Structure of an AMPK complex in an inactive, ATP-bound state
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Yan, Yan↗
SEARCH · Engineering Papers
Results for “ATPS”
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A novel chloroplast super-complex consisting of the ATP synthase and photosystem I reaction center
Not Available
59 BASIC BIOLOGICAL SCIENCES↗
Rapid Acceleration of Diagnostics: RADx (Tech + ATP)
This virtual symposium was held to highlight the impact the U.S. Department of Energy’s (DOE) National Virtual Biotechnology Laboratory (NVBL) has had utilizing the unique capabilities of the DOE to tackle the science and technology challenges associated with COVID-19, and to discuss areas in which the NVBL can have impact in the future. With perspectives from Chris Fall, Director, Office of Science, DOE, and William A. Bookless, Principal Deputy Administrator of the National Nuclear Security Administration, the event featured Presentations from lead investigators reporting progress in: Epidemiological modeling, Therapeutics, Testing, Understanding transport of the virus, and Solving issues around supply chain challenges. In addition, keynote speakers from outside the NVBL discussed the upcoming science and technology needs in computing, testing and surveillance, and vaccines. The event was aimed at the S&T community, media, and general public.
Tromberg, Bruce J.↗
Optimization of ATP Synthase c–Rings for Oxygenic Photosynthesis
Not Available
59 BASIC BIOLOGICAL SCIENCES↗
Re‐evaluating the energy balance of the many routes of carbon flow through and from photorespiration
Photorespiration is an essential process related to photosynthesis that is initiated following the oxygenation reaction catalyzed by rubisco, the initial enzyme of the Calvin–Benson–Bassham cycle. This reaction produces an inhibitory intermediate that is recycled back into the Calvin–Benson–Bassham cycle by photorespiration which requires the use of energy and the release of a portion of the carbon as CO 2 . The energy use and CO 2 release of canonical photorespiration form a foundation for biochemical models used to describe and predict leaf carbon exchange and energy use (ATP and NAPDH). The ATP and NADPH demand of canonical photorespiration is thought to be different than that of the Calvin–Benson–Bassham cycle, requiring increased flexibility in the ratio of ATP and NADPH from the light reactions. Photorespiration requires many reactions across the chloroplasts, mitochondria and peroxisomes and involves many intermediates. Growing evidence indicates that these intermediates do not all stay in photorespiration as typically assumed and instead feed into other aspects of metabolism and leave as glycine, serine, and methylene‐THF. Here we discuss how alternative flux through and from canonical photorespiration alters the ATP and NADPH requirements of metabolism following rubisco oxygenation using additional derivations of biochemical models of leaf photosynthesis and energetics. Using these new derivations, we determine that the ATP and NADPH demands of photorespiration are highly sensitive to alternative flux in ways that fundamentally changes how photorespiration contributes to the ratio of total ATP and NADPH demand. Specifically, alternative flows of carbon through photorespiration could reduce ATP and NADPH demand ratio to values below what is produced from linear electron transport.
59 BASIC BIOLOGICAL SCIENCES↗
Visualizing the enzyme mechanism of mevalonate diphosphate decarboxylase
Mevalonate diphosphate decarboxylases (MDDs) catalyze the ATP-dependent-Mg 2+ -decarboxylation of mevalonate-5-diphosphate (MVAPP) to produce isopentenyl diphosphate (IPP), which is essential in both eukaryotes and prokaryotes for polyisoprenoid synthesis. The substrates, MVAPP and ATP, have been shown to bind sequentially to MDD. Here we report crystals in which the enzyme remains active, allowing the visualization of conformational changes in Enterococcus faecalis MDD that describe sequential steps in an induced fit enzymatic reaction. Initial binding of MVAPP modulates the ATP binding pocket with a large loop movement. Upon ATP binding, a phosphate binding loop bends over the active site to recognize ATP and bring the molecules to their catalytically favored configuration. Positioned substrates then can chelate two Mg 2+ ions for the two steps of the reaction. Closure of the active site entrance brings a conserved lysine to trigger dissociative phosphoryl transfer of γ-phosphate from ATP to MVAPP, followed by the production of IPP.
