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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 469 records · Page 26
All sourcesNASA NTRSDOE OSTI
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 OSTI2023

Kinetic Control via Binding Sites within the Confined Space of Metal Metalloporphyrin-Frameworks for Enhanced Shape-Selectivity Catalysis

One striking feature of enzyme is its controllable ability to trap substrates via synergistic or cooperative binding in the enzymatic pocket, which renders the shape-selectivity of product by the confined spatial environment. The success of shape-selective catalysis relies on the ability of enzyme to tune the thermodynamics and kinetics for chemical reactions. In emulation of enzyme's ability, we showcase herein a targeting strategy with the substrate being anchored on the internal pore wall of metal-organic frameworks (MOFs), taking full advantage of the sterically kinetic control to achieve shape-selectivity for the reactions. For this purpose, a series of binding site-accessible metal metalloporphyrin-frameworks (MMPFs) have been investigated to shed light on the nature of enzyme-mimic catalysis. They exhibit a different density of binding sites that are well arranged into the nanospace with corresponding distances of opposite binding sites. Furthermore, such a structural specificity results in a facile switch in selectivity from an exclusive formation of the thermodynamically stable product to the kinetic product. Thus, the proposed targeting strategy, based on the combination of porous materials and binding events, paves a new way to develop highly efficient heterogeneous catalysts for shifting selectivity.

32 ENERGY CONSERVATION, CONSUMPTION, AND UTILIZATI↗
DOE OSTI2021

Controlled Organization of Inorganic Materials Using Biological Molecules for Activating Therapeutic Functionalities

Precise control over the assembly of biocompatible three-dimensional (3D) nanostructures would allow for programmed interactions within the cellular environment. Nucleic acids can be used as programmable crosslinkers to direct the assembly of quantum dots (QDs) and tuned to demonstrate different interparticle binding strategies. In this work, morphologies of self-assembled QDs are evaluated via gel electrophoresis, transmission electron microscopy, small-angle X-ray scattering, and dissipative particle dynamics simulations, with all results being in good agreement. The controlled assembly of 3D QD organizations is demonstrated in cells via the colocalized emission of multiple assembled QDs, and their immunorecognition is assessed via enzyme-linked immunosorbent assays. RNA interference inducers are also embedded into the interparticle binding strategy to be released in human cells only upon QD assembly, which is demonstrated by specific gene silencing. The programmability and intracellular activity of QD assemblies offer a strategy for nucleic acids to imbue the structure and therapeutic function into the formation of complex networks of nanostructures, while the photoluminescent properties of the material allow for optical tracking in cells in vitro.

36 MATERIALS SCIENCE↗
DOE OSTI2025

Understanding and Harnessing the Robustness of Undomesticated Yarrowia lipolytica Strains for Biosynthesis of Designer Bioesters (Final Report)

This project seeks to elucidate and harness the exceptional robustness of novel and undomesticated Y. lipolytica isolates, which were identified from a genetic diversity screening for compatibility with bioenergy development. Bioenergy-relevant isolates were further developed as microbial platforms for efficient conversion of undetoxified biomass hydrolysates into designer bio-esters continuously recovered by solvent extraction. The project has three major goals. Goal 1. Elucidate and enhance the endogenous metabolism of Y. lipolytica for superior growth, sugar utilization, and lipid accumulation in undetoxified biomass hydrolysates under hypoxic conditions. Goal 2. Understand and enhance the underlying mechanism of exceptional tolerance of Y. lipolytica to organic solvents. Goal 3. Elucidate and rewire endogenous metabolism of the most robust Y. lipolytica strains for effective conversion of accumulating lipids to designer bio-esters. Significant progress has been made toward completing all research goals. We elucidated and optimized the robustness of Y. lipolytica by utilizing mixed C5 and C6 sugars in switchgrass hydrolysates (SGH) for lipid production (Aim 1). We conducted extensive omics analysis to investigate how genetic diversity among Yarrowia strains, derived from natural isolates or developed through adaptive laboratory evolution, influences lipid production when utilizing SGH. In Aim 2, novel mechanisms and underlying genetics were discovered that enabled Yarrowia strains to thrive in cultures containing high ionic liquid (IL) concentrations. Amongst other novel findings, it was found that sterols strengthened cell membranes to confer IL toxicity resistance, specifically via increased ergosterol content upon exposure to IL. In Aim 3, mechanistic studies elucidated how Y. lipolytica utilized intracellular lipids and alkanes/alkenes, leading to our discovery of novel enzymes and pathways for making short-chain esters. Most notably, thermostable chloramphenicol transferases were repurposed to function as alcohol acetyltransferases in Y. lipolytica, as well as the Gram-negative and Gram-positive bacteria Escherichia coli and thermophile Clostridium thermocellum, respectively.

