf8901-(Asp248)–AlF 4 –OH 2 •Co 2+ transition-state mimetic suggests an associative mechanism in which Asp195 and Asp363 orient and activate the water nucleophile. Whereas deletion of the duf89 gene elicited a phenotype in which expression of phosphate homeostasis gene pho1 was derepressed, deleting duf8901 did not, thereby hinting that the DUF89 paralogs have distinct functional repertoires in vivo.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗
DOE OSTI2026

Synergistic cellulase–xylanase formulations for enhanced dewatering and fiber bonding toward energy-efficient and sustainable paper and packaging production

A mechanistic understanding of the synergistic effects of enzymes on cellulosic fibers dewatering and fiber bonding is essential for advancing energy-efficiency and lightweight production of paper and packaging materials. This study investigates the impact of varying cellulase and xylanase formulations on equilibrium moisture content (EMC) after pressing and tensile strength of cellulosic fiber webs using a factorial experimental design. Nine custom enzyme formulations were evaluated at controlled dosages ranging from ∼20 to 76 FPU/mL cellulase and ∼500–1130 IU/mL xylanase. Response surface modeling revealed a significant synergy, particularly at 20–40 FPU/mL cellulase combined with ≥ 1000 IU/mL xylanase. Under these conditions, EMC decreased by up to 3.6% compared with the untreated refined control, while tensile index gains of up to 16% were statistically significant for optimized blends (p < 0.05). The interaction between cellulase and xylanase was also significant for the tensile response (p = 0.010). Protein efficiency analysis showed that optimized formulations containing 30–40% less protein outperformed the commercial benchmark. The nonlinear synergy between cellulase and xylanase is attributed to their complementary substrate specificities. Endoglucanase- and β‑glucosidase‑rich cellulases hydrolyze internal β‑1,4‑glycosidic bonds in amorphous cellulose, loosening fiber walls and increasing flexibility, while xylanases target hemicellulose, primarily xylan-rich domains, enhancing porosity and improve cellulase accessibility. Tailoring enzyme formulations at low loadings overcomes traditional trade-offs between strength and dewatering, enabling cost-effective, energy-efficient, low-carbon solutions for sustainable packaging and hygiene products.

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

2-D soil zymography: Accounting for the spatial variation of pH

Soil zymography is commonly used to quantify spatial distribution of hydrolytic enzyme activities on soil and plant root surfaces. It is recommended to adjust pH in zymography substrates and calibration solutions with respect to soil/root pH. However, pH values may vary greatly within a few mm of plant rhizosphere, potentially altering the distribution of pH in zymography membranes. Despite the fact that the effect of pH on the calibration of zymography membranes is generally known, its potential impact on zymography results is unaccounted for in processing zymography images and calculations of enzyme activity. In this study we assessed the effect of pH variations on the persistency of the methylumbelliferone (MUF) calibration. The studied pH values ranged from 4.5 to 7.5. The MUF calibration curves greatly deviated from that at a reference pH of 6.5, with a marked nonlinear increase of deviation with greater membrane brightness. We suggest that the problem can be partially alleviated by reducing the membrane incubation time. However, such deviations suggest the need for a more comprehensive resolution via mapping pH and using pH-specific calibrations to process zymography images. Here, we developed a MATLAB code to implement a pixel-based correction of enzyme activity for pH in processing time-lapse zymography images.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2023

Engineering Escherichia coli for selective 1-decanol production using the reverse β-oxidation (rBOX) pathway