59 BASIC BIOLOGICAL SCIENCES↗
Kinetic and substrate complex characterization of RamA, a corrinoid protein reductive activase from Methanosarcina barkeri
ABSTRACT In microbial corrinoid-dependent methyltransferase systems, adventitious Co(I)-corrinoid oxidation halts catalysis and necessitates repair by ATP-dependent reductive activases. RamA, an activase with a C-terminal ferredoxin domain with two [4Fe-4S] clusters from methanogenic archaea, has been far less studied than the bacterial activases bearing an N-terminal ferredoxin domain with one [2Fe-2S] cluster. These differences suggest RamA might prove to have other distinctive characteristics. Here, we examine RamA kinetics and the stoichiometry of the corrinoid protein:RamA complex. Like bacterial activases, K+ stimulates RamA. Potassium stimulation had been questioned due to differences in the primary structure of bacterial and methanogen activases. Unlike one bacterial activase, ATP is not inhibitory allowing the first determination of apparent kinetic parameters for any corrinoid activase. Unlike bacterial activases, a single RamA monomer complexes a single corrinoid protein monomer. Alanine replacement of a RamA serine residue corresponding to the serine of one bacterial activase which ligates the corrinoid cobalt during complex formation led to only moderate changes in the kinetics of RamA. These results reveal new differences in the two types of corrinoid activases, and provide direct evidence for the proposal that corrinoid activases act as catalytic monomers, unlike other enzymes that couple ATP hydrolysis to difficult reductions.
Microbiology↗
Caveat mutator: alanine substitutions for conserved amino acids in RNA ligase elicit unexpected rearrangements of the active site for lysine adenylylation
Naegleria gruberi RNA ligase (NgrRnl) exemplifies the Rnl5 family of adenosine triphosphate (ATP)-dependent polynucleotide ligases that seal 3'-OH RNA strands in the context of 3'-OH/5'-PO 4 nicked duplexes. Like all classic ligases, NgrRnl forms a covalent lysyl–AMP intermediate. A two-metal mechanism of lysine adenylylation was established via a crystal structure of the NgrRnl•ATP•(Mn 2+ ) 2 Michaelis complex. Here we conducted an alanine scan of active site constituents that engage the ATP phosphates and the metal cofactors. We then determined crystal structures of ligase-defective NgrRnl-Ala mutants in complexes with ATP/Mn 2+ . The unexpected findings were that mutations K170A, E227A, K326A and R149A (none of which impacted overall enzyme structure) triggered adverse secondary changes in the active site entailing dislocations of the ATP phosphates, altered contacts to ATP, and variations in the numbers and positions of the metal ions that perverted the active sites into off-pathway states incompatible with lysine adenylylation. Each alanine mutation elicited a distinctive off-pathway distortion of the ligase active site. Our results illuminate a surprising plasticity of the ligase active site in its interactions with ATP and metals. More broadly, they underscore a valuable caveat when interpreting mutational data in the course of enzyme structure-function studies.