09 BIOMASS FUELS↗
NASA NTRS1985

Quantitation of Na+, K+-atpase Enzymatic Activity in Tissues of the Mammalian Vestibular System

In order to quantify vestibular Na(+), K(+)-ATPase, a microassay technique was developed which is sufficiently sensitive to measure the enzymatic activity in tissue from a single animal. The assay was used to characterize ATPase in he vestibular apparatus of the Mongolian gerbil. The quantitative procedure employs NPP (5 mM) as synthetic enzyme substrate. The assay relies upon spectrophotometric measurement (410 nm) of nitrophenol (NP) released by enzymatic hydrolysis of the substrate. Product formation in the absence of ouabain reflects both specific (Na(+), K(+)-ATPase) and non-specific (Mg(++)-ATPase) enzymatic activity. By measuring the accumulation of reaction product (NP) at three-minute intervals during the course of incubation, it is found that the overall enzymatic reaction proceeds linearly for at least 45 minutes. It is therefore possible to determine two separate reaction rates from a single set of tissues. Initial results indicate that total activity amounts to 53.3 + or - 11.2 (S.E.M.) nmol/hr/mg dry tissue, of which approximately 20% is ouabain-sensitive.

Kerr, T. P.↗
NASA NTRS2017

Comparison of Two Bioinformatics Tools Used to Characterize the Microbial Diversity and Predictive Functional Attributes of Microbial Mats from Lake Obersee, Antarctica

In this study, using NextGen sequencing of the collective 16S rRNA genes obtained from two sets of samples collected from Lake Obersee, Antarctica, we compared and contrasted two bioinformatics tools, PICRUSt and Tax4Fun. We then developed an R script to assess the taxonomic and predictive functional profiles of the microbial communities within the samples. Taxa such as Pseudoxanthomonas, Planctomycetaceae, Cyanobacteria Subsection III, Nitrosomonadaceae, Leptothrix, and Rhodobacter were exclusively identified by Tax4Fun that uses SILVA database; whereas PICRUSt that uses Greengenes database uniquely identified Pirellulaceae, Gemmatimonadetes A1-B1, Pseudanabaena, Salinibacterium and Sinibacteraceae. Predictive functional profiling of the microbial communities using Tax4Fun and PICRUSt separately revealed common metabolic capabilities, while also showing specific functional IDs not shared between the two approaches. Combining these functional predictions using a customized R script revealed a more inclusive metabolic profile, such as hydrolases, oxidoreductases, transferases; enzymes involved in carbohydrate and amino acid metabolisms; and membrane transport proteins known for nutrient uptake from the surrounding environment. Our results present the first molecular-phylogenetic characterization and predictive functional profiles of the microbial mat communities in Lake Obersee, while demonstrating the efficacy of combining both the taxonomic assignment information and functional IDs using the R script created in this study for a more streamlined evaluation of predictive functional profiles of microbial communities.

