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At least 73 records · Page 4

The nitrosoamphetamine metabolite is accommodated in the active site of human hemoglobin: Spectroscopy and crystal structure

Amphetamine-based (Amph) drugs are metabolized in humans to their hydroxylamine (AmphNHOH) and nitroso (AmphNO) derivatives. The latter metabolites are known to bind to the Fe centers of cytochrome P450 and other heme enzymes to inhibit their activities. Although these AmphNHOH/AmphNO metabolites are present in vivo, their interactions with the blood protein hemoglobin (Hb) and the muscle protein (Mb) have been largely discounted due to a perception that the relatively small heme active sites of Hb and Mb will not be able to accommodate the large AmphNO group. Here, we report the 2.15 Å resolution X-ray crystal structure of the AmphNO adduct of adult human hemoglobin as the Hb [ α -Fe III (H 2 O)][ β -Fe II (AmphNO)] derivative. We show that the binding of AmphNO to the β subunit is enabled by an E helix movement and stabilization of ligand binding by H-bonding with the distal His63 residue. We also observe an AmphNHOH group in the Xe2 pocket in close proximity to the α heme site in this derivative. Additionally, UV–vis spectroscopy was used to characterize this and related wt and mutant Mb adducts. Importantly, our X-ray crystal structure of this Hb-nitrosoamphetamine complex represents the first crystal structure of a wild-type heme protein adduct of any amphetamine metabolite. Our results provide a framework for further studies of AmphNHOH/AmphNO interactions with Hb and Mb as viable processes that potentially contribute to the overall biological inorganic chemistry of amphetamine drugs.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗

Electrochemical Detection of Oxycodone and Its Main Metabolites with Nafion-Coated Single-Walled Carbon Nanotube Electrodes

Oxycodone is a strong opioid frequently used as an analgesic. Although proven efficacious in the management of moderate to severe acute pain and cancer pain, use of oxycodone imposes a risk of adverse effects such as addiction, overdose, and death. Fast and accurate determination of oxycodone blood concentration would enable personalized dosing and monitoring of the analgesic as well as quick diagnostics of possible overdose in emergency care. However, in addition to the parent drug, several metabolites are always present in the blood after a dose of oxycodone, and to date, there is no electrochemical data available on any of these metabolites. In this paper, a single-walled carbon nanotube (SWCNT) electrode and a Nafion-coated SWCNT electrode were used, for the first time, to study the electrochemical behavior of oxycodone and its two main metabolites, noroxycodone and oxymorphone. Both electrode types could selectively detect oxycodone in the presence of noroxycodone and oxymorphone. However, we have previously shown that addition of a Nafion coating on top of the SWCNT electrode is essential for direct measurements in complex biological matrices. Therefore, in this work, the Nafion/SWCNT electrode was further characterized and used for measuring clinically relevant concentrations of oxycodone in buffer solution. The limit of detection for oxycodone with the Nafion/SWCNT sensor was 85 nM, and the linear range was 0.5–10 μM in buffer solution. Furthermore, this study shows that the fabricated Nafion/SWCNT sensor has potential to be applied in clinical concentration measurements.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗

PeakDecoder enables machine learning-based metabolite annotation and accurate profiling in multidimensional mass spectrometry measurements

Multidimensional measurements using state-of-the-art separations and mass spectrometry provide advantages in untargeted metabolomics analyses for studying biological and environmental bio-chemical processes. However, the lack of rapid analytical methods and robust algorithms for these heterogeneous data has limited its application. Here, we develop and evaluate a sensitive and high-throughput analytical and computational workflow to enable accurate metabolite profiling. Our workflow combines liquid chromatography, ion mobility spectrometry and data-independent acquisition mass spectrometry with PeakDecoder, a machine learning-based algorithm that learns to distinguish true co-elution and co-mobility from raw data and calculates metabolite identification error rates. We apply PeakDecoder for metabolite profiling of various engineered strains of Aspergillus pseudoterreus, Aspergillus niger, Pseudomonas putida and Rhodosporidium toruloides. Results, validated manually and against selected reaction monitoring and gas-chromatography platforms, show that 2683 features could be confidently annotated and quantified across 116 microbial sample runs using a library built from 64 standards.

59 BASIC BIOLOGICAL SCIENCES↗

Metabolite releasing polymers control dendritic cell function by modulating their energy metabolism

Metabolites control immune cell functions, and delivery of these metabolites in a sustained manner may be able to modulate function of the immune cells. In this study, alpha-ketoglutarate (aKG) and diol based polymeric-microparticles (termed paKG MPs) were synthesized to provide sustained release of aKG and promote an immunosuppressive cellular phenotype. Notably, after association with dendritic cells (DCs), paKG MPs modulated the intracellular metabolic-profile/pathways, and decreased glycolysis and mitochondrial respiration in vitro. These metabolic changes resulted in modulation of MHC-II, CD86 expression in DCs, and altered the frequency of regulatory T cells (Tregs), and T-helper type-1/2/17 cells in vitro. In conclusion, this unique strategy of intracellular delivery of key-metabolites in a sustained manner provides a new direction in immunometabolism field-based immunotherapy with potential applications in different diseases associated with immune disorders.

36 MATERIALS SCIENCE↗

Atomic view of photosynthetic metabolite permeability pathways and confinement in synthetic carboxysome shells

Carboxysomes are protein microcompartments found in cyanobacteria, whose shell encapsulates rubisco at the heart of carbon fixation in the Calvin cycle. Carboxysomes are thought to locally concentrate CO 2 in the shell interior to improve rubisco efficiency through selective metabolite permeability, creating a concentrated catalytic center. However, permeability coefficients have not previously been determined for these gases, or for Calvin-cycle intermediates such as bicarbonate (HCO$^–_3$), 3-phosphoglycerate, or ribulose-1,5-bisphosphate. Starting from a high-resolution cryogenic electron microscopy structure of a synthetic β-carboxysome shell, we perform unbiased all-atom molecular dynamics to track metabolite permeability across the shell. The synthetic carboxysome shell structure, lacking the bacterial microcompartment trimer proteins and encapsulation peptides, is found to have similar permeability coefficients for multiple metabolites, and is not selectively permeable to HCO$^–_3$ relative to CO 2 . To resolve how these comparable permeabilities can be reconciled with the clear role of the carboxysome in the CO 2 -concentrating mechanism in cyanobacteria, complementary atomic-resolution Brownian Dynamics simulations estimate the mean first passage time for CO 2 assimilation in a crowded model carboxysome. Despite a relatively high CO 2 permeability of approximately 10 -2 cm/s across the carboxysome shell, the shell proteins reflect enough CO 2 back toward rubisco that 2,650 CO 2 molecules can be fixed by rubisco for every 1 CO 2 molecule that escapes under typical conditions. The permeabilities determined from all-atom molecular simulation are key inputs into flux modeling, and the insight gained into carbon fixation can facilitate the engineering of carboxysomes and other bacterial microcompartments for multiple applications.

59 BASIC BIOLOGICAL SCIENCES↗

Mammalian Nudix proteins cleave nucleotide metabolite caps on RNAs

We recently reported the presence of nicotinamide adenine dinucleotide (NAD)-capped RNAs in mammalian cells and a role for DXO and the Nudix hydrolase Nudt12 in decapping NAD-capped RNAs (deNADding) in cells. Analysis of 5'caps has revealed that in addition to NAD, mammalian RNAs also contain other metabolite caps including flavin adenine dinucleotide (FAD) and dephosphoCoA (dpCoA). In the present study we systematically screened all mammalian Nudix proteins for their potential deNADing, FAD cap decapping (deFADding) and dpCoA cap decapping (deCoAping) activity. We demonstrate that Nudt16 is a novel deNADding enzyme in mammalian cells. Additionally, we identified seven Nudix proteins—Nudt2, Nudt7, Nudt8, Nudt12, Nudt15, Nudt16 and Nudt19, to possess deCoAping activity in vitro. Moreover, our screening revealed that both mammalian Nudt2 and Nudt16 hydrolyze FAD-capped RNAs in vitro with Nudt16 regulating levels of FAD-capped RNAs in cells. All decapping activities identified hydrolyze the metabolite cap substrate within the diphosphate linkage. Crystal structure of human Nudt16 in complex with FAD at 2.7 Å resolution provide molecular insights into the binding and metal-coordinated hydrolysis of FAD by Nudt16. In summary, our study identifies novel cellular deNADding and deFADding enzymes and establishes a foundation for the selective functionality of the Nudix decapping enzymes on non-canonical metabolite caps.