59 BASIC BIOLOGICAL SCIENCES↗
Data for "Quantifying the Propagation of Parametric Uncertainty on Flux Balance Analysis"
In the repository are example scripts that perform uncertainty injection and propagation to flux balance analysis with outputs for a small sample size (for demonstration purpose only). For proper analysis, user should download the scripts and run for a large sample size (e.g., 10,000 samples). If you use the scripts, please cite the following Metabolic Engineering article: “Quantifying the propagation of parametric uncertainty on flux balance analysis” (https://doi.org/10.1016/j.ymben.2021.10.012) There are two subdirectories: /uncFBA/uncBiom: injection of normally distributed noise to biomass precursor coeffcients and ATP maintenance (growth-associated ATP maintenance (GAM) and non-growth associated ATP maintenance (NGAM)) /uncFBA/uncRHS: departure from steady-state by adding noise drawn from normal distribution to the RHS terms of mass balance constraints
Metabolomics↗
Above-threshold multiphoton photoemission from noble metal surfaces
Exciting solids with intense femtosecond laser pulses prompts electrons of the interrogated material to respond in a highly nonlinear manner, as is evident in the emission of high-order harmonic radiation and photoelectrons with kinetic energies well above that of the driving photons. Such high-field interactions can be resolved, for example, in above-threshold multiphoton photoemission (ATP) spectroscopy. In this work, we interrogate the nonlinear photoelectric responses of the pristine copper, silver, and gold noble metal surfaces in (111) and (100) crystal orientations in the perturbative regime. Using multiphoton photoemission spectroscopy (mPP) excited by finely tuned optical fields, we characterize enhancement of the mPP and ATP yields from (111) surfaces in selected k||-momentum ranges when the occupied Shockley surface (SS) states are (near-)resonantly coupled by multiphoton transitions to image potential (IP) intermediate states in the excitation process. The ATP signal from the IP states of (111) surfaces is largely defined by their formation through polarization of SS electrons; this observation is contrasted with ATP experiments from the Ag(100) surface, for which the SS becomes an unoccupied resonance and the IP states can only be excited from bands with significantly more bulk character. In addition, based on the optical power and nonlinear order-dependent mPP spectra, we provide evidence for ATP being a one-step, rather than a sequential process, as previously postulated.
74 ATOMIC AND MOLECULAR PHYSICS↗
Integrative Modeling and Analysis of Fungal Central Carbon Metabolism
Over a thousand fungal genomes have been sequenced, yet manually curated genome-scale metabolic models (GEMs) are available for only a limited number of species. Moreover, these models have often been developed independently, leading to inconsistencies in namespaces, compartment definitions, and pathway representations that hinder comparative analysis, the systematic reuse of prior curation efforts, and the integration of consolidated metabolic knowledge. Here, we present the Consolidated Fungal Core Metabolism Model (CFCMM), constructed by integrating thirteen published fungal models spanning Ascomycota, Mucoromycota, and both Crabtree-positive and Crabtree-negative yeasts. We harmonized metabolites and reactions into a non-redundant shared ModelSEED ontological space, standardized compartmentalization, and refined gene–protein–reaction (GPR) rules. Using pathway-level visualization and systematic gap detection, we further improved the integrated network through literature-guided curation to correct stoichiometry, stereospecificity, and pathway architecture. Orthologous protein family reconstruction and functional annotation workflows were used to validate and inform GPR associations, with particular emphasis on ambiguous enzyme superfamilies and membrane-associated components. Using the resulting CFCMM, we built high-quality central carbon core models for each fungus and performed flux balance analysis to quantify ATP-yield variation under aerobic and anaerobic conditions, explicitly evaluating scenarios driven by differences in electron transport chain (ETC) composition. Simulations reproduced the expected fermentative yield of approximately 2 mmol ATP per mmol glucose under anaerobic conditions and separated the thirteen fungi into two bioenergetic groups under aerobic respiration based on Complex I status, with predicted yields of approximately 30 versus 22 mmol ATP per mmol glucose. Forcing flux through the alternative oxidase bypass further reduced ATP yields to approximately 12 and 4 mmol ATP per mmol glucose in Complex I-containing and Complex I-lacking fungi, respectively. Collectively, this work provides a manually curated, ModelSEED-consistent, and extensible fungal core metabolic template, deployed in DOE KBase as a resource for automated reconstruction of central carbon core models from any sequenced fungal genome. In addition, the CFCMM provides modular components for developing GEMs with more accurate energy predictions and enables robust comparative analyses of fungal bioenergetics and core metabolic diversity
