Engineering Papers⌕ Search

SEARCH · Engineering Papers

Results for “ATP”

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.

Quote a phrase for an exact phrase match. Source license links do not imply unrestricted reuse.

At least 1,009 records · Page 56
All sourcesNASA NTRSDOE OSTI
DOE OSTI2026

Comparative mitogenomics of kingdom Fungi – evolutionary insights and metagenomic applications

Mitochondria are essential components of eukaryotic cells, responsible for ATP production through oxidative phosphorylation. Despite their biological importance, unique challenges have hindered the adoption of automated mitochondrial genome (mitogenome) annotation methods, obstructing mitochondrial comparative genomics in a broad evolutionary context. Using Fungi as a study system and a Joint Genome Institute (JGI) annotated high-quality reference set, we observed broad patterns of mitochondrial evolution across the kingdom. We found that the median fungal mitogenome size is 58 kb and identified exceptionally large examples over 1 Mb in Pezizomycetes. All 14 expected oxidative phosphorylation protein-coding genes, plus rps3, were generally conserved. We found evidence of major evolutionary transitions within the Ascomycota, including the transfer of mitochondrially encoded atp8 and atp9 to the nuclear genomes across the Pezizomycotina and shifts in mitogenome tRNA patterns across the kingdom. We found substantial concordance between mitochondrial and nuclear evolution, enabling us to document 3131 total fungal mitogenomes from JGI-derived metagenomic datasets. We also identified 6467 total undeclared mitogenomes embedded in Genbank fungal nuclear assemblies. We provide interactive tools for mitogenome analysis through the JGI MycoCosm platform. Collectively, this work generated nearly 10 000 new fungal mitogenome annotations, providing a foundation and resources for future exploration of comparative fungal mitogenomics.

Ahrendt, Steven R. [USDOE Joint Genome Institute (↗
DOE OSTI2014

A tRNA splicing operon: Archease endows RtcB with dual GTP/ATP cofactor specificity and accelerates RNA ligation

Archease is a 16-kDa protein that is conserved in all three domains of life. In diverse bacteria and archaea, the genes encoding Archease and the tRNA ligase RtcB are localized into an operon. Here we provide a rationale for this operon organization by showing that Archease and RtcB from Pyrococcus horikoshii function in tandem, with Archease altering the catalytic properties of the RNA ligase. RtcB catalyzes the GTP and Mn(II)-dependent joining of either 2',3'-cyclic phosphate or 3'-phosphate termini to 5'-hydroxyl termini. We find that catalytic concentrations of Archease are sufficient to activate RtcB, and that Archease accelerates both the RNA 3'-P guanylylation and ligation steps. In addition, we show that Archease can alter the NTP specificity of RtcB such that ATP, dGTP or ITP is used efficiently. Moreover, RtcB variants that have inactivating substitutions in the guanine-binding pocket can be rescued by the addition of Archease. We also present a 1.4 Å-resolution crystal structure of P. horikoshii Archease that reveals a metal-binding site consisting of conserved carboxylates located at the protein tip. Substitution of the Archease metal-binding residues drastically reduced Archease-dependent activation of RtcB. Thus, evolution has sought to co-express archease and rtcB by creating a tRNA splicing operon.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2021

Phloem unloading via the apoplastic pathway is essential for shoot distribution of root-synthesized cytokinins

Abstract Root-synthesized cytokinins are transported to the shoot and regulate the growth, development, and stress responses of aerial tissues. Previous studies have demonstrated that Arabidopsis (Arabidopsis thaliana) ATP binding cassette (ABC) transporter G family member 14 (AtABCG14) participates in xylem loading of root-synthesized cytokinins. However, the mechanism by which these root-derived cytokinins are distributed in the shoot remains unclear. Here, we revealed that AtABCG14-mediated phloem unloading through the apoplastic pathway is required for the appropriate shoot distribution of root-synthesized cytokinins in Arabidopsis. Wild-type rootstocks grafted to atabcg14 scions successfully restored trans-zeatin xylem loading. However, only low levels of root-synthesized cytokinins and induced shoot signaling were rescued. Reciprocal grafting and tissue-specific genetic complementation demonstrated that AtABCG14 disruption in the shoot considerably increased the retention of root-synthesized cytokinins in the phloem and substantially impaired their distribution in the leaf apoplast. The translocation of root-synthesized cytokinins from the xylem to the phloem and the subsequent unloading from the phloem is required for the shoot distribution and long-distance shootward transport of root-synthesized cytokinins. This study revealed a mechanism by which the phloem regulates systemic signaling of xylem-mediated transport of root-synthesized cytokinins from the root to the shoot.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2022