1-Decanol has great value in the pharmaceutical and fragrance industries and plays an important role in the chemical industry. Here, in this study, we engineered Escherichia coli to selectively synthesize 1-decanol by using enzymes of the core reverse β-oxidation (rBOX) pathway and termination module with overlapping chain-length specificity. Through screening for acyl-CoA reductase termination enzymes and proper regulation of rBOX pathway expression, a 1-decanol titer of 1.4 g/L was achieved. Further improvements were realized by engineering pyruvate dissimilation to ensure the generation of NADH through pyruvate dehydrogenase (PDH) and reducing byproduct synthesis via a tailored YigI thioesterase knockout, increasing 1-decanol titer to 1.9 g/L. The engineered strain produced about 4.4 g/L 1-decanol with a yield of 0.21 g/g in 36 hours in a bi-phasic fermentation that used a dodecane overlay to increase 1-decanol transport and reduce its toxicity. Adjustment of pathway expression (varying inducer concentration) and cell growth (oxygen availability) enabled 1-decanol production at 6.1 g/L (0.26 g/g yield) and 10.05 g/L (0.2 g/g yield) using rich in shake flasks and bioreactor, respectively. Remarkably, the use of minimal medium resulted in 1-decanol production at 100% specificity at 2.8 g/L (0.14 g/g yield) and a per cell mass yield higher than rich medium. These 1-decanol titers, yields and purity are at least 10-fold higher than others reported to date and the engineered strain shows great potential for industrial production. Taken together, our findings suggest that using rBOX pathway and termination enzymes of proper chain-length specificity in combination with optimal chassis engineering should be an effective approach for the selective production of alcohols.

1-decanol↗
DOE OSTI2019

Enhanced Softwood Cellulose Accessibility by H 3 PO 4 Pretreatment: High Sugar Yield without Compromising Lignin Integrity

Softwood lignocellulose is a potential feedstock for the production of biofuels and bioproducts. However, the highly cross-linked nature of softwood lignocellulose restricts enzyme access to its sugars. Thus, harsh pretreatment conditions (180–280 °C) and/or high enzyme loading are required to unlock sugars. These requirements negatively affect the economic viability of softwoods in biorefineries. Here we show that H 3 PO 4 pretreatment of pine and Douglas fir under a mild reaction temperature (50 °C) and atmospheric pressure enabled a high (~80%) glucan digestibility with low enzyme loading (5 filter paper units (FPU)/g glucan). The dissolution and regeneration of softwoods disrupted the hydrogen bonding between cellulose chains, thereby increasing the cellulose accessibility to cellulase (CAC) values by ~38-fold (from ~0.4 to 15 m 2 /g biomass). Examination of H 3 PO 4 -pretreated softwoods by cross-polarization/magic angle spin (CP/MAS), 13 C- nuclear magnetic resonance (NMR), and Fourier-transform infrared spectroscopy (FTIR) revealed that breaking of the orderly hydrogen bonding of crystalline cellulose caused the increase in CAC (higher than 11 m 2 /g biomass), which, in turn, was responsible for the high glucan digestibility of pretreated softwoods. The H 3 PO 4 pretreatment process was feedstock independent. Finally, 2D 13 C– 1 H heteronuclear single quantum coherence (HSQC) NMR showed that the lignin was depolymerized but not condensed; thus, the lignin can be available for producing high-value products.

09 BIOMASS FUELS↗
DOE OSTI2025

Role of the Mobile Active Site Flap in IMP Dehydrogenase Inhibitor Binding

Inosine 5′-monophosphate dehydrogenase (IMPDH) is a promising antibiotic target. This enzyme catalyzes the NAD-dependent oxidation of inosine 5′-monophosphate (IMP) to xanthosine 5′-monophosphate (XMP), which is the rate-limiting step in guanine nucleotide biosynthesis. Bacterial IMPDH-specific inhibitors have been developed that bind to the NAD + site. These inhibitors display varied affinities to different bacterial IMPDHs that are not easily rationalized by X-ray crystal structures of enzyme–inhibitor complexes. Inspection of X-ray crystal structures of 25 enzyme–inhibitor complexes, including 10 newly described, suggested that a mobile active site flap may be a structural determinant of inhibitor potency. Saturation transfer difference NMR experiments also suggested that the flap may contact the inhibitors to varying extents in different IMPDHs. Flap residue Leu413 contacted some inhibitors but was not structured in the crystal structures of other inhibitor complexes. The substitution of Leu413 with Phe or Ala in Bacillus anthracis IMPDH had inhibitor-selective effects, suggesting residue 413 could be a structural determinant of affinity. Curiously, the Ala substitution increased the potency of most inhibitors, even those that contacted Leu413 in the crystal structures. Presteady-state and steady-state kinetics experiments showed that the Leu413Ala substitution had comparable effects on inhibitor binding to the noncovalent E·IMP complex and the covalent intermediate E-XMP*, suggesting that the flap had similar interactions in both complexes. These results demonstrate that contacts do not necessarily indicate favorable interactions, and poorly structured mobile regions should not be discounted when assessing binding determinants.

