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Results for “Enzyme Activation”

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 37 records · Page 2
All sourcesNASA NTRSDOE OSTI
DOE OSTI2023

4'-Deoxypyridoxine disrupts vitamin B 6 homeostasis in Escherichia coli K12 through combined inhibition of cumulative B 6 uptake and PLP-dependent enzyme activity

Pyridoxal 5'-phosphate (PLP) is the active form of vitamin B 6 and a cofactor for many essential metabolic processes such as amino acid biosynthesis and one carbon metabolism. 4’-deoxypyridoxine (4dPN) is a long known B 6 antimetabolite but its mechanism of action was not totally clear. By exploring different conditions in which PLP metabolism is affected in the model organism Escherichia coli K12, we showed that 4dPN cannot be used as a source of vitamin B 6 as previously claimed and that it is toxic in several conditions where vitamin B 6 homeostasis is affected, such as in a B 6 auxotroph or in a mutant lacking the recently discovered PLP homeostasis gene, yggS . In addition, we found that 4dPN sensitivity is likely the result of multiple modes of toxicity, including inhibition of PLP-dependent enzyme activity by 4’-deoxypyridoxine phosphate (4dPNP) and inhibition of cumulative pyridoxine (PN) uptake. These toxicities are largely dependent on the phosphorylation of 4dPN by pyridoxal kinase (PdxK).

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2020

Patterns in extracellular enzyme activity and microbial diversity in deep-sea Mediterranean sediments

Deep-sea sediments are populated by diverse microbial communities that derive their nutritional requirements from the degradation of organic matter. Extracellular hydrolytic enzymes play a key role in the survival of microbes by enabling them to access and degrade complex organic compounds that are found in seafloor sediments. Despite their importance, extracellular enzymatic activity is poorly characterized at water depths greater than a few hundred meters where physical properties, such as pressure and temperature, create a unique environment for influencing enzyme behavior. In this work, we investigated microbial communities and enzyme activities in surface sediment collected at four sampling stations in the central Mediterranean Sea at water depths ranging from 800 to 2200 m. Fluorometric assays revealed that extracellular hydrolytic activity varied according to substrate type and water depth which suggests that the distributions of these enzymes within this basin are not homogenous. Furthermore, enzyme activities indicated substantial demand for phosphomonoesters and proteins, with measurable but much lower demand for polysaccharides. Barcoded amplicon sequencing of bacterial and archaeal SSU genes revealed that microbial communities varied across sampling stations and some groups displayed water-depth related trends. Our results demonstrate that heterotrophic capabilities of microbes in deep-sea Mediterranean sediments can differ substantially even within the same region.

16S rRNA↗
DOE OSTI2021

Development and evaluation of an agar capture system (ACS) for high-throughput screening of insoluble particulate substrates with bacterial growth and enzyme activity assays

In this work, we describe a method for containing insoluble particulates for use as substrates in either bacterial growth or enzyme assays. This method was designed for high-throughput screening of environmental or engineered bacteria. Benchmarking this method with several model bacteria uncovered phenotypes not observable with the particulate substrates alone.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2020

Continental-scale patterns of extracellular enzyme activity in the subsoil: an overlooked reservoir of microbial activity

Abstract Chemical stabilization of microbial-derived products such as extracellular enzymes (EE) onto mineral surfaces has gained attention as a possibly important mechanism leading to the persistence of soil organic carbon (SOC). While the controls on EE activities and their stabilization in the surface soil are reasonably well-understood, how these activities change with soil depth and possibly diverge from those at the soil surface due to distinct physical, chemical, and biotic conditions remains unclear. We assessed EE activity to a depth of 1 m (10 cm increments) in 19 soil profiles across the Critical Zone Observatory Network, which represents a wide range of climates, soil orders, and vegetation types. For all EEs, activities per mass of soil correlated positively with microbial biomass (MB) and SOC, and all three of these variables decreased logarithmically with depth ( p < 0.05). Across all sites, over half of the potential EE activities per mass soil consistently occurred below 20 cm for all measured EEs. Activities per unit MB or SOC were substantially higher at depth (soils below 20 cm accounted for 80% of whole-profile EE activity), suggesting an accumulation of stabilized (i.e. mineral sorbed) EEs in subsoil horizons. The pronounced enzyme stabilization in subsurface horizons was corroborated by mixed-effects models that showed a significant, positive relationship between clay concentration and MB-normalized EE activities in the subsoil. Furthermore, the negative relationships between soil C, N, and P and C-, N-, and P-acquiring EEs found in the surface soil decoupled below 20 cm, which could have also been caused by EE stabilization. This finding suggests that EEs may not reflect soil nutrient availabilities deeper in the soil profile. Taken together, our results suggest that deeper soil horizons hold a significant reservoir of EEs, and that the controls of subsoil EEs differ from their surface soil counterparts.

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

Assessment of enzyme active site positioning and tests of catalytic mechanisms through X-ray–derived conformational ensembles

How enzymes achieve their enormous rate enhancements remains a central question in biology, and our understanding to date has impacted drug development, influenced enzyme design, and deepened our appreciation of evolutionary processes. While enzymes position catalytic and reactant groups in active sites, physics requires that atoms undergo constant motion. Numerous proposals have invoked positioning or motions as central for enzyme function, but a scarcity of experimental data has limited our understanding of positioning and motion, their relative importance, and their changes through the enzyme’s reaction cycle. To examine positioning and motions and test catalytic proposals, we collected “room temperature” X-ray crystallography data for Pseudomonas putida ketosteroid isomerase (KSI), and we obtained conformational ensembles for this and a homologous KSI from multiple PDB crystal structures. Here, ensemble analyses indicated limited change through KSI’s reaction cycle. Active site positioning was on the 1- to 1.5-Å scale, and was not exceptional compared to noncatalytic groups. The KSI ensembles provided evidence against catalytic proposals invoking oxyanion hole geometric discrimination between the ground state and transition state or highly precise general base positioning. Instead, increasing or decreasing positioning of KSI’s general base reduced catalysis, suggesting optimized Ångstrom-scale conformational heterogeneity that allows KSI to efficiently catalyze multiple reaction steps. Ensemble analyses of surrounding groups for WT and mutant KSIs provided insights into the forces and interactions that allow and limit active-site motions. Most generally, this ensemble perspective extends traditional structure–function relationships, providing the basis for a new era of “ensemble–function” interrogation of enzymes.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2024

A combinatorially complete epistatic fitness landscape in an enzyme active site

Protein engineering often targets binding pockets or active sites which are enriched in epistasis—nonadditive interactions between amino acid substitutions—and where the combined effects of multiple single substitutions are difficult to predict. Few existing sequence-fitness datasets capture epistasis at large scale, especially for enzyme catalysis, limiting the development and assessment of model-guided enzyme engineering approaches. We present here a combinatorially complete, 160,000-variant fitness landscape across four residues in the active site of an enzyme. Assaying the native reaction of a thermostable β-subunit of tryptophan synthase (TrpB) in a nonnative environment yielded a landscape characterized by significant epistasis and many local optima. These effects prevent simulated directed evolution approaches from efficiently reaching the global optimum. There is nonetheless wide variability in the effectiveness of different directed evolution approaches, which together provide experimental benchmarks for computational and machine learning workflows. The most-fit TrpB variants contain a substitution that is nearly absent in natural TrpB sequences—a result that conservation-based predictions would not capture. Thus, although fitness prediction using evolutionary data can enrich in more-active variants, these approaches struggle to identify and differentiate among the most-active variants, even for this near-native function. Overall, this work presents a large-scale testing ground for model-guided enzyme engineering and suggests that efficient navigation of epistatic fitness landscapes can be improved by advances in both machine learning and physical modeling.

biocatalysis↗
DOE OSTI2021

A multiplexed nanostructure-initiator mass spectrometry (NIMS) assay for simultaneously detecting glycosyl hydrolase and lignin modifying enzyme activities

Lignocellulosic biomass is composed of three major biopolymers: cellulose, hemicellulose and lignin. Analytical tools capable of quickly detecting both glycan and lignin deconstruction are needed to support the development and characterization of efficient enzymes/enzyme cocktails. Previously we have described nanostructure-initiator mass spectrometry-based assays for the analysis of glycosyl hydrolase and most recently an assay for lignin modifying enzymes. Here we integrate these two assays into a single multiplexed assay against both classes of enzymes and use it to characterize crude commercial enzyme mixtures. Application of our multiplexed platform based on nanostructure-initiator mass spectrometry enabled us to characterize crude mixtures of laccase enzymes from fungi Agaricus bisporus (Ab) and Myceliopthora thermophila (Mt) revealing activity on both carbohydrate and aromatic substrates. Using time-series analysis we determined that crude laccase from Ab has the higher GH activity and that laccase from Mt has the higher activity against our lignin model compound. Inhibitor studies showed a significant reduction in Mt GH activity under low oxygen conditions and increased activities in the presence of vanillin (common GH inhibitor). Ultimately, this assay can help to discover mixtures of enzymes that could be incorporated into biomass pretreatments to deconstruct diverse components of lignocellulosic biomass.

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

Enzyme activities predicted by metabolite concentrations and solvent capacity in the cell

Experimental measurements or computational model predictions of the post-translational regulation of enzymes needed in a metabolic pathway is a difficult problem. Consequently, regulation is mostly known only for well-studied reactions of central metabolism in various model organisms. In this study, we use two approaches to predict enzyme regulation policies and investigate the hypothesis that regulation is driven by the need to maintain the solvent capacity in the cell. The first predictive method uses a statistical thermodynamics and metabolic control theory framework while the second method is performed using a hybrid optimization–reinforcement learning approach. Efficient regulation schemes were learned from experimental data that either agree with theoretical calculations or result in a higher cell fitness using maximum useful work as a metric. As previously hypothesized, regulation is herein shown to control the concentrations of both immediate and downstream product concentrations at physiological levels. Model predictions provide the following two novel general principles: (1) the regulation itself causes the reactions to be much further from equilibrium instead of the common assumption that highly non-equilibrium reactions are the targets for regulation; and (2) the minimal regulation needed to maintain metabolite levels at physiological concentrations maximizes the free energy dissipation rate instead of preserving a specific energy charge. The resulting energy dissipation rate is an emergent property of regulation which may be represented by a high value of the adenylate energy charge. In addition, the predictions demonstrate that the amount of regulation needed can be minimized if it is applied at the beginning or branch point of a pathway, in agreement with common notions. The approach is demonstrated for three pathways in the central metabolism of E. coli (gluconeogenesis, glycolysis-tricarboxylic acid (TCA) and pentose phosphate-TCA) that each require different regulation schemes. It is shown quantitatively that hexokinase, glucose 6-phosphate dehydrogenase and glyceraldehyde phosphate dehydrogenase, all branch points of pathways, play the largest roles in regulating central metabolism.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2020

Kinetic modeling of microbial growth, enzyme activity, and gene deletions: An integrated model of β-glucosidase function in Cellvibrio japonicus

Understanding the complex growth and metabolic dynamics in microorganisms requires advanced kinetic models containing both metabolic reactions and enzymatic regulation to predict phenotypic behaviors under different conditions and perturbations. Most current kinetic models lack gene expression dynamics and are separately calibrated to distinct media, which consequently makes them unable to account for genetic perturbations or multiple substrates. This challenge limits our ability to gain a comprehensive understanding of microbial processes towards advanced metabolic optimizations that are desired for many biotechnology applications. Here, we present an integrated computational and experimental approach for the development and optimization of mechanistic kinetic models for microbial growth and metabolic and enzymatic dynamics. Our approach integrates growth dynamics, gene expression, protein secretion, and gene-deletion phenotypes. We applied this methodology to build a dynamic model of the growth kinetics in batch culture of the bacterium Cellvibrio japonicus grown using either cellobiose or glucose media. The model parameters were inferred from an experimental data set using an evolutionary computation method. The resulting model was able to explain the growth dynamics of C. japonicus using either cellobiose or glucose media and was also able to accurately predict the metabolite concentrations in the wild-type strain as well as in β-glucosidase gene deletion mutant strains. We validated the model by correctly predicting the non-diauxic growth and metabolite consumptions of the wild-type strain in a mixed medium containing both cellobiose and glucose, made further predictions of mutant strains growth phenotypes when using cellobiose and glucose media, and demonstrated the utility of the model for designing industrially-useful strains. Importantly, the model is able to explain the role of the different β-glucosidases and their behavior under genetic perturbations. Here, this integrated approach can be extended to other metabolic pathways to produce mechanistic models for the comprehensive understanding of enzymatic functions in multiple substrates.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2025

Cholesterol-dependent enzyme activity of human TSPO1

The amino acid sequence of the tryptophan-rich sensory proteins (TSPO) is substantially conserved throughout all kingdoms of life. Human mitochondrial TSPO1 (HsTSPO1) binds to porphyrins and steroids, although its interactions with these molecules remains unknown.HsTSPO1 is associated with numerous physiological and pathological disorders, but the underlying molecular mechanisms are unknown. Here, we disclose the finding of human mitochondrial TSPO as a cholesterol-dependent protoporphyrin IX oxygenase. The results of our biochemical characterization are consistent with structural data and evolutionary analysis. The dependence ofHsTSPO1 activity on cholesterol may be the result of the coevolution of this membrane protein with the membrane system. Our study provides a molecular foundation for comprehending the various roles played by mitochondrial TSPO in normal physiological and pathological situations.

Science & Technology - Other Topics↗
DOE OSTI2025

Unlocking soybean meal pectin recalcitrance using a multi-enzyme cocktail approach

Pectin is a complex plant heteropolysaccharide whose structure and function differ depending on its source. In animal feed, breaking down pectin is essential, as its presence increases feed viscosity and reduces nutrient absorption. Soybean meal, a protein-rich poultry feed ingredient, contains significant amounts of pectin, the structure of which remains unclear. Consequently, the enzyme activities required to degrade soybean meal pectin and how they interact are still open questions. In this study, we produced 15 recombinant fungal carbohydrate-active enzymes (CAZymes) identified from fungal secretomes acting on pectin. After observing that these enzymes were not active on soybean meal pectin when used alone, we developed a semi-miniaturized method to evaluate their effect as multi-activity cocktails. We designed and tested 12 enzyme pools, containing up to 15 different CAZymes, using several hydrolysis markers. Thanks to our multiactivity enzymatic approach combined with a Pearson correlation matrix, we identified 10 fungal CAZymes efficient on soybean meal pectin, 9 of which originate from Talaromyces versatilis. Based on enzyme specificity and linkage analysis, we propose a structural model for soybean meal pectin. Our findings underscore the importance of combining CAZymes to improve the degradation of agricultural co-products.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2022

Thermophilic Geobacillus WSUCF1 Secretome for Saccharification of Ammonia Fiber Expansion and Extractive Ammonia Pretreated Corn Stover

A thermophilic Geobacillus bacterial strain, WSUCF1 contains different carbohydrate-active enzymes (CAZymes) capable of hydrolyzing hemicellulose in lignocellulosic biomass. We used proteomic, genomic, and bioinformatic tools, and genomic data to analyze the relative abundance of cellulolytic, hemicellulolytic, and lignin modifying enzymes present in the secretomes. Results showed that CAZyme profiles of secretomes varied based on the substrate type and complexity, composition, and pretreatment conditions. The enzyme activity of secretomes also changed depending on the substrate used. The secretomes were used in combination with commercial and purified enzymes to carry out saccharification of ammonia fiber expansion (AFEX)-pretreated corn stover and extractive ammonia (EA)-pretreated corn stover. When WSUCF1 bacterial secretome produced at different conditions was combined with a small percentage of commercial enzymes, we observed efficient saccharification of EA-CS, and the results were comparable to using a commercial enzyme cocktail (87% glucan and 70% xylan conversion). It also opens the possibility of producing CAZymes in a biorefinery using inexpensive substrates, such as AFEX-pretreated corn stover and Avicel, and eliminates expensive enzyme processing steps that are used in enzyme manufacturing. Implementing in-house enzyme production is expected to significantly reduce the cost of enzymes and biofuel processing cost.

Bhalla, Aditya↗