Respiratory energy demands and scope for demand expansion and destruction

Photosynthesis is the primary energy input to plants, but most plant metabolic processes are powered much or all of the time by ATP and NAD(P)H that come from respiratory oxidation of photosynthetically produced carbohydrates, that is, “dark respiration” (Amthor, 1994). The processes of growth, nutrient uptake and assimilation, active transport, and maintenance are thus all clients of respiration and compete for their share of a respiratory energy budget that is capped by photosynthate income. The economic principle of opportunity cost applies to these clients’ competing demands: spending respiratory energy on process A means missing the benefit of investing that energy in process B (Mahmoudabadi et al., 2019). This principle is key to assessing prospects for crop improvement by metabolic engineering. As de Lorenzo (2015) put it: “metabolism…frames and ultimately resolves whether a given genetic program (existing…or engineered) can be deployed or not.” Foreign or reconfigured native processes bolted on to a crop-plant’s metabolic chassis must compete for respiratory energy with native ones without crashing the energy economy. A proviso on plant carbon budgets is that photosynthetic carbon fixation (“source activity”) can in certain cases increase to meet increased carbon demand (“sink activity”), that is, budget envelopes are not always fixed (Smith et al., 2018). However, as there is some consensus that productivity is most often limited or co-limited by carbon supply (Ainsworth and Long, 2005; Körner, 2015; Sonnewald and Fernie, 2018), we make this our basal assumption in the analyses below.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2023

Chloroplast phosphate transporter CrPHT4-7 regulates phosphate homeostasis and photosynthesis in Chlamydomonas

Abstract In eukaryotic cells, phosphorus is assimilated and utilized primarily as phosphate (Pi). Pi homeostasis is mediated by transporters that have not yet been adequately characterized in green algae. This study reports on PHOSPHATE TRANSPORTER 4-7 (CrPHT4-7) from Chlamydomonas reinhardtii, a member of the PHT4 transporter family, which exhibits remarkable similarity to AtPHT4;4 from Arabidopsis (Arabidopsis thaliana), a chloroplastic ascorbate transporter. Using fluorescent protein tagging, we show that CrPHT4-7 resides in the chloroplast envelope membrane. Crpht4-7 mutants, generated by the CRISPR/Cas12a-mediated single-strand templated repair, show retarded growth, especially in high light, reduced ATP level, strong ascorbate accumulation, and diminished non-photochemical quenching in high light. On the other hand, total cellular phosphorous content was unaffected, and the phenotype of the Crpht4-7 mutants could not be alleviated by ample Pi supply. CrPHT4-7-overexpressing lines exhibit enhanced biomass accumulation under high light conditions in comparison with the wild-type strain. Expressing CrPHT4-7 in a yeast (Saccharomyces cerevisiae) strain lacking Pi transporters substantially recovered its slow growth phenotype, demonstrating that CrPHT4-7 transports Pi. Even though CrPHT4-7 shows a high degree of similarity to AtPHT4;4, it does not display any substantial ascorbate transport activity in yeast or intact algal cells. Thus, the results demonstrate that CrPHT4-7 functions as a chloroplastic Pi transporter essential for maintaining Pi homeostasis and photosynthesis in C. reinhardtii.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2024

The structural OFF and ON states of myosin can be decoupled from the biochemical super- and disordered-relaxed states