Conformation↗
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 OSTI2025

Harnessing the Power of Machine Learning and Omics to Identify Environmental Regulation on Microbial Functional Composition for Soil C, N, and P Cycling

Microbial enzyme-mediated soil organic matter (SOM) decomposition regulates many key ecosystem functions, such as elemental cycling, soil carbon sequestration, and soil fertility. However, representing microbial processes in Earth system models (ESMs) remains challenging due to a limited understanding of the spatial patterns of diverse microbial functions responsible for soil carbon (C), nitrogen (N), and phosphorus (P) cycling as well as the underlying mechanisms regulating their relative abundances across various environments. We collected published metagenomics data across the continental US (CONUS) to identify hundreds of microbial genes involved in soil C, N, and P cycling and grouped them into eight enzyme functional classes (EFCs). Each EFC represented a group of gene-encoded potential enzymes that decompose similar soil compounds. By integrating the abundances of omics-informed EFCs with the corresponding environmental information, we trained a machine learning (ML) model to identify key edaphic, climate, and vegetation factors regulating the abundances of each EFC. Quantitative analysis of effects of these factors revealed that the spatial distribution of eight EFCs for soil C, N, and P cycling across CONUS reflected potential resource optimization strategies of microbial communities under nutrient limitation, preferential organic-mineral associations, and climatological stresses. This insight, together with the interpreted ML tool and the CONUS-level benchmark for EFCs abundances, paves the way for parameterizing environmental-regulated microbial functional dynamics in biogeochemical models.

machine learning↗
DOE OSTI2021

Direct detection of coupled proton and electron transfers in human manganese superoxide dismutase

Human manganese superoxide dismutase is a critical oxidoreductase found in the mitochondrial matrix. Concerted proton and electron transfers are used by the enzyme to rid the mitochondria of O 2 •– . The mechanisms of concerted transfer enzymes are typically unknown due to the difficulties in detecting the protonation states of specific residues and solvent molecules at particular redox states. Here, neutron diffraction of two redox-controlled manganese superoxide dismutase crystals reveal the all-atom structures of Mn 3+ and Mn 2+ enzyme forms. The structures deliver direct data on protonation changes between oxidation states of the metal. Observations include glutamine deprotonation, the involvement of tyrosine and histidine with altered pK a s, and four unusual strong-short hydrogen bonds, including a low barrier hydrogen bond. We report a concerted proton and electron transfer mechanism for human manganese superoxide dismutase from the direct visualization of active site protons in Mn 3+ and Mn 2+ redox states.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2023

Molecular basis of β-lactam antibiotic resistance of ESKAPE bacterium E. faecium Penicillin Binding Protein PBP5

Abstract Penicillin-binding proteins (PBPs) are essential for the formation of the bacterial cell wall. They are also the targets of β-lactam antibiotics. In Enterococcus faecium , high levels of resistance to β-lactams are associated with the expression of PBP5, with higher levels of resistance associated with distinct PBP5 variants. To define the molecular mechanism of PBP5-mediated resistance we leveraged biomolecular NMR spectroscopy of PBP5 – due to its size (>70 kDa) a challenging NMR target. Our data show that resistant PBP5 variants show significantly increased dynamics either alone or upon formation of the acyl-enzyme inhibitor complex. Furthermore, these variants also exhibit increased acyl-enzyme hydrolysis. Thus, reducing sidechain bulkiness and expanding surface loops results in increased dynamics that facilitates acyl-enzyme hydrolysis and, via increased β-lactam antibiotic turnover, facilitates β-lactam resistance. Together, these data provide the molecular basis of resistance of clinical E. faecium PBP5 variants, results that are likely applicable to the PBP family.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2023