Hyunmin Koo↗
DOE OSTI2022

Effect of Electroacupuncture at Wushu Acupoints of the Cardiopulmonary Meridian on the Autophagy in Rats with Acute Myocardial Ischemia

Wushu acupoints are the five acupoints distributed below the human elbow and knee joint. They are all located on the same meridian and divided into five categories: Jing, Ying, Shu, Jing, and He. It has been shown that electroacupuncture (EA) at Shenmen point of heart meridian can improve acute myocardial ischemia (AMI) early. However, it is still unclear if all the Wushu acupoints of the heart meridian can improve AMI. Hence, this study emphasizes Wushu acupoints of heart meridian, compares them with Wushu acupoints of lung meridian, and studies the therapeutic effect of EA at Wushu acupoints on AMI and its possible mechanism. It also discusses the specificity of the heart meridian to heart disease. The AMI model is established by ligation of the left anterior descending coronary artery. The detection methods like the physiological recorder, TTC staining, ELISA, and so forth were used to determine the ECG, myocardial infarct size, serum myocardial enzymes, and myocardial tissue-related protein expression in rats. The heart rate (HR) and ST segment along with creatine kinase (CK), creatine kinase isoenzymes (CK-MB), lactate dehydrogenase (LDH), and myocardial infarctions increased after the induction with AMI. Furthermore, the expressions of PINK1 and Parkin protein also showed an increase. However, EA at Wushu acupoints in the heart meridian can reverse the above changes, whereas EA at the lung meridian exhibits limited effect. It is depicted that the heart meridian has a relatively specific relationship with the heart in a diseased state.

Zhu, Chao↗
DOE OSTI2025

Understanding the dynamic nature of plant lipid anabolic and catabolic metabolism is key to sustainable oilseed engineering

Plant-derived oils are essential sources of reduced carbon and various fatty acid (FA) structures for food, biofuels, and the oleochemical industry. Despite extensive efforts, engineering mainstream oilseed crops to produce high levels of industrially valuable unusual FAs (UFAs) remains challenging. This review synthesizes recent advances in the understanding of lipid metabolic networks, emphasizing how species-specific regulation of FA synthesis, activation, and delivery influences triacylglycerol (TAG) assembly to govern the efficiency of UFA accumulation. Key insights reveal that acyl flux through anabolic and catabolic branches of lipid metabolism is tightly controlled by enzyme substrate selectivities, diacylglycerol (DAG) pool compartmentalization, and metabolic context, including lipid remodeling and degradation pathways. Engineering success is often constrained by incompatibilities between UFA biosynthetic enzymes and endogenous host metabolism, leading to flux imbalances, futile cycles, and undesired phenotypes. We highlight emerging strategies to overcome these barriers, such as the use of UFA-selective acyltransferases, coordinated manipulation of DAG source pools, suppression of competing endogenous enzymes, and exploitation of TAG remodeling mechanisms. This integrated synthesis provides a conceptual framework for logic-based engineering of oilseeds with enhanced UFA content by offering new avenues for sustainable biomanufacturing of valuable lipids.

acyltransferase specificity↗
DOE OSTI2022

Mechanistic investigation of SARS-CoV-2 main protease to accelerate design of covalent inhibitors

Targeted covalent inhibition represents one possible strategy to block the function of SARS-CoV-2 Main Protease (M PRO ), an enzyme that plays a critical role in the replication of the novel SARS-CoV-2. Toward the design of covalent inhibitors, we built a covalent inhibitor dataset using deep learning models followed by high throughput virtual screening of these candidates against M PRO . Two top-ranking inhibitors were selected for mechanistic investigations—one with an activated ester warhead that has a piperazine core and the other with an acrylamide warhead. Specifically, we performed a detailed analysis of the free energetics of covalent inhibition by hybrid quantum mechanics/molecular mechanics simulations. Cleavage of a fragment of the non-structured protein (NSP) from the SARS-CoV-2 genome was also simulated for reference. Simulations show that both candidates form more stable enzyme-inhibitor (E-I) complexes than the chosen NSP. It was found that both the NSP fragment and the activated ester inhibitor react with CYS145 of M PRO in a concerted manner, whereas the acrylamide inhibitor follows a stepwise mechanism. Most importantly, the reversible reaction and the subsequent hydrolysis reaction from E-I complexes are less probable when compared to the reactions with an NSP fragment, showing promise for these candidates to be the base for efficient M PRO inhibitors.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2023