59 BASIC BIOLOGICAL SCIENCES↗

Drought Shifts Sorghum Root Metabolite and Microbiome Profiles and Enriches for Pipecolic Acid

Plant-associated microbial communities shift in composition as a result of environmental perturbations, such as drought. It has been shown that Actinobacteria are enriched in plant roots and rhizospheres during drought stress. However, the correlations between microbiome dynamics and plant response to drought are poorly understood. Here we apply a combination of bacterial community composition analysis and plant metabolite profiling in Sorghum bicolor roots, rhizospheres, and soil during drought and drought recovery to investigate potential contributions of host metabolism to shifts in bacterial composition. Our results provide a detailed view of metabolic shifts across the plant root during drought and show that the response to rewatering differs between root and soil; additionally, we identify drought-responsive metabolites that are highly correlated with the observed changes in Actinobacteria abundance. Furthermore, we find that pipecolic acid is a drought-enriched metabolite in sorghum roots, and that exogenous application of pipecolic acid inhibits root growth. Finally, we show that this activity functions independent of the systemic acquired resistance pathway and has the potential to impact Actinobacterial taxa within the root microbiome.

165 RRNA↗

Switchgrass Rhizosphere Metabolite Chemistry Driven by Nitrogen Availability

Plants and soil microorganisms interact closely in the rhizosphere where plants may exchange carbon (C) for functional benefits from the microbial community. For example, the bioenergy crop, switchgrass (Panicum virgatum) is thought to exchange root-exuded C for nitrogen (N) fixed by diazotrophs (free-living N-fixers). However, this interaction is not well characterized and it is not known how or if switchgrass responds to diazotrophs or their activity. To explore this question, we assessed rhizosphere metabolite chemistry of switchgrass grown in a hydroponic system under two N levels and under inoculated or uninoculated conditions. Plants were grown with the inoculum Azotobacter vinelandii DJ for 3 days before harvest. We found switchgrass root exudate chemistry to be driven by N availability. Total metabolite concentrations were generally greater under high N versus low N and unaffected by inoculation. Examination of rhizosphere chemical fingerprints indicates metabolite chemistry was also driven strongly by N availability with a greater relative abundance of carbohydrates under high N and greater relative abundance of organic acids under low N. We also found evidence of changes in rhizosphere chemical fingerprints by inoculation treatment. However, we found little evidence of N treatment and inoculation interaction effects that suggests this response is not directly mediated by N availability.

59 BASIC BIOLOGICAL SCIENCES↗

Siderophores and secondary metabolites produced by Ganoderma adspersum

Ganoderma adspersum is a white-rot wood-degrading basidiomycete of ecological, biotechnological and medicinal interest. In addition to its role in lignin degradation, it produces bioactive metabolites with reported antimicrobial and antioxidant activities. However, the mechanisms of iron acquisition, including siderophore-mediated pathways, remain poorly characterized in Ganoderma species. Improved understanding of these systems is essential to elucidate their contributions to fungal physiology, secondary metabolism and ecological adaptation. In this study, the genome of G. adspersum was sequenced for the first time and screened for genes that may be involved in the production of secondary metabolites. A gene cluster was identified as potentially involved in iron uptake. In particular, genes related to non-ribosomal peptide synthetases were detected next to a gene encoding a monooxygenase and indicated a potential hydroxamate-family siderophore. Liquid chromatography (LC)-ES-MS analysis of secondary metabolites secreted by G. adspersum into the growth medium under iron-limiting conditions revealed a group of previously undescribed siderophores. Genome and MS/MS analysis suggested that these structures might be related to the coprinoferrin family of siderophores. Aside from siderophores, the genome and LC-MS analysis revealed G. adspersum to be a prolific producer of a variety of triterpenoids and sesquiterpenoids, in agreement with previous findings. This is the first description of the genome sequence of G. adspersum and its siderophores.