Heterogeneity in M. tuberculosis β-lactamase inhibition by Sulbactam

Abstract For decades, researchers have elucidated essential enzymatic functions on the atomic length scale by tracing atomic positions in real-time. Our work builds on possibilities unleashed by mix-and-inject serial crystallography (MISC) at X-ray free electron laser facilities. In this approach, enzymatic reactions are triggered by mixing substrate or ligand solutions with enzyme microcrystals. Here, we report in atomic detail (between 2.2 and 2.7 Å resolution) by room-temperature, time-resolved crystallography with millisecond time-resolution (with timepoints between 3 ms and 700 ms) how the Mycobacterium tuberculosis enzyme BlaC is inhibited by sulbactam (SUB). Our results reveal ligand binding heterogeneity, ligand gating, cooperativity, induced fit, and conformational selection all from the same set of MISC data, detailing how SUB approaches the catalytic clefts and binds to the enzyme noncovalently before reacting to a trans- enamine. This was made possible in part by the application of singular value decomposition to the MISC data using a program that remains functional even if unit cell parameters change up to 3 Å during the reaction.

74 ATOMIC AND MOLECULAR PHYSICS↗
DOE OSTI2020

Generalized enzymatic mechanism of catalysis by tetrameric L -asparaginases from mesophilic bacteria

The mechanism of catalysis by the L -glutaminase-asparaginase from Pseudomonas 7A (PGA) was investigated using structural, mass spectrometry, and kinetic data. We had previously proposed mechanism of hydrolysis of L -Asn by the type II L-asparaginase from E. coli (EcAII), but that work was limited to just one enzyme. Based on results presented in this report, we postulate that all homotetrameric L -asparaginases from mesophilic bacteria utilize a common ping-pong mechanism of catalysis consisting of two subsequent nucleophilic substitutions. Several new structures of non-covalent complexes of PGA with different substrates, as well as structures of covalent acyl-enzyme intermediates of PGA with canonical substrates ( L -Asp and L -Glu) and an opportunistic ligand, a citrate anion, were determined. The results of kinetic experiments monitored by high-resolution LC/MS, when combined with new structural data, clearly show that the reaction catalyzed by L -glutaminase-asparaginases proceeds through formation of a covalent intermediate, as observed previously for EcAII. Additionally, by showing that the same mechanism applies to L -Asn and L -Gln, we postulate that it is common for all these structurally related enzymes.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2021

Structural characterization of a GNAT family acetyltransferase from Elizabethkingia anophelis bound to acetyl-CoA reveals a new dimeric interface

General control non-repressible 5 (GCN5)-related N-acetyltransferases (GNATs) catalyse the acetylation of a diverse range of substrates, thereby orchestrating a variety of biological processes within prokaryotes and eukaryotes. GNAT enzymes can catalyze the transfer of an acetyl group from acetyl coenzyme A to substrates such as aminoglycoside antibiotics, amino acids, polyamines, peptides, vitamins, catecholamines, and large macromolecules including proteins. Although GNATs generally exhibit low to moderate sequence identity, they share a conserved catalytic fold and conserved structural motifs. In this current study we characterize the high-resolution X-ray crystallographic structure of a GNAT enzyme bound with acetyl-CoA from Elizabethkingia anophelis , an important multi-drug resistant bacterium. The tertiary structure is comprised of six α-helices and nine β-strands, and is similar with other GNATs. We identify a new and uncharacterized GNAT dimer interface, which is conserved in at least two other unpublished GNAT structures. This suggests that GNAT enzymes can form at least five different types of dimers, in addition to a range of other oligomers including trimer, tetramer, hexamer, and dodecamer assemblies. The high-resolution structure presented in this study is suitable for future in-silico docking and structure–activity relationship studies.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2024