Light‐Driven Metabolic Pathways in Non‐Photosynthetic Biohybrid Bacteria

Biomanufacturing via microorganisms relies on carbon substrates for molecular feedstocks and a source of energy to carry out enzymatic reactions. This creates metabolic bottlenecks and lowers the efficiency for substrate conversion. Nanoparticle biohybridization with proteins and whole cell surfaces can bypass the need for redox cofactor regeneration for improved secondary metabolite production in a non-specific manner. Here we propose using nanobiohybrid organisms (Nanorgs), intracellular protein-nanoparticle hybrids formed through the spontaneous coupling of core-shell quantum dots (QDs) with histidine-tagged enzymes in non-photosynthetic bacteria, for light-mediated control of bacterial metabolism. Furthermore, this proved to eliminate metabolic constrictions and replace glucose with light as the source of energy in Escherichia coli, with an increase in growth by 1.7-fold in 75 % reduced nutrient media. Metabolomic tracking through carbon isotope labeling confirmed flux shunting through targeted pathways, with accumulation of metabolites downstream of respective targets. Finally, application of Nanorgs with the Ehrlich pathway improved isobutanol titers/yield by 3.9-fold in 75 % less sugar from E. coli strains with no genetic alterations. These results demonstrate the promise of Nanorgs for metabolic engineering and low-cost biomanufacturing.

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

An Integral Activity-Based Protein Profiling Method for Higher Throughput Determination of Protein Target Sensitivity to Small Molecules

Activity-based protein profiling (ABPP) is a chemoproteomic technique that uses small molecule probes to label active enzymes selectively and covalently in complex proteomes. Competitive ABPP, which involves treatment of the active proteome with an analyte of interest, is especially powerful for profiling how small molecules impact specific protein activities. Advances in higher throughput workflows have made it possible to generate extensive competitive ABPP data across diverse biological samples, making this approach highly appealing for characterizing shared and unique proteins affected by perturbations such as drug or chemical exposures. To use the competitive ABPP approach effectively to understand potential adverse effects of chemicals of concern (CoC), a wide range of concentrations may be needed, particularly for chemicals that lack potency or toxicity data. In this work, we present an integral competitive ABPP method that enables target sensitivity determination for different organophosphate (OP) pesticides as model toxicants. Using previously developed OP-ABPs, we optimized conditions for tandem mass tag (TMT) multiplexing of ABPP samples and compared conventional competitive ABPP involving samples at discrete paraoxon concentrations to pooled samples across that same concentration range. We then expanded our approach to compare protein target sensitivities toward two additional OP pesticides, chlorpyrifos oxon and malaoxon. The results showed that differences in integral intensities for the pooled competition sample can be used to evaluate the relative sensitivity of specific proteins without increasing the overall number of samples. For 8 CoC concentrations of interest, this strategy reduced the number of TMT plexes and the corresponding number of LC–MS/MS analyses 3-fold. In conclusion, we envision the integral ABPP (IABPP) method will provide a means to screen diverse chemicals more rapidly to identify both high and low sensitivity protein targets.