Reyes, Carolina [Laboratory for Cellulose and Wood↗

Comparative transcriptomics and metabolomics reveal specialized metabolite drought stress responses in switchgrass ( Panicum virgatum )

Summary Switchgrass ( Panicum virgatum ) is a bioenergy model crop valued for its energy efficiency and drought tolerance. The related monocot species rice ( Oryza sativa ) and maize ( Zea mays ) deploy species‐specific, specialized metabolites as core stress defenses. By contrast, specialized chemical defenses in switchgrass are largely unknown. To investigate specialized metabolic drought responses in switchgrass, we integrated tissue‐specific transcriptome and metabolite analyses of the genotypes Alamo and Cave‐in‐Rock that feature different drought tolerance. The more drought‐susceptible Cave‐in‐Rock featured an earlier onset of transcriptomic changes and significantly more differentially expressed genes in response to drought compared to Alamo. Specialized pathways showed moderate differential expression compared to pronounced transcriptomic alterations in carbohydrate and amino acid metabolism. However, diterpenoid‐biosynthetic genes showed drought‐inducible expression in Alamo roots, contrasting largely unaltered triterpenoid and phenylpropanoid pathways. Metabolomic analyses identified common and genotype‐specific flavonoids and terpenoids. Consistent with transcriptomic alterations, several root diterpenoids showed significant drought‐induced accumulation, whereas triterpenoid abundance remained predominantly unchanged. Structural analysis verified select drought‐responsive diterpenoids as oxygenated furanoditerpenoids. Drought‐dependent transcriptome and metabolite profiles provide the foundation to understand the molecular mechanisms underlying switchgrass drought responses. Accumulation of specialized root diterpenoids and corresponding pathway transcripts supports a role in drought stress tolerance.

transcriptomics↗

Quorum sensing modulates microbial community structure through regulation of secondary metabolites

Bacteria are recognized for their diverse metabolic capabilities, yet the impact of microbe-microbe interactions on multispecies community structure and dynamics is poorly understood. Cell-to-cell signaling in the form of quorum sensing (QS) often regulates secondary metabolite production and microbial interactions. Here, we examine how acylhomoserine lactone (AHL)-mediated QS impacts microbial community structure in a 10-member synthetic community of isolates from Populus deltoides . To explore the role of QS in microbial community structure and dynamics, we disrupted AHL signaling by exogenous addition of AiiA-lactonase, an enzyme that cleaves the lactone ring. Microbial community structure resulting from signal inactivation, as measured by 16S rRNA amplicon sequencing and secondary metabolite production, was assessed after successive passaging of the community. Further, we investigated the impact of quorum quenching on specific microbe-microbe interactions using pairwise inhibition assays. Our results indicate that AHL inactivation alters the relative abundance of dominant community members at later passages but does not impact the overall membership in the community. Quorum quenching significantly alters the metabolic profile in lactonase-treated communities. This metabolic alteration impacts microbe-microbe interactions through decreased inhibition of other community members. Together, these results indicate that QS impacts microbial community structure through the regulation of secondary metabolites in dominant members and that the membership of microbial communities can be relatively stable despite changes in metabolic profiles.

59 BASIC BIOLOGICAL SCIENCES↗

Metabolic Feedback Inhibition Influences Metabolite Secretion by the Human Gut Symbiont Bacteroides thetaiotaomicron

ABSTRACT Microbial metabolism and trophic interactions between microbes give rise to complex multispecies communities in microbe-host systems. Bacteroides thetaiotaomicron ( B. theta ) is a human gut symbiont thought to play an important role in maintaining host health. Untargeted nuclear magnetic resonance metabolomics revealed B. theta secretes specific organic acids and amino acids in defined minimal medium. Physiological concentrations of acetate and formate found in the human intestinal tract were shown to cause dose-dependent changes in secretion of metabolites known to play roles in host nutrition and pathogenesis. While secretion fluxes varied, biomass yield was unchanged, suggesting feedback inhibition does not affect metabolic bioenergetics but instead redirects carbon and energy to CO 2 and H 2 . Flux balance analysis modeling showed increased flux through CO 2 -producing reactions under glucose-limiting growth conditions. The metabolic dynamics observed for B. theta , a keystone symbiont organism, underscores the need for metabolic modeling to complement genomic predictions of microbial metabolism to infer mechanisms of microbe-microbe and microbe-host interactions. IMPORTANCE Bacteroides is a highly abundant taxon in the human gut, and Bacteroides thetaiotaomicron ( B. theta ) is a ubiquitous human symbiont that colonizes the host early in development and persists throughout its life span. The phenotypic plasticity of keystone organisms such as B. theta is important to understand in order to predict phenotype(s) and metabolic interactions under changing nutrient conditions such as those that occur in complex gut communities. Our study shows B. theta prioritizes energy conservation and suppresses secretion of “overflow metabolites” such as organic acids and amino acids when concentrations of acetate are high. Secreted metabolites, especially amino acids, can be a source of nutrients or signals for the host or other microbes in the community. Our study suggests that when metabolically stressed by acetate, B. theta stops sharing with its ecological partners.

59 BASIC BIOLOGICAL SCIENCES↗

Untargeted metabolite data from a root surface in a rhizobox

Raw data is provided from samples analyzed using separate reverse phase chromatographic methods on a high performance liquid chromatograph with mass spectrometry. These porewater samples were collected from a microdialysis which generated samples along the surface of a growing A. sative root (all_hc). This data was used to answer questions connecting rhizosphere metabolite (putatively identified metabolites, hc_putative_norm) changes over time (root growth) with changes in the surrounding rhizosphere biogeochemistry (DOC, redox, pH). Rhizosphere biogeochemistry values are provided in the hc_putative_norm file as averages over their respective range of time that they were collected at. The hc_putative_norm file also contains all normalized values over only the intensity values collected for putatively identified metabolites.

54 ENVIRONMENTAL SCIENCES↗

Uses of Multi-Objective Flux Analysis for Optimization of Microbial Production of Secondary Metabolites

Secondary metabolites are not essential for the growth of microorganisms, but they play a critical role in how microbes interact with their surroundings. In addition to this important ecological role, secondary metabolites also have a variety of agricultural, medicinal, and industrial uses, and thus the examination of secondary metabolism of plants and microbes is a growing scientific field. While the chemical production of certain secondary metabolites is possible, industrial-scale microbial production is a green and economically attractive alternative. This is even more true, given the advances in bioengineering that allow us to alter the workings of microbes in order to increase their production of compounds of interest. This type of engineering requires detailed knowledge of the “chassis” organism’s metabolism. Since the resources and the catalytic capacity of enzymes in microbes is finite, it is important to examine the tradeoffs between various bioprocesses in an engineered system and alter its working in a manner that minimally perturbs the robustness of the system while allowing for the maximum production of a product of interest. The in silico multi-objective analysis of metabolism using genome-scale models is an ideal method for such examinations.

59 BASIC BIOLOGICAL SCIENCES↗

Data from: Switchgrass rhizosphere metabolite chemistry driven by nitrogen availability

Plants and soil microorganisms interact closely in the rhizosphere where plants may exchange carbon (C) for functional benefits from the microbial community. For example, the bioenergy crop, switchgrass (Panicum virgatum) is thought to exchange root-exuded C for nitrogen (N) fixed by diazotrophs (free-living N-fixers). However, this interaction is not well characterized and it is not known how or if switchgrass responds to diazotrophs or their activity. To explore this question, we assessed rhizosphere metabolite chemistry of switchgrass grown in a hydroponic system under two N levels and under inoculated or uninoculated conditions. Plants were grown with the inoculum Azotobacter vinelandii DJ for three days before harvest. We found switchgrass root exudate chemistry to be driven by N availability. Total metabolite concentrations were generally greater under high N versus low N and unaffected by inoculation. Examination of rhizosphere chemical fingerprints indicates metabolite chemistry was also driven strongly by N availability with a greater relative abundance of carbohydrates under high N and greater relative abundance of organic acids under low N. We also found evidence of changes in rhizosphere chemical fingerprints by inoculation treatment. However, we found little evidence of N treatment and inoculation interaction effects which suggests this response is not directly mediated by N availability.