Abstract There is a growing awareness that both thick-filament and classical thin-filament regulations play central roles in modulating muscle contraction. Myosin ATPase assays have demonstrated that under relaxed conditions, myosin may reside either in a high-energy-consuming disordered-relaxed (DRX) state available for binding actin to generate force or in an energy-sparing super-relaxed (SRX) state unavailable for actin binding. X-ray diffraction studies have shown that the majority of myosin heads are in a quasi-helically ordered OFF state in a resting muscle and that this helical ordering is lost when myosin heads are turned ON for contraction. It has been assumed that myosin heads in SRX and DRX states are equivalent to the OFF and ON states, respectively, and the terms have been used interchangeably. In this study, we use X-ray diffraction and ATP turnover assays to track the structural and biochemical transitions of myosin heads, respectively, induced with either omecamtiv mecarbil (OM) or piperine in relaxed porcine myocardium. We find that while OM and piperine induce dramatic shifts of myosin heads from the OFF to the ON state, there are no appreciable changes in the population of myosin heads in the SRX and DRX states in both unloaded and loaded preparations. Our results show that biochemically defined SRX and DRX can be decoupled from structurally defined OFF and ON states. In summary, while SRX/DRX and OFF/ON transitions can be correlated in some cases, these two phenomena are measured using different approaches, reflect different properties of the thick filament, and should be investigated and interpreted separately.

Jani, Vivek P. (ORCID:0000000238114973)↗
DOE OSTI2019

Attempts to develop an enzyme converting DHIV to KIV

Abstract Dihydroxy-acid dehydratase (DHAD) catalyzes the dehydration of R-2,3-dihydroxyisovalerate (DHIV) to 2-ketoisovalerate (KIV) using an Fe-S cluster as a cofactor, which is sensitive to oxidation and expensive to synthesize. In contrast, sugar acid dehydratases catalyze the same chemical reactions using a magnesium ion. Here, we attempted to substitute the high-cost DHAD with a cost-efficient engineered sugar acid dehydratase using computational protein design (CPD). First, we tried without success to modify the binding pocket of a sugar acid dehydratase to accommodate the smaller, more hydrophobic DHIV. Then, we used a chemically activated substrate analog to react with sugar acid dehydratases or other enolase superfamily enzymes. Mandelate racemase from Pseudomonas putida (PpManR) and the putative sugar acid dehydratase from Salmonella typhimurium (StPutD) showed beta-elimination activity towards chlorolactate (CLD). CPD combined with medium-throughput selection improved the PpManR kcat/KM for CLD by four-fold. However, these enzyme variants did not show dehydration activity towards DHIV. Lastly, assuming phosphorylation could also be a good activation mechanism, we found that mevalonate-3-kinase (M3K) from Picrophilus torridus (PtM3K) exhibited adenosine triphosphate (ATP) hydrolysis activity when mixed with DHIV, indicating phosphorylation activity towards DHIV. Engineering PpManR or StPutD to accept 3-phospho-DHIV as a substrate was performed, but no variants with the desired activity were obtained.

Oki, Kenji↗
DOE OSTI2020

Transporters and ascorbate–glutathione metabolism for differential cadmium accumulation and tolerance in two contrasting willow genotypes

Salix matsudana Koidz is a low cadmium (Cd)-accumulating willow, whereas its cultivated variety, Salix matsudana var. matsudana f. umbraculifera Rehd., is a high Cd-accumulating and tolerant willow (HCW). The physiological and molecular mechanisms underlying differential Cd accumulation and tolerance in the two Salix species are poorly understood. In this study, we confirmed that the differential Cd translocation capacity from roots to the shoots leads to the differential Cd accumulation in their aboveground parts between these two willow genotypes. Cadmium accumulation happens preferentially in the transport pathway, and Cd is mainly located in the vacuolar, cell wall and intercellular space in HCW bark by cadmium location analysis at tissue and subcellular levels. Comparative transcriptome analysis revealed that higher expressions of several metal transporter genes (ATP-binding cassette transporters, K + transporters/channels, yellow stripe-like proteins, zinc-regulated transporter/iron-regulated transporter-like proteins, etc.) are involved in root uptake and translocation capacity in HCW; meanwhile, ascorbate–glutathione metabolic pathways play essential roles in Cd detoxification and higher tolerance of the Cd-accumulator HCW. These results lay the foundation for further understanding the molecular mechanisms of Cd accumulation in woody plants and provide new insights into molecular-assisted-screening woody plant varieties for phytoremediation.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2025

Unraveling Bacterial Adaptation Strategies in the Microbiome Shaped by the Chemical Environment of the Plant Rhizosphere