59 BASIC BIOLOGICAL SCIENCES↗
Bioinformatics Investigations of Universal Stress Proteins from Mercury-Methylating Desulfovibrionaceae
The presence of methylmercury in aquatic environments and marine food sources is of global concern. The chemical reaction for the addition of a methyl group to inorganic mercury occurs in diverse bacterial taxonomic groups including the Gram-negative, sulfate-reducing Desulfovibrionaceae family that inhabit extreme aquatic environments. The availability of whole-genome sequence datasets for members of the Desulfovibrionaceae presents opportunities to understand the microbial mechanisms that contribute to methylmercury production in extreme aquatic environments. We have applied bioinformatics resources and developed visual analytics resources to categorize a collection of 719 putative universal stress protein (USP) sequences predicted from 93 genomes of Desulfovibrionaceae. We have focused our bioinformatics investigations on protein sequence analytics by developing interactive visualizations to categorize Desulfovibrionaceae universal stress proteins by protein domain composition and functionally important amino acids. We identified 651 Desulfovibrionaceae universal stress protein sequences, of which 488 sequences had only one USP domain and 163 had two USP domains. The 488 single USP domain sequences were further categorized into 340 sequences with ATP-binding motif and 148 sequences without ATP-binding motif. The 163 double USP domain sequences were categorized into (1) both USP domains with ATP-binding motif (3 sequences); (2) both USP domains without ATP-binding motif (138 sequences); and (3) one USP domain with ATP-binding motif (21 sequences). We developed visual analytics resources to facilitate the investigation of these categories of datasets in the presence or absence of the mercury-methylating gene pair (hgcAB). Future research could utilize these functional categories to investigate the participation of universal stress proteins in the bacterial cellular uptake of inorganic mercury and methylmercury production, especially in anaerobic aquatic environments.
Isokpehi, Raphael D. (ORCID:0000000268770840)↗
An argument for using anaerobes as microbial cell factories to advance synthetic biology and biomanufacturing
Anaerobes thrive in the absence of oxygen and are an untapped reservoir of biotechnological potential. Therefore, bioprospecting efforts focused on anaerobic microbial diversity could rapidly uncover new enzymes, pathways, and chassis organisms to drive biotechnology innovation. Despite their potential utility, anaerobic fermenters are viewed as inefficient from a biochemical perspective because their metabolisms produce fewer ATP (~2) per molecule of glucose processed than heterotrophic respirers (~32–38 ATP). While aerobes excel at ATP generation, they are often less efficient than anaerobes at processes that compete with ATP generation for cellular resources. This perspective highlights how anaerobic adaptations are advantageous for synthetic biology and biomanufacturing applications through the engineering of microbial cell factories. We further highlight emerging applications of anaerobic bioprocessing, including the use of anaerobic metabolisms for lignocellulosic bioprocessing, human and environmental health, and value-added bioproduction.
59 BASIC BIOLOGICAL SCIENCES↗
On the co-nucleation of adjoining twin pairs at grain boundaries in hexagonal close-packed materials
Here this work addresses the formation of adjoining twin pairs (ATPs) at grain boundaries (GBs) in hexagonal close-packed (hcp) metals from the perspective of the co-nucleation (CN) of pairs of deformation twins. A continuum defect mechanics model is proposed to investigate the energetic feasibility of the CN of ATPs resulting from the dissociation of GB dislocations. Application of the model to a set of GBs in Mg reveals that CN is largely preferred over the nucleation of a single twin variant for low misorientation angle GBs, which is consistent with previous experimental analyses. The CN events are subsequently analyzed considering the GB character, and the twin system alignment ($m$') and Schmid factors to obtain better insights into the conditions governing the formation of ATPs via CN. The likelihood of CN even at low $m$' values reveals that CN events could be responsible for the formation of ATP pairs in these scenarios.
36 MATERIALS SCIENCE↗
Probing remdesivir nucleotide analogue insertion to SARS-CoV-2 RNA dependent RNA polymerase in viral replication
Remdesivir (RDV) prodrug can be metabolized into a triphosphate form nucleotide analogue (RDV-TP) to bind and insert into the active site of viral RNA dependent RNA polymerase (RdRp) to further interfere with viral genome replication. Here in this work, we computationally studied how RDV-TP binds and inserts to the SARS-CoV-2 RdRp active site, in comparison with natural nucleotide substrate adenosine triphosphate (ATP). To do that, we first constructed atomic structural models of an initial binding complex (active site open) and a substrate insertion complex (active site closed), based on high-resolution cryo-EM structures determined recently for SARS-CoV-2 RdRp or non-structural protein (nsp) 12, in complex with accessory protein factors nsp7 and nsp8. By conducting all-atom molecular dynamics simulation with umbrella sampling strategies on the nucleotide insertion between the open and closed state RdRp complexes, our studies show that RDV-TP can initially bind in a comparatively stabilized state to the viral RdRp active site, as it primarily forms base stacking with the template uracil nucleotide (nt +1), which under freely fluctuations supports a low free energy barrier of the RDV-TP insertion (~1.5 kcal mol -1 ). In comparison, the corresponding natural substrate ATP binds initially to the RdRp active site in Watson–Crick base pairing with the template nt, and inserts into the active site with a medium low free energy barrier (~2.6 kcal mol -1 ), when the fluctuations of the template nt are well quenched. The simulations also show that the initial base stacking of RDV-TP with the template can be specifically stabilized by motif C-S759, S682 (near motif B) with the base, and motif G-K500 with the template backbone. Although the RDV-TP insertion can be hindered by motif F-R555/R553 interaction with the triphosphate, the ATP insertion seems to be facilitated by such interactions. The inserted RDV-TP and ATP can be further distinguished by specific sugar interaction with motif B-T687 and motif A-D623, respectively.
36 MATERIALS SCIENCE↗
Biochemical properties of glycerol kinase from the hypersaline-adapted archaeon Haloferax volcanii
ABSTRACT Extremophilic microorganisms are promising candidates for industrial and analytical biocatalysis.Haloferax volcanii, a halophilic archaeon that prefers glycerol over glucose, channels this substrate into central metabolism through glycerol kinase (GK). Here, we report the biochemical properties ofH. volcaniiGK and its potential for biotechnological applications. An N-terminal His-tagged GK was functionalin vivoand yielded 3 mg/L culture—4.5 times more enzyme than a C-terminal StrepII-tagged version. Size exclusion chromatography revealed a glycerol-induced oligomeric shift from homodimer to a dimer-dominant state with detectable tetramer. The purified enzyme showed robust activity across broad pH and salinity ranges, with optimal activity at 100 mM NaCl and 50°C–60°C. It retained catalytic activity in 5%–10% dimethyl sulfoxide (DMSO) and crude glycerol containing methanol. His-GK was freeze-thaw stable and thermotolerant in 2 M NaCl buffers. In the absence of ligands, the enzyme’s melting temperature (T m ) was 80°C. Glycerol increased the T m to 85°C, and combinations with MgCl₂ (84°C) or ATP (88°C) provided further stabilization. The highest T m (89°C) occurred with all three ligands, suggesting a cumulative stabilizing effect. Kinetic analyses revealed positive cooperativity for glycerol, ATP, and Mg² + ; Mn² + and Co² + also supported the activity.H. volcaniiGK is the first known GK to exhibit positive cooperativity with glycerol and ATP. Its high stability and substrate flexibility support its use in biodiesel waste valorization,in vitrobiocatalysis, and biosensor development—applications demanding robust, specific, and stable enzymes. IMPORTANCE This study reveals thatH. volcaniiGK exhibits positive cooperativity for glycerol, ATP, and Mg² + , a kinetic feature not previously reported for glycerol kinases. This behavior enables steep, switch-like responses to small substrate changes, offering unique advantages for biosensor design. Importantly,H. volcaniiGK also maintains high activity under extreme salinity, temperature, broad pH, and solvent conditions that typically limit enzyme use in industrial and environmental applications. These traits make this GK an ideal candidate for enzyme-based biosensors, which often suffer from poor tolerance to pH, solvent, and thermal stress. Its robustness supports its use in cross-linked enzyme crystals, an immobilization method that enhances enzyme stability and reusability under harsh conditions. Moreover, GKs are already employed in Mg² + detection kits; however,H. volcaniiGK’s ability to tolerate and respond to diverse divalent cations (e.g., Co² + , Mn² + ) broadens their potential for pollutant detection and environmental monitoring. These features collectively positionH. volcaniiGK as a valuable biocatalyst for biosensing,in vitrodiagnostics, and biotechnological applications requiring both precision and durability.
Biotechnology & Applied Microbiology↗
Biophysical and Biochemical Characterization of TP0037, a D-Lactate Dehydrogenase, Supports an Acetogenic Energy Conservation Pathway in Treponema pallidum
A longstanding conundrum in Treponema pallidum biology concerns how the spirochete generates sufficient energy to fulfill its complex pathogenesis processes during human syphilitic infection. For decades, it has been assumed that the bacterium relies solely on glucose catabolism (via glycolysis) for generation of its ATP. However, the organism’s robust motility, believed to be essential for human tissue invasion and dissemination, would require abundant ATP likely not provided by the parsimony of glycolysis. As such, additional ATP generation, either via a chemiosmotic gradient, substrate-level phosphorylation, or both, likely exists in T. pallidum. Along these lines, we have hypothesized that T. pallidum exploits an acetogenic energy conservation pathway that relies on the redox chemistry of flavins. Central to this hypothesis is the apparent existence in T. pallidum of an acetogenic pathway for the conversion of d-lactate to acetate. Herein we have characterized the structural, biophysical, and biochemical properties of the first enzyme (d-lactate dehydrogenase [d-LDH]; TP0037) predicted in this pathway. Binding and enzymatic studies showed that recombinant TP0037 consumed d-lactate and NAD+ to produce pyruvate and NADH. The crystal structure of TP0037 revealed a fold similar to that of other d-acid dehydrogenases; residues in the cofactor-binding and active sites were homologous to those of other known d-LDHs. The crystal structure and solution biophysical experiments revealed the protein’s propensity to dimerize, akin to other d-LDHs. This study is the first to elucidate the enzymatic properties of T. pallidum’s d-LDH, thereby providing new compelling evidence for a flavin-dependent acetogenic energy conservation (ATP-generating) pathway in T. pallidum.
59 BASIC BIOLOGICAL SCIENCES↗
Structures of Fission Yeast Inositol Pyrophosphate Kinase Asp1 in Ligand-Free, Substrate-Bound, and Product-Bound States
Expression of the fission yeast Schizosaccharomyces pombe phosphate regulon is sensitive to the intracellular level of the inositol pyrophosphate signaling molecule 1,5-IP 8 . IP 8 dynamics are determined by Asp1, a bifunctional enzyme consisting of an N-terminal kinase domain and a C-terminal pyrophosphatase domain that catalyze IP 8 synthesis and catabolism, respectively. Here, we report structures of the Asp1 kinase domain, crystallized with two protomers in the asymmetric unit, one of which was complexed with ligands (ADPNP, ADP, or ATP; Mg 2+ or Mn 2+ ; IP 6 , 5-IP 7 , or 1,5-IP 8 ) and the other which was ligand-free. The ligand-free enzyme adopts an “open” conformation that allows ingress of substrates and egress of products. ADPNP, ADP, and ATP and associated metal ions occupy a deep phospho-donor pocket in the active site. IP 6 or 5-IP 7 engagement above the nucleotide favors adoption of a “closed” conformation, in which surface protein segments undergo movement and a disordered-to-ordered transition to form an inositol polyphosphate-binding site. In a structure mimetic of the kinase Michaelis complex, the anionic 5-IP 7 phosphates are encaged by an ensemble of nine cationic amino acids: Lys43, Arg223, Lys224, Lys260, Arg274, Arg285, Lys290, Arg293, and Lys341. Alanine mutagenesis of amino acids that contact the adenosine nucleoside of the ATP donor underscored the contributions of Asp258 interaction with the ribose 3'-OH and of Glu248 with adenine-N 6 . Changing Glu248 to Gln elicited a gain of function whereby the kinase became adept at using GTP as phosphate donor. Wild-type Asp1 kinase can utilize N 6 -benzyl-ATP as phosphate donor.
59 BASIC BIOLOGICAL SCIENCES↗