09 BIOMASS FUELS↗

Assessing the Impact of Measurement Precision on Metabolite Identification Probability in Multidimensional Mass Spectrometry-Based, Reference-Free Metabolomics

Identification of compounds with minimal ambiguity remains a central challenge in mass spectrometry-based metabolomics. Conventional compound identification relies on comparing analytical signatures (e.g., mass-to-charge ratio, collision cross section, tandem mass spectra) against reference data obtained from measurements of authentic chemical standards. The breadth of annotatable compounds using this approach is necessarily limited by availability of authentic standards, analytical throughput, and resolving power of the separations that underly the measurements. The maturation of computational methods, both theory-driven and artificial intelligence/machine learning-based, for prediction of various molecular properties relevant to multidimensional mass spectrometry measurements has opened the door to a new “reference-free” paradigm of compound annotation. Through augmenting existing reference data for molecular properties with computational predictions, the universe of identifiable chemical species can be expanded significantly beyond its current limits. An unexplored aspect of this novel approach is understanding how to gauge confidence in resulting annotations, especially as the compound search space is expanded. Intuitively, the confidence of a compound annotation is related to the inherent discriminatory power of the molecular properties used for identification, as well as the precision with which the properties are measured or predicted. In this work, we characterize this relationship between measurement precision and identification probability in a systematic and quantitative fashion for a defined region of chemical space that includes organic small molecule metabolites. Importantly, this work establishes a framework for conducting metabolite identification probability analysis that enables others to quantify this relationship for their own compounds and properties of interest.

Metabolite Identification↗

Metabolite release by nitrifiers facilitates metabolic interactions in the ocean

Microbial chemoautotroph-heterotroph interactions may play a pivotal role in the cycling of carbon in the deep ocean, reminiscent of phytoplankton-heterotroph associations in surface waters. Nitrifiers are the most abundant chemoautotrophs in the global ocean, yet very little is known about nitrifier metabolite production, release, and transfer to heterotrophic microbial communities. To elucidate which organic compounds are released by nitrifiers and potentially available to heterotrophs, we characterized the exo- and endometabolomes of the ammonia-oxidizing archaeon Nitrosopumilus adriaticus CCS1 and the nitrite-oxidizing bacterium Nitrospina gracilis Nb-211. Nitrifier endometabolome composition was not a good predictor of exometabolite availability, indicating that metabolites were predominately released by mechanisms other than cell death/lysis. Although both nitrifiers released labile organic compounds, N. adriaticus preferentially released amino acids, particularly glycine, suggesting that its cell membranes might be more permeable to small, hydrophobic amino acids. We further initiated co-culture systems between each nitrifier and a heterotrophic alphaproteobacterium, and compared exometabolite and transcript patterns of nitrifiers grown axenically to those in co-culture. In particular, B vitamins exhibited dynamic production and consumption patterns in nitrifier-heterotroph co-cultures. We observed an increased production of vitamin B 2 and the vitamin B 12 lower ligand dimethylbenzimidazole by N. adriaticus and N. gracilis, respectively. In contrast, the heterotroph likely produced vitamin B 5 in co-culture with both nitrifiers and consumed the vitamin B 7 precursor dethiobiotin when grown with N. gracilis. Our results indicate that B vitamins and their precursors could play a particularly important role in governing specific metabolic interactions between nitrifiers and heterotrophic microbes in the ocean.

54 ENVIRONMENTAL SCIENCES↗

Metabolite Damage and Damage Control in a Minimal Genome

Analysis of the genes retained in the minimized Mycoplasma JCVI-Syn3A genome established that systems that repair or preempt metabolite damage are essential to life. Several genes known to have such functions were identified and experimentally validated, including 5-formyltetrahydrofolate cycloligase, coenzyme A (CoA) disulfide reductase, and certain hydrolases. Furthermore, we discovered that an enigmatic YqeK hydrolase domain fused to NadD has a novel proofreading function in NAD synthesis and could double as a MutT-like sanitizing enzyme for the nucleotide pool. Finally, we combined metabolomics and cheminformatics approaches to extend the core metabolic map of JCVI-Syn3A to include promiscuous enzymatic reactions and spontaneous side reactions. This extension revealed that several key metabolite damage control systems remain to be identified in JCVI-Syn3A, such as that for methylglyoxal.

59 BASIC BIOLOGICAL SCIENCES↗