The rhizosphere is a dynamic environment where rhizodeposits that include primary and secondary metabolites and mucilage serve as nutrient sources for soil microorganisms, attracting them toward plant roots. However, understanding how these microbes specifically respond to plant root chemical signals has been hindered by the challenges of disentangling physical and chemical interactions between the microbes and plant roots. To address this, we implemented an innovative filter-based experimental setup on plant roots that creates a physical barrier while facilitating the exchange of chemical signals. The proteomic analysis of 10 Populus root-associated bacterial strains grown in the presence or absence of a plant in either individual or mixed community conditions provided detailed insights into the functional responses of these strains to the root chemical environment. Additionally, this approach allowed us to discern the impact of root exudates on overall community dynamics. In particular, metaproteomic analyses revealed that each of these 10 microbial members responds uniquely to the presence of the plant, with Bacillus and Pantoea exhibiting the most dramatic favorable impact. Proteomic examination revealed the details of metabolism fine-tuning, including processes such as chemotaxis and ATP-binding cassette transporter proteins. This study demonstrates the application of a filter-based experimental setup to study microbial responses to plant chemicals and sheds light on adaptation strategies employed by various bacterial strains for survival in the rhizosphere.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗
DOE OSTI2016

Simultaneously Revealing All Lysine Acylations in an Organism to Elucidate Substrate Metabolism

Within the past 8 years, lysine acetylation knowledge has extended greatly beyond one modification important for chromatin maintenance and transcription regulation to the whole family of reversible conserved short‐chain acylations emerging as an important regulatory mechanism coordinating metabolism in response to changes in nutrient availability. Inspired by these lysine acylation discoveries, we devise a mass spectrometry (MS) method to simultaneously reveal and evaluate all lysine acylations in an organism. Hypothesizing that an organism's pool of acyl‐CoA esters could suggest potential acyl‐lysine modifications hidden within MS/MS datasets, we chose two syntrophic bacteria, Syntrophomonas wolfei and Syntrophus aciditrophicus, for the study. They activate fatty acid and/or aromatic substrates with coenzyme A and stepwise convert acyl‐CoAs down to acetyl‐CoA for ATP production, thus containing a variety of acyl‐CoA intermediates. Different from the current approach of immuno‐enriching acylated peptide prior to MS, our shotgun non‐enrichment MS approach allows a quick, non immuno‐bias, economical and quantitative one‐step assessment of all lysine acylations within an organism, and the modification stoichiometries and their relative levels. We also showed characteristic ions are reliable to detect and confirm lysine acylations, establishing seven lysine acylations first seen in any organism and doubling the number of known short‐chain acylations. Our lysine acylation studies suggest a metabolism regulation mechanism in these syntrophs living at the thermodynamic limit of feasibility. This proteomic approach can be applied to any organism having a complex profile of acyl‐CoAs, especially anaerobic bacteria degrading fatty acids and aromatic compounds and gut bacteria producing butyrate. Support or Funding Information This work is funded by the US Department of Energy (UCLA Institute of Genomics and Proteomics, DE‐FC03‐02ER63421 to J. Loo) and the National Institutes of Health (R01GM085402 to J. Loo and R. Ogorzalek Loo).

Nguyen, Hong Hanh T.↗
DOE OSTI2016

Studies of the PfkB Family of Proteins in Arabidopsis thaliana

The phosphofructokinase B (PkfB) family of carbohydrate kinases is a large group that includes members with specificity for a diverse array of carbohydrate substrates, including fructose, ribose, fructose‐6‐phosphate, tagatose‐6‐phosphate, adenosine, myo‐inositol, and others. Although there are 21 genes encoding PfkB family members in the higher plant Arabidopsis thaliana , only a few have been studied in detail. Two Arabidopsis proteins with fructokinase activity were identified and characterized via native PAGE and in‐gel assays, though the genes were not identified. Another class of PfkB proteins identified in Arabidopsis lacks demonstrated enzymatic activity, but is necessary for the accumulation of photosynthetic transcripts in chloroplasts. The fructokinase‐like proteins 1 and 2 (FLN1 and FLN2, respectively) and NARA5 all belong to this class, although the mechanism of how NARA5 exerts it's phenotype remains elusive. FLN1 and 2 have been identified in proteomics studies as members of a large protein complex that interacts with, and likely regulates, the prokaryotic‐type plastid RNA polymerase which specifically transcribes photosynthetic genes in the plastid. We have found that FLN1 and FLN2 interact with each other in an ATP‐dependent manner both in vitro and in vivo . Phenotypic analysis suggests that the presence of both proteins is necessary for proper levels of transcription. Our data on the interaction between FLN1 and FLN2 suggests a mechanism where the FLN proteins act as a biochemical switch to activate transcription under conditions that favor their binding to each other. We have also begun characterization of the remainder of the Arabidopsis PfkB family and have identified a group of seven highly similar Arabidopsis PfkB proteins. These seven proteins were expressed in E. coli and enzymatic assays performed. All are active as fructokinases with high specificity for fructose. T‐DNA mediated inactivation of five of the seven genes resulted in no overt phenotype which hints at possible redundancy between some of these gene products. Using GFP fusions, we have determined that all localize to the cytosol except one, which localizes to plastids. Higher order mutants and biochemical analysis, along with expression analysis will allow us to tease apart the differences and the importance of each of these family members. We have characterized another PfkB protein that appears to be the sole Arabidopsis ribokinase (AtRBSK). Its biochemical properties and cofactor requirements are similar to that of known mammalian and prokaryotic RBSKs. T‐DNA insertion into AtRBSK results in the hyper‐accumulation of ribose in both the roots and leaves of 10 day‐old seedlings compared to wild type. Arabidopsis lacking AtRBSK grow poorly on media supplemented with ribose as the only sugar. We are currently dissecting the pathway that leads to the production of ribose in plants to put AtRBSK into a biological context. Our studies of the PfkB family in Arabidopsis are yielding promising results and will continue to further our understanding of many fundamental processes in plant biology ranging from central metabolism to the regulation of transcription in chloroplasts. Support or Funding Information The authors gratefully acknowledge support from the Chemical Sciences, Geosciences and Biosciences Division, Office of Basic Energy Sciences, Office of Science, US. Department of Energy (contracts DE‐FG02–09ER16077 and DE‐SC0002175) to JC

Riggs, John W.↗
DOE OSTI2016

ABCA1 agonist peptides for the treatment of disease

Purpose of review The review summarizes information pertaining to the preclinical development of new apolipoprotein (apo) E mimetic peptides that stimulate cellular cholesterol efflux. Recent findings Small α-helical peptides based on the C-terminal domain of apoE have been developed for therapeutic applications. These peptides stimulate cellular cholesterol efflux via the ATP-binding cassette transporter A1 (ABCA1) with high potency, like native apolipoproteins on a molar basis. This potent activity has been related to the unique ability of these peptides to maintain α-helix structure upon dilution. Recent structure-activity studies improving the safety features of these mimetic peptides have greatly improved their potential for clinical use. Structural features of the class A α-helix motif that induce muscle toxicity and hypertriglyceridemia have been identified. These may have implications for the design of other HDL mimetic peptides. Summary ABCA1 is an integral membrane protein that plays a central role in biology. Its principal function is to mediate the efflux of cholesterol and phospholipid from cells to extracellular apo, preventing a build-up of excess cholesterol in membranes. This process generates HDL particles that perform a variety of functions to protect against disease. A number of these functions can be viewed as directly or indirectly supporting ABCA1 activity, thus constituting a positive feedback system to optimize cellular lipid efflux responses and disease prevention. Consequently, therapeutic approaches that mimic the activities of apos may prove highly effective to combat disease. One such approach involves the use of peptides. The broad biological relevance of ABCA1 suggests these apo mimetic peptides may be useful for the treatment of a number of diseases, such as atherosclerosis, diabetes, and Alzheimer's disease.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2016

MultispeQ Beta: a tool for large-scale plant phenotyping connected to the open PhotosynQ network

Large-scale high-throughput plant phenotyping (sometimes called phenomics) is becoming increasingly important in plant biology and agriculture and is essential to cutting-edge plant breeding and management approaches needed to meet the food and fuel needs for the next century. Currently, the application of these approaches is severely limited by the availability of appropriate instrumentation and by the ability to communicate experimental protocols, results and analyses. To address these issues, we have developed a low-cost, yet sophisticated open-source scientific instrument designed to enable communities of researchers, plant breeders, educators, farmers and citizen scientists to collect high-quality field data on a large scale. The MultispeQ provides measurements in the field or laboratory of both, environmental conditions (light intensity and quality, temperature, humidity, CO 2 levels, time and location) and useful plant phenotypes, including photosynthetic parameters—photosystem II quantum yield (Φ II ), non-photochemical exciton quenching (NPQ), photosystem II photoinhibition, light-driven proton translocation and thylakoid proton motive force, regulation of the chloroplast ATP synthase and potentially many others—and leaf chlorophyll and other pigments. Plant phenotype data are transmitted from the MultispeQ to mobile devices, laptops or desktop computers together with key metadata that gets saved to the PhotosynQ platform (https://photosynq.org) and provides a suite of web-based tools for sharing, visualization, filtering, dissemination and analyses. We present validation experiments, comparing MultispeQ results with established platforms, and show that it can be usefully deployed in both laboratory and field settings. We present evidence that MultispeQ can be used by communities of researchers to rapidly measure, store and analyse multiple environmental and plant properties, allowing for deeper understanding of the complex interactions between plants and their environment.

47 OTHER INSTRUMENTATION↗
DOE OSTI2024

Evolution of the regulatory subunits for the heteromeric acetyl-CoA carboxylase

The committed step for de novo fatty acid (FA) synthesis is the ATP-dependent carboxylation of acetyl-coenzyme A catalysed by acetyl-CoA carboxylase (ACCase). In most plants, ACCase is a multi-subunit complex orthologous to prokaryotes. However, unlike prokaryotes, the plant and algal orthologues are comprised both catalytic and additional dedicated regulatory subunits. Novel regulatory subunits, biotin lipoyl attachment domain-containing proteins (BADC) and carboxyltransferase interactors (CTI) (both three-gene families inArabidopsis) represent new effectors specific to plants and certain algal species. The evolutionary history of these genes in autotrophic eukaryotes remains elusive, making it an ongoing area of research. Analyses of potential protein–protein and co-occurrence interactions, informed by gene network patterns using the STRING database, inArabidopsis thalianaandChlamydomonas reinhardtiiunveil intricate gene associations with ACCase, suggesting a complex interplay between FA synthesis and other cellular processes. Among both species, a higher number of co-expressed genes was identified inArabidopsis, indicating a wider potential regulatory network of ACCase in plants. This review investigates the extent to which these genes arose in autotrophic eukaryotes and provides insights into their evolutionary trajectory. This article is part of the theme issue ‘The evolution of plant metabolism’.

Life Sciences & Biomedicine - Other Topics↗
DOE OSTI2025

Cryo-EM structure of a methanogen nitrogenase-PII protein supercomplex

Abstract Nitrogenases are metalloenzymes that catalyze the reduction of atmospheric dinitrogen to ammonia, sustaining the global nitrogen cycle. While bacterial nitrogenase has been extensively characterized, the architecture and regulation of archaeal nitrogenases remain unknown despite longstanding evidence of nitrogen fixation in methanogens. Here we report a 3.1 Å cryo-electron microscopy structure of a native nitrogenase–PII protein supercomplex from Methanosarcina acetivorans. The structure reveals an unprecedented assembly of three NifDK heterotetramers bridged by six NifI1,2 heterotrimeric PII complexes, which sterically block NifH association and lock the enzyme in an inactive state. The NifI complexes display asymmetric binding of ADP and 2-oxoglutarate, coupling nitrogenase inhibition directly to cellular energy and nitrogen status. Addition of 2-oxoglutarate and ATP releases the NifI complexes, stimulating a threefold increase in NifDK activity in vitro. This higher-order architecture uncovers a previously unrecognized regulatory strategy in methanogens, in which PII proteins drive nitrogenase oligomerization to control activity. The discovery that nitrogenase activity may be modulated through direct assembly into higher-order structures opens new avenues for exploring nitrogenase evolution, regulation, and biotechnological applications. One sentence summary Discovery of a nitrogenase-PII protein supercomplex in methanogens, uncovering a metabolite-gated assembly mechanism for nitrogenase inhibition.

Nitrogenase, Electron Transfer, Methanogen↗
DOE OSTI2014

Structural and mechanistic insights into Mcm2-7 double-hexamer assembly and function

Eukaryotic cells license each DNA replication origin during G1 phase by assembling a prereplication complex that contains a Mcm2–7 (minichromosome maintenance proteins 2–7) double hexamer. During S phase, each Mcm2–7 hexamer forms the core of a replicative DNA helicase. However, the mechanisms of origin licensing and helicase activation are poorly understood. The helicase loaders ORC–Cdc6 function to recruit a single Cdt1–Mcm2–7 heptamer to replication origins prior to Cdt1 release and ORC–Cdc6–Mcm2–7 complex formation, but how the second Mcm2–7 hexamer is recruited to promote double-hexamer formation is not well understood. Here, structural evidence for intermediates consisting of an ORC–Cdc6–Mcm2–7 complex and an ORC–Cdc6–Mcm2–7–Mcm2–7 complex are reported, which together provide new insights into DNA licensing. Detailed structural analysis of the loaded Mcm2–7 double-hexamer complex demonstrates that the two hexamers are interlocked and misaligned along the DNA axis and lack ATP hydrolysis activity that is essential for DNA helicase activity. Moreover, we show that the head-to-head juxtaposition of the Mcm2–7 double hexamer generates a new protein interaction surface that creates a multisubunit-binding site for an S-phase protein kinase that is known to activate DNA replication. The data suggest how the double hexamer is assembled and how helicase activity is regulated during DNA licensing, with implications for cell cycle control of DNA replication and genome stability.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2013

A systems level predictive model for global gene regulation of methanogenesis in a hydrogenotrophic methanogen

Methanogens catalyze the critical methane-producing step (called methanogenesis) in the anaerobic decomposition of organic matter. Here, we present the first predictive model of global gene regulation of methanogenesis in a hydrogenotrophic methanogen, Methanococcus maripaludis. We generated a comprehensive list of genes (protein-coding and noncoding) for M. maripaludis through integrated analysis of the transcriptome structure and a newly constructed Peptide Atlas. The environment and gene-regulatory influence network (EGRIN) model of the strain was constructed from a compendium of transcriptome data that was collected over 58 different steady-state and time-course experiments that were performed in chemostats or batch cultures under a spectrum of environmental perturbations that modulated methanogenesis. Analyses of the EGRIN model have revealed novel components of methanogenesis that included at least three additional protein-coding genes of previously unknown function as well as one noncoding RNA. We discovered that at least five regulatory mechanisms act in a combinatorial scheme to intercoordinate key steps of methanogenesis with different processes such as motility, ATP biosynthesis, and carbon assimilation. Through a combination of genetic and environmental perturbation experiments we have validated the EGRIN-predicted role of two novel transcription factors in the regulation of phosphate-dependent repression of formate dehydrogenase—a key enzyme in the methanogenesis pathway. The EGRIN model demonstrates regulatory affiliations within methanogenesis as well as between methanogenesis and other cellular functions.

Biochemistry & Molecular Biology↗
DOE OSTI2014

Elements Required for an Efficient NADP-Malic Enzyme Type C4 Photosynthesis

Abstract C4 photosynthesis has higher light, nitrogen, and water use efficiencies than C3 photosynthesis. Although the basic anatomical, cellular, and biochemical features of C4 photosynthesis are well understood, the quantitative significance of each element of C4 photosynthesis to the high photosynthetic efficiency are not well defined. Here, we addressed this question by developing and using a systems model of C4 photosynthesis, which includes not only the Calvin-Benson cycle, starch synthesis, sucrose synthesis, C4 shuttle, and CO2 leakage, but also photorespiration and metabolite transport between the bundle sheath cells and mesophyll cells. The model effectively simulated the CO2 uptake rates, and the changes of metabolite concentrations under varied CO2 and light levels. Analyses show that triose phosphate transport and CO2 leakage can help maintain a high photosynthetic rate by balancing ATP and NADPH amounts in bundle sheath cells and mesophyll cells. Finally, we used the model to define the optimal enzyme properties and a blueprint for C4 engineering. As such, this model provides a theoretical framework for guiding C4 engineering and studying C4 photosynthesis in general.

09 BIOMASS FUELS↗