activity-based probes↗
DOE OSTI2022

A distinct RNA recognition mechanism governs Np 4 decapping by RppH

Dinucleoside tetraphosphates, often described as alarmones because their cellular concentration increases in response to stress, have recently been shown to function in bacteria as precursors to nucleoside tetraphosphate (Np 4 ) RNA caps. Removal of this cap is critical for initiating 5' end-dependent degradation of those RNAs, potentially affecting bacterial adaptability to stress; however, the predominant Np 4 decapping enzyme in proteobacteria, ApaH, is inactivated by the very conditions of disulfide stress that enable Np 4 -capped RNAs to accumulate to high levels. Here, in this study, we show that, in Escherichia coli cells experiencing such stress, the RNA pyrophosphohydrolase RppH assumes a leading role in decapping those transcripts, preferring them as substrates over their triphosphorylated and diphosphorylated counterparts. Unexpectedly, this enzyme recognizes Np 4 -capped 5' ends by a mechanism distinct from the one it uses to recognize other 5' termini, resulting in a one-nucleotide shift in substrate specificity. The unique manner in which capped substrates of this kind bind to the active site of RppH positions the δ-phosphate, rather than the β-phosphate, for hydrolytic attack, generating triphosphorylated RNA as the primary product of decapping. Consequently, a second RppH-catalyzed deprotection step is required to produce the monophosphorylated 5' terminus needed to stimulate rapid RNA decay. The unconventional manner in which RppH recognizes Np 4 -capped 5' ends and its differential impact on the rates at which such termini are deprotected as a prelude to RNA degradation could have major consequences for reprogramming gene expression during disulfide stress.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2023

Biochemical and structural characterization of a sphingomonad diarylpropane lyase for cofactorless deformylation

Lignin valorization is being intensely pursued via tandem catalytic depolymerization and biological funneling to produce single products. In many lignin depolymerization processes, aromatic dimers and oligomers linked by carbon–carbon bonds remain intact, necessitating the development of enzymes capable of cleaving these compounds to monomers. Recently, the catabolism of erythro -1,2-diguaiacylpropane-1,3-diol ( erythro -DGPD), a ring-opened lignin-derived β-1 dimer, was reported in Novosphingobium aromaticivorans . The first enzyme in this pathway, LdpA (formerly LsdE), is a member of the nuclear transport factor 2 (NTF-2)-like structural superfamily that converts erythro -DGPD to lignostilbene through a heretofore unknown mechanism. In this study, we performed biochemical, structural, and mechanistic characterization of the N. aromaticivorans LdpA and another homolog identified in Sphingobium sp. SYK-6, for which activity was confirmed in vivo. For both enzymes, we first demonstrated that formaldehyde is the C 1 reaction product, and we further demonstrated that both enantiomers of erythro -DGPD were transformed simultaneously, suggesting that LdpA, while diastereomerically specific, lacks enantioselectivity. We also show that LdpA is subject to a severe competitive product inhibition by lignostilbene. Three-dimensional structures of LdpA were determined using X-ray crystallography, including substrate-bound complexes, revealing several residues that were shown to be catalytically essential. We used density functional theory to validate a proposed mechanism that proceeds via dehydroxylation and formation of a quinone methide intermediate that serves as an electron sink for the ensuing deformylation. Overall, this study expands the range of chemistry catalyzed by the NTF-2-like protein family to a prevalent lignin dimer through a cofactorless deformylation reaction.

09 BIOMASS FUELS↗
DOE OSTI2026

Application of Peracetic Acid in Poultry Processing: Effects of Treatment Dynamics and Emerging Risk of Resistance Development in Salmonella spp.

Nontyphoidal Salmonella is a leading cause of foodborne illness, with poultry representing a major source. Peracetic acid (PAA), a widely adopted antimicrobial in poultry processing, offers advantages over traditional disinfectants but has sparked interest in its combined use with other antimicrobials and potential resistance. This review evaluates the efficacy of PAA in mitigating Salmonella in combination with other food-grade antimicrobials, explores possible synergism, efficacy under varying treatment parameters, resistance development against PAA, and its role in resistance evolution. While PAA demonstrates broad-spectrum efficacy, its performance varies with environmental parameters; higher temperatures generally enhance antimicrobial action but also accelerate PAA degradation. Organic matter diminishes PAA efficacy by reactive quenching. Variability in concentration and contact time further influences outcomes. Despite its oxidative mode of action and presumed low risk for resistance, emerging studies indicate that Salmonella can develop adaptive tolerance and potential cross/coresistance following repeated or sublethal exposure to PAA. These adaptations may involve genetic upregulation of oxidative stress response pathways, efflux systems, and modifications in cell membrane integrity, raising concerns about the long-term sustainability of PAA use. Additionally, combinatorial treatments (e.g., PAA with UV-C, enzymes, or organic acids) show promise in enhancing efficacy while mitigating resistance risks. Despite recognition of PAA's safety and effectiveness, knowledge gaps remain regarding standardized resistance definitions, serotype-specific tolerance, and optimal intervention strategies in commercial settings. Therefore, there is a need for standardized testing protocols, robust studies on potential resistance, and further exploration of synergistic PAA applications to ensure sustained poultry product safety and public health protection.

37 - INORGANIC, ORGANIC, PHYSICAL AND ANALYTICAL C↗
NASA NTRS2009

Selective functionalization of carbon nanotube tips allowing fabrication of new classes of nanoscale sensing and manipulation tools

Embodiments in accordance with the present invention relate to techniques for the growth and attachment of single wall carbon nanotubes (SWNT), facilitating their use as robust and well-characterized tools for AFM imaging and other applications. In accordance with one embodiment, SWNTs attached to an AFM tip can function as a structural scaffold for nanoscale device fabrication on a scanning probe. Such a probe can trigger, with nanometer precision, specific biochemical reactions or conformational changes in biological systems. The consequences of such triggering can be observed in real time by single-molecule fluorescence, electrical, and/or AFM sensing. Specific embodiments in accordance with the present invention utilize sensing and manipulation of individual molecules with carbon nanotubes, coupled with single-molecule fluorescence imaging, to allow observation of spectroscopic signals in response to mechanically induced molecular changes. Biological macromolecules such as proteins or DNA can be attached to nanotubes to create highly specific single-molecule probes for investigations of intermolecular dynamics, for assembling hybrid biological and nanoscale materials, or for developing molecular electronics. In one example, electrical wiring of single redox enzymes to carbon nanotube scanning probes allows observation and electrochemical control over single enzymatic reactions by monitoring fluorescence from a redox-active cofactor or the formation of fluorescent products. Enzymes ''nanowired'' to the tips of carbon nanotubes in accordance with embodiments of the present invention, may enable extremely sensitive probing of biological stimulus-response with high spatial resolution, including product-induced signal transduction.

Wade, Lawrence A.↗
DOE OSTI2023

Comparative Genomics and Transcriptomics Analyses Reveal Divergent Plant Biomass-Degrading Strategies in Fungi

Plant biomass is one of the most abundant renewable carbon sources, which holds great potential for replacing current fossil-based production of fuels and chemicals. In nature, fungi can efficiently degrade plant polysaccharides by secreting a broad range of carbohydrate-active enzymes (CAZymes), such as cellulases, hemicellulases, and pectinases. Due to the crucial role of plant biomass-degrading (PBD) CAZymes in fungal growth and related biotechnology applications, investigation of their genomic diversity and transcriptional dynamics has attracted increasing attention. In this project, we systematically compared the genome content of PBD CAZymes in six taxonomically distant species, Aspergillus niger, Aspergillus nidulans, Penicillium subrubescens, Trichoderma reesei, Phanerochaete chrysosporium, and Dichomitus squalens, as well as their transcriptome profiles during growth on nine monosaccharides. Considerable genomic variation and remarkable transcriptomic diversity of CAZymes were identified, implying the preferred carbon source of these fungi and their different methods of transcription regulation. In addition, the specific carbon utilization ability inferred from genomics and transcriptomics was compared with fungal growth profiles on corresponding sugars, to improve our understanding of the conversion process. This study enhances our understanding of genomic and transcriptomic diversity of fungal plant polysaccharide-degrading enzymes and provides new insights into designing enzyme mixtures and metabolic engineering of fungi for related industrial applications.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2020

Identification of a Novel Cobamide Remodeling Enzyme in the Beneficial Human Gut Bacterium Akkermansia muciniphila

The beneficial human gut bacterium Akkermansia muciniphila provides metabolites to other members of the gut microbiota by breaking down host mucin, but most of its other metabolic functions have not been investigated. A. muciniphila strain Muc T is known to use cobamides, the vitamin B 12 family of cofactors with structural diversity in the lower ligand. However, A. muciniphila Muc T is unable to synthesize cobamides de novo, and the specific forms that can be used by A. muciniphila have not been examined. We found that the levels of growth of A. muciniphila Muc T were nearly identical with each of seven cobamides tested, in contrast to nearly all bacteria that had been studied previously. Unexpectedly, this promiscuity is due to cobamide remodeling—the removal and replacement of the lower ligand—despite the absence of the canonical remodeling enzyme CbiZ in A. muciniphila. We identified a novel enzyme, CbiR, that is capable of initiating the remodeling process by hydrolyzing the phosphoribosyl bond in the nucleotide loop of cobamides. CbiR does not share similarity with other cobamide remodeling enzymes or B12-binding domains and is instead a member of the apurinic/apyrimidinic (AP) endonuclease 2 enzyme superfamily. We speculate that CbiR enables bacteria to repurpose cobamides that they cannot otherwise use in order to grow under cobamide-requiring conditions; this function was confirmed by heterologous expression of cbiR in Escherichia coli. Homologs of CbiR are found in over 200 microbial taxa across 22 phyla, suggesting that many bacteria may use CbiR to gain access to the diverse cobamides present in their environment.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2020

Novel Mode of Molybdate Inhibition of Desulfovibrio vulgaris Hildenborough

Sulfate-reducing microorganisms (SRM) are found in multiple environments and play a major role in global carbon and sulfur cycling. Because of their growth capabilities and association with metal corrosion, controlling the growth of SRM has become of increased interest. One such mechanism of control has been the use of molybdate (MoO 4 2− ), which is thought to be a specific inhibitor of SRM. The way in which molybdate inhibits the growth of SRM has been enigmatic. It has been reported that molybdate is involved in a futile energy cycle with the sulfate-activating enzyme, sulfate adenylyl transferase (Sat), which results in loss of cellular ATP. However, we show here that a deletion of this enzyme in the model SRM, Desulfovibrio vulgaris Hildenborough, remained sensitive to molybdate. We performed several subcultures of the ∆ sat strain in the presence of increasing concentrations of molybdate and obtained a culture with increased resistance to the inhibitor (up to 3 mM). The culture was re-sequenced and three single nucleotide polymorphisms (SNPs) were identified that were not present in the parental strain. Two of the SNPs seemed unlikely candidates for molybdate resistance due to a lack of conservation of the mutated residues in homologous genes of closely related strains. The remaining SNP was located in DVU2210, a protein containing two domains: a YcaO-like domain and a tetratricopeptide-repeat domain. The SNP resulted in a change of a serine residue to arginine in the ATP-hydrolyzing motif of the YcaO-like domain. Deletion mutants of each of the three genes apparently enriched with SNPs in the presence of inhibitory molybdate and combinations of these genes were generated in the Δ sat and wild-type strains. Strains lacking both sat and DVU2210 became more resistant to molybdate. Deletions of the other two genes in which SNPs were observed did not result in increased resistance to molybdate. YcaO-like proteins are distributed across the bacterial and archaeal domains, though the function of these proteins is largely unknown. The role of this protein in D. vulgaris is unknown. Due to the distribution of YcaO-like proteins in prokaryotes, the veracity of molybdate as a specific SRM inhibitor should be reconsidered.

59 BASIC BIOLOGICAL SCIENCES↗