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

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

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2020

Unusual zwitterionic catalytic site of SARS–CoV-2 main protease revealed by neutron crystallography

The main protease (3CL M pro ) from SARS–CoV-2, the etiological agent of COVID-19, is an essential enzyme for viral replication. 3CL M pro possesses an unusual catalytic dyad composed of Cys 145 and His 41 residues. A critical question in the field has been what the protonation states of the ionizable residues in the substrate-binding active-site cavity are; resolving this point would help understand the catalytic details of the enzyme and inform rational drug development against this pernicious virus. Here, we present the room-temperature neutron structure of 3CL M pro , which allowed direct determination of hydrogen atom positions and, hence, protonation states in the protease. We observe that the catalytic site natively adopts a zwitterionic reactive form in which Cys 145 is in the negatively charged thiolate state and His 41 is doubly protonated and positively charged, instead of the neutral unreactive state usually envisaged. The neutron structure also identified the protonation states, and thus electrical charges, of all other amino acid residues and revealed intricate hydrogen-bonding networks in the active-site cavity and at the dimer interface. The fine atomic details present in this structure were made possible by the unique scattering properties of the neutron, which is an ideal probe for locating hydrogen positions and experimentally determining protonation states at near-physiological temperature. Our observations provide critical information for structure-assisted and computational drug design, allowing precise tailoring of inhibitors to the enzyme's electrostatic environment.

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

Genomics-enabled analysis of specialized metabolism in bioenergy crops: Current progress and challenges

Plants produce a staggering diversity of specialized small molecule metabolites that play vital roles in mediating environmental interactions and stress adaptation. This chemical diversity derives from dynamic biosynthetic pathway networks that are often species-specific and operate under tight spatiotemporal and environmental control. A growing divide between demand and environmental challenges in food and bioenergy crop production have intensified research on these complex metabolite networks and their contribution to crop fitness. High-throughput omics technologies provide access to ever-increasing data resources for investigating plant metabolism. However, the efficiency of using such system-wide data to decode the gene and enzyme functions controlling specialized metabolism has remained limited; due largely to the recalcitrance of many plants to genetic approaches and the lack of ‘user-friendly’ biochemical tools for studying the diverse enzyme classes involved in specialized metabolism. With emphasis on terpenoid metabolism in the bioenergy crop switchgrass as an example, this review aims to illustrate current advances and challenges in the application of DNA synthesis and synthetic biology tools for accelerating the functional discovery of genes, enzymes and pathways in plant specialized metabolism. These technologies have accelerated knowledge development on the biosynthesis and physiological roles of diverse metabolite networks across many ecologically and economically important plant species and can provide resources for application to precision breeding and natural product metabolic engineering.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2022

Microbial community shifts correspond with suppression of decomposition 25 years after liming of acidic forest soils

Microbial community structure and function regularly covary with soil pH, yet effects of these interactions on soil carbon are rarely tested experimentally within natural ecosystems. We investigated the enduring (25 year) impacts of liming on microbial community structure and decomposition at an acidic northern hardwood forest, where experimental liming increased pH one unit and surprisingly doubled the organic carbon stocks of the forest floor. Here we show that this increase in carbon storage corresponded with restructuring of the bacterial and fungal communities that drive decomposition. In the Oe horizon, liming reduced the activities of five extracellular enzymes that mediate decomposition, while the Oa horizon showed an especially large (64%) reduction in the activity of a sixth, peroxidase, which is an oxidative enzyme central to lignocellulose degradation. Decreased enzyme activities corresponded with loss of microbial taxa important for lignocellulose decay, including large reductions in the dominant ectomycorrhizal genera Russula and Cenococcum, saprotrophic and wood decaying fungi, and Actinobacteria (Thermomonosporaceae). These results demonstrate the importance of pH as a dominant regulator of microbial community structure and illustrate how changes to this structure can produce large, otherwise unexpected increases in carbon storage in forest soils.

59 BASIC BIOLOGICAL SCIENCES↗