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NASA NTRS2003

Structure, Function, Self-Assembly and Origin of Simple Membrane Proteins

Integral membrane proteins perform such essential cellular functions as transport of ions, nutrients and waste products across cell walls, transduction of environmental signals, regulation of cell fusion, recognition of other cells, energy capture and its conversion into high-energy compounds. In fact, 30-40% of genes in modem organisms codes for membrane proteins. Although contemporary membrane proteins or their functional assemblies can be quite complex, their transmembrane fragments are usually remarkably simple. The most common structural motif for these fragments is a bundle of alpha-helices, but occasionally it could be a beta-barrel. In a series of molecular dynamics computer simulations we investigated self-organizing properties of simple membrane proteins based on these structural motifs. Specifically, we studied folding and insertion into membranes of short, nonpolar or amphiphatic peptides. We also investigated glycophorin A, a peptide that forms sequence-specific dimers, and a transmembrane aggregate of four identical alpha-helices that forms an efficient and selective voltage-gated proton channel was investigated. Many peptides are attracted to water-membrane interfaces. Once at the interface, nonpolar peptides spontaneously fold to a-helices. Whenever the sequence permits, peptides that contain both polar and nonpolar amino also adopt helical structures, in which polar and nonpolar amino acid side chains are immersed in water and membrane, respectively. Specific identity of side chains is less important. Helical peptides at the interface could insert into the membrane and adopt a transmembrane conformation. However, insertion of a single helix is unfavorable because polar groups in the peptide become completely dehydrated upon insertion. The unfavorable free energy of insertion can be regained by spontaneous association of peptides in the membrane. The first step in this process is the formation of dimers, although the most common are aggregates of 4-7 helices. The helices could arrange themselves such that they formed pores capable of transporting ions and small molecules across membranes. Stability of transmembrane aggregates of simple proteins is often only marginal and, therefore, it can be regulated by environmental signals or small sequence modifications in the region of interhelical interactions. A key step in the earliest evolution of membrane proteins was the emergence of selectivity for specific substrates. Many channels could become selective if one or only a few properly chosen amino acids are properly placed along the channel, acting as filters or gates. This is a convenient evolutionary solution because it does not require imposing conditions on the whole sequence.

Pohorille, Andrew↗

NASA NTRS2003

An Examination of Issues Related to a Europa Subsurface Component for the JIMO Mission

The Galileo Europa data set served to revolutionize our view of Europa. In particular the strong evidence of a large, cold, salty Ocean beneath 5-30 km of ice has profoundly altered the significance of Europa in our thinking, especially of context of habitability in the solar system. While much remains to be learned from spacecraft observations of several sorts, there are significant questions answerable only by in-situ techniques; these relate to the formation of Europa, the nature of its ocean, and the prospects for life in its ocean, sediments, and ice. We feel that wide-ranging discussion of an in-situ subsurface mission to Europa, as part of JIMO, should proceed. The science objective of the mission is to characterize the icy shell of Europa to resolve its provenance, estimate the composition of brine of the Europa ocean, and search for evidence of Earth-like life. Probably anyone would agree that an in-situ mission to Europa would be of great value, but he or she would also immediately take the position that such a mission is utterly impractical. We take the position here of defining the least complex mission that can nonetheless justify its cost and to argue that such a mission is realistic enough that it should be seriously considered. Our mission thinking has been: 1) Soft landing. A soft lander is required on a site sufficiently flat to offer a stable platform; no further site selectivity is required. 2) Subsurface exploration. The Europa subsurface must be examined. Surficial processes on Europa arguably have exposed the upper 200 m of shell to chemical effects from the Jovian radiation belts as well as cometary infall, etc; to examine native ice we must descend below that point to, for discussion, 300 m. At that depth we argue that the ice is characteristic of ice at depth and possibly is effectively sea ice. 3) Science data. A few simple measurements at various depths and at 300 m constitute a scientifically successful mission. Measurements would include analysis of meltwater for a few inorganic ions and amino acids and an optical examination of the borehole wall. 4) Communication. Transmission of data to an orbiter is essential, but we will constrain the landed mission to a daily communication over a few days. 5) Subsurface access. Drilling to 300 m is a significant challenge; it can be addressed by several means: Thermal Probe (Cryobot) which permits water to refreeze above the vehicle. This is our tentative choice with plutonium as the fuel to generate thermal energy for drilling and electrical power for operations. Open Hole Drill, a thermal system in which the meltwater is removed for greater thermal efficiency. Meltwater removal on Europa is both a complexity and a risk, but analysis is improved. Mechanical Drilling in which cutting or grinding generates ice chips which are removed. This is too complex at Europa temperatures. The measurement objectives for the mission will be: Obj. 1: Determine the concentration of simple inorganic salts in the Europa Ice Shell and, by extrapolation, of the ocean. These data will also validate spaceborne sensors. Obj. 2: Determine the nature and abundance of amino acids in the ice such that cometary infall material in the upper ice can be compared to material at depth. Obj. 3: Optically examine the ice to resolve inclusion structure, particulate content, and stratification. Access to 300 m depth is a significant if not audacious plan; we are aware that this has not been done on any planetary body. Our approach is the use of a plutonium heat source; to overcome Europa's surface temperature and to melt ice a significant amount of plutonium is needed, and significant shielding and other protective steps will be required. The quantity of plutonium is a key concern. The mission will require subsurface collection and processing of samples for in situ analysis, calling for a miniature, high pressure micro-sampling system designed to meet needs of instruments that require low presses for operation. The inlet system itself collects a micro-sample in the external high pressure environment, then transfers it into a protected low pressure environment for analysis.

Carsey, F. D.↗

DOE OSTI2017

Refactoring the Genetic Code for Increased Evolvability

ABSTRACT The standard genetic code is robust to mutations during transcription and translation. Point mutations are likely to be synonymous or to preserve the chemical properties of the original amino acid. Saturation mutagenesis experiments suggest that in some cases the best-performing mutant requires replacement of more than a single nucleotide within a codon. These replacements are essentially inaccessible to common error-based laboratory engineering techniques that alter a single nucleotide per mutation event, due to the extreme rarity of adjacent mutations. In this theoretical study, we suggest a radical reordering of the genetic code that maximizes the mutagenic potential of single nucleotide replacements. We explore several possible genetic codes that allow a greater degree of accessibility to the mutational landscape and may result in a hyperevolvable organism that could serve as an ideal platform for directed evolution experiments. We then conclude by evaluating the challenges of constructing such recoded organisms and their potential applications within the field of synthetic biology. IMPORTANCE The conservative nature of the genetic code prevents bioengineers from efficiently accessing the full mutational landscape of a gene via common error-prone methods. Here, we present two computational approaches to generate alternative genetic codes with increased accessibility. These new codes allow mutational transitions to a larger pool of amino acids and with a greater extent of chemical differences, based on a single nucleotide replacement within the codon, thus increasing evolvability both at the single-gene and at the genome levels. Given the widespread use of these techniques for strain and protein improvement, along with more fundamental evolutionary biology questions, the use of recoded organisms that maximize evolvability should significantly improve the efficiency of directed evolution, library generation, and fitness maximization.

59 BASIC BIOLOGICAL SCIENCES↗

NASA NTRS2012

Strategies for Distinguishing Abiotic Chemistry from Martian Biochemistry in Samples Returned from Mars

A key goal in the search for evidence of extinct or extant life on Mars will be the identification of chemical biosignatures including complex organic molecules common to all life on Earth. These include amino acids, the monomer building blocks of proteins and enzymes, and nucleobases, which serve as the structural basis of information storage in DNA and RNA. However, many of these organic compounds can also be formed abiotically as demonstrated by their prevalence in carbonaceous meteorites [1]. Therefore, an important challenge in the search for evidence of life on Mars will be distinguishing between abiotic chemistry of either meteoritic or martian origin from any chemical biosignatures from an extinct or extant martian biota. Although current robotic missions to Mars, including the 2011 Mars Science Laboratory (MSL) and the planned 2018 ExoMars rovers, will have the analytical capability needed to identify these key classes of organic molecules if present [2,3], return of a diverse suite of martian samples to Earth would allow for much more intensive laboratory studies using a broad array of extraction protocols and state-of-theart analytical techniques for bulk and spatially resolved characterization, molecular detection, and isotopic and enantiomeric compositions that may be required for unambiguous confirmation of martian life. Here we will describe current state-of-the-art laboratory analytical techniques that have been used to characterize the abundance and distribution of amino acids and nucleobases in meteorites, Apollo samples, and comet- exposed materials returned by the Stardust mission with an emphasis on their molecular characteristics that can be used to distinguish abiotic chemistry from biochemistry as we know it. The study of organic compounds in carbonaceous meteorites is highly relevant to Mars sample return analysis, since exogenous organic matter should have accumulated in the martian regolith over the last several billion years and the analytical techniques previously developed for the study of extraterrestrial materials can be applied to martian samples.

Glavin, D. P.↗

NASA NTRS2015

Indigenous Aliphatic Amines in the Aqueously Altered Orgueil Meteorite

The CI1 Orgueil meteorite is a highly aqueously altered carbonaceous chondrite. Ithas been extensively studied, and despite its extensive degree of aqueous alteration andsome documented instances of contamination, several indigenous organic compoundsincluding amino acids, carboxylic acids, and nucleobases have been detected in its carbon-rich matrix. We recently developed a novel gas chromatographic method for theenantiomeric and compound-specific isotopic analyses of meteoritic aliphatic monoamines inextracts and have now applied this method to investigate the monoamine content inOrgueil. We detected 12 amines in Orgueil, with concentrations ranging from 1.1 to332 nmol g1of meteorite and compared this amine content in Orgueil with that of theCM2 Murchison meteorite, which experienced less parent-body aqueous alteration.Methylamine is four times more abundant in Orgueil than in Murchison. As with otherspecies, the amine content in Orgueil extracts shows less structural diversity than that inMurchison extracts. We measured the compound-specific stable carbon isotopic ratios(d13C) for 5 of the 12 monoamines detected in Orgueil and found a range ofd13C valuesfrom–20 to+59&. Thesed13C values fall into the range of other meteoritic organiccompounds, although they are13C-depleted relative to their counterparts extracted from theMurchison meteorite. In addition, we measured the enantiomeric composition for the chiralmonoamines (R)- and (S)-sec-butylamine in Orgueil, and found it was racemic withinexperimental error, in contrast with theL-enantiomeric excess found for its amino acidstructural analog isovaline. The racemic nature ofsec-butylamine in Orgueil wascomparable to that previously observed in Murchison, and to other CM2 and CR2carbonaceous chondrites measured in this work (ALH 83100 [CM1/2], LON 94101 [CM2],LEW 90500 [CM2], LAP 02342 [CR2], and GRA 95229 [CR2]). These results allow us toplace some constraints on the effects of aqueous alteration observed over the monoamineconcentrations in Orgueil and Murchison, and to evaluate the primordial syntheticrelationships between meteoritic monoamines and amino acids.

Jose C Aponte↗

DOE OSTI2025

A semidominant point mutation of Mediator tail subunit MED5b in Arabidopsis leads to altered enrichment of H3K27me3 and reduced expression of targets of MYC2

Abstract The Mediator complex coordinates regulatory input for transcription driven by RNA polymerase II in eukaryotes. reduced epidermal fluorescence4-3 (ref4-3) is a semidominant mutation that results in a single amino acid substitution in the Mediator tail subunit Med5b. Previous characterization of ref4-3 revealed altered expression of a variety of loci in Arabidopsis, including those contributing to phenylpropanoid biosynthesis. Examination of existing RNA-seq data indicated that loci enriched for the transcriptionally repressive chromatin modification H3K27me3 are overrepresented among genes that are misregulated in ref4-3. We used ChIP-seq and RNA-seq to examine the possibility that perturbation of H3K27me3 homeostasis in ref4-3 plants contributed to altered transcript levels. We observed that ref4-3 results in a modest global reduction of H3K27me3 at enriched loci and that this reduction is not dependent on gene expression; however, altered H3K27me3 was not strongly predictive of altered expression in ref4-3 plants. Instead, our analyses revealed a substantial enrichment of targets of the MYC2 transcriptional regulator among genes that exhibit decreased expression in ref4-3. Consistent with previous characterization of ref4-3, we observed that ref4-3-dependent decreased expression of MYC2 targets can be suppressed by loss of another Mediator tail subunit, MED25. This observation is consistent with previous biochemical characterization of MYC2. Our data highlight the diverse and distinct impacts that a single amino acid change in the tail subunit of Mediator can have on transcriptional circuits and raise the prospect that Mediator directly contributes to H3K27me3 homeostasis in plants.

Long, Jiaxin (ORCID:0000000335150706)↗

DOE OSTI2018

Asparagine synthetase gene regulatory network and plant nitrogen metabolism

This DOE project concerns the molecular mechanisms by which plants coordinate carbon (C) and nitrogen (N) metabolism. Specifically, it focuses on a histone methyltransferase protein that targets C/N-metabolism genes for chromatin modifications. How the dynamic changes of chromatin regulate energy metabolism in response to developmental and environmental cues is a new area of study. We have filled this knowledge-gap with our studies of SDG8, a histone methyltransferase, that targets light, C and N metabolism genes. We aimed to understand how plants allocate C resources to assimilate inorganic N onto C-skeletons -- the most energy-intensive pathway in plants – in order to balance energy production and consumption in a changing environment. In order to achieve more C-efficient N-storage under low energy conditions, plants convert assimilated N from “reactive” Gln (C5:N2) into “inert” Asn (C4:N2) through the enzyme ASN1. To identify regulatory factors in this pathway, we used the ASN1 promoter in a genetic selection and discovered SDG8, a histone methyltransferase. We discovered that histone modification by SDG8 coordinates the expression of genes involved in energy generation (light-harvesting and C fixation) with energy consumption (N assimilation and sulfur metabolism). We used this opportunity to explore how histone modifications control energy metabolism genome-wide. Our aims focus on various levels of the regulation of energy related genes at the level of: 1) Chromatin; 2) Transcription; and 3) Metabolites of the C/N regulatory network. Specifically, we asked: 1) How does histone methylation regulate energy metabolism during the N response? 2) How does histone methylation control transcription of the energy metabolism genes? And 3) how does the histone methyltransferase SDG8 and other TF regulators affect C/N metabolism and N use efficiency? Our study indicates that N supply triggers genome-wide changes in chromatin modification via the histone methyltransferase SDG8 which impacts on gene expression, RNA processing, chlorophyll synthesis, N uptake and metabolism. Our study thus revealed a previously overlooked layer of chromatin regulation during nutrient signaling. In broad terms, our study provides a framework to understand how chromatin modifications influence responses to environmental changes and provides unique opportunities for breeding and engineering resilient crops with better performance in the current climate model. Accomplishments AIM 1 CHROMATIN: Role of histone methylation in the ASN1 energy metabolism network. This aim focuses on understanding the role of chromatin modification in response to the environment. We studied the targeting mechanism of histone methylation by SDG8, in particular the role of the CW domain in reading histone methylation status. We also began to study the effect of nitrogen-treatment on genome-wide chromatin modifications in WT and sdg8 mutant plants. AIM 2 TRANSCRIPTION: Transcription control of the ASN1 energy metabolism network. This aim investigated whether SDG8 and the histone modification it mediates affects mRNA processing. We performed genome-wide profiling that uncovered that histone methylation by SDG8 affects the co-transcriptional RNA processing of downstream genes involved in energy and photosynthesis. AIM 3 METABOLISM: A gene regulatory network controlling energy, C/N metabolism and N use efficiency. In Aim 3A, we showed that SDG8 affects the chromatin modification and expression of a transcription factor, CCT, which in turn affects the regulation of carbon responsive genes. In Aim 3B we showed that C and N metabolism is perturbed in the sdg8 mutant through differences in the chlorophyll content, free amino acids, and N uptake. Project activities In Aim 1, we uncovered that the histone methyltransferase SDG8 affects chromatin modification in response to N treatments. The N treatments causes changes in the H3K36me3 distribution off ~200 genes in WT plants, while the N-triggered changes in H3K36me3 of these genes is abrogated in the sdg8-5 mutant. The functional annotation of these 200 genes shows significant enrichment of N-related pathways. Overall, our study suggests that SDG8 mediates chromatin-level adaption of functionally relevant genes in response to changes in N. In Aim 2, we tested if SDG8 plays a role in transcriptional elongation and/or RNA processing. We detected more N-triggered isoform shifting events in the sdg8-5 mutant than in WT, which provides supports for SDG8 being required for proper RNA processing in response to N signaling. In Aim 3, we identified the gene network controlled by a CCT transcription factor under the epigenetic control of SDG8. Isoform abundance of the CCT protein is regulated by SDG8, which leads to an altered response of energy related genes to N level changes. Using physiological, metabolic and genetic approaches, we found that responses to N, including chlorophyll accumulation, N uptake and free amino acids, are altered in sdg8 mutants. Finally, this project provided training to a high school student who performed experiments on the complex interaction of photosynthesis and N availability. The student was named a semi-finalist in both the Regeneron and Siemens National Science competitions for her work.

59 BASIC BIOLOGICAL SCIENCES↗

DOE OSTI2018

Antigenic evolution of H3N2 influenza A viruses in swine in the United States from 2012 to 2016

Background Six amino acid positions (145, 155, 156, 158, 159, and 189, referred to as the antigenic motif; H3 numbering) in the globular head region of hemagglutinin (HA1 domain) play an important role in defining the antigenic phenotype of swine Clade IV (C-IV) H3N2 IAV, containing an H3 from a late 1990s human-to-swine introduction. We hypothesized that antigenicity of a swine C-IV H3 virus could be inferred based upon the antigenic motif if it matched a previously characterized antigen with the same motif. An increasing number of C-IV H3 genes encoding antigenic motifs that had not been previously characterized were observed in the U.S. pig population between 2012 and 2016. Objectives A broad panel of contemporary H3 viruses with uncharacterized antigenic motifs was selected across multiple clades within C-IV to assess the impact of HA1 genetic diversity on the antigenic phenotype. Methods Hemagglutination inhibition (HI) assays were performed with isolates selected based on antigenic motif, tested against a panel of swine antisera, and visualized by antigenic cartography. Results A previously uncharacterized motif with low but sustained circulation in the swine population demonstrated a distinct phenotype from those previously characterized. Antigenic variation increased for viruses with similar antigenic motifs, likely due to amino acid substitutions outside the motif. Conclusions Although antigenic motifs were largely associated with antigenic distances, substantial diversity among co-circulating viruses poses a significant challenge for effective vaccine development. Continued surveillance and antigenic characterization of circulating strains is critical for improving vaccine efforts to control C-IV H3 IAV in U.S. swine.

Infectious Diseases↗

DOE OSTI2021

Spatio-temporal control of phenylpropanoid biosynthesis by inducible complementation of a cinnamate 4-hydroxylase mutant

Abstract Cinnamate 4-hydroxylase (C4H) is a cytochrome P450-dependent monooxygenase that catalyzes the second step of the general phenylpropanoid pathway. Arabidopsis reduced epidermal fluorescence 3 (ref3) mutants, which carry hypomorphic mutations in C4H, exhibit global alterations in phenylpropanoid biosynthesis and have developmental abnormalities including dwarfing. Here we report the characterization of a conditional Arabidopsis C4H line (ref3-2pOpC4H), in which wild-type C4H is expressed in the ref3-2 background. Expression of C4H in plants with well-developed primary inflorescence stems resulted in restoration of fertility and the production of substantial amounts of lignin, revealing that the developmental window for lignification is remarkably plastic. Following induction of C4H expression in ref3-2pOpC4H, we observed rapid and significant reductions in the levels of numerous metabolites, including several benzoyl and cinnamoyl esters and amino acid conjugates. These atypical conjugates were quickly replaced with their sinapoylated equivalents, suggesting that phenolic esters are subjected to substantial amounts of turnover in wild-type plants. Furthermore, using localized application of dexamethasone to ref3-2pOpC4H, we show that phenylpropanoids are not transported appreciably from their site of synthesis. Finally, we identified a defective Casparian strip diffusion barrier in the ref3-2 mutant root endodermis, which is restored by induction of C4H expression.

Plant Sciences↗

DOE OSTI2014

Crystal structure of B acillus anthracis virulence regulator AtxA and effects of phosphorylated histidines on multimerization and activity: AtxA multimerization, phosphorylation and activity

The Bacillus anthracis virulence regulator AtxA controls transcription of the anthrax toxin genes and capsule biosynthetic operon. AtxA activity is elevated during growth in media containing glucose and CO2/bicarbonate, and there is a positive correlation between the CO2/bicarbonate signal, AtxA activity and homomultimerization. AtxA activity is also affected by phosphorylation at specific histidines. We show that AtxA crystallizes as a dimer. Distinct folds associated with predicted DNA-binding domains (HTH1 and HTH2) and phosphoenolpyruvate: carbohydrate phosphotransferase system-regulated domains (PRD1 and PRD2) are apparent. We tested AtxA variants containing single and double phosphomimetic (HisAsp) and phosphoablative (HisAla) amino acid changes for activity in B.anthracis cultures and for protein-protein interactions in cell lysates. Reduced activity of AtxA H199A, lack of multimerization and activity of AtxAH379D variants, and predicted structural changes associated with phosphorylation support a model for control of AtxA function. We propose that (i) in the AtxA dimer, phosphorylation of H199 in PRD1 affects HTH2 positioning, influencing DNA-binding; and (ii) phosphorylation of H379 in PRD2 disrupts dimer formation. The AtxA structure is the first reported high-resolution full-length structure of a PRD-containing regulator, and can serve as a model for proteins of this family, especially those that link virulence to bacterial metabolism.

59 BASIC BIOLOGICAL SCIENCES↗

NASA NTRS2005

Improved Charge-Transfer Fluorescent Dyes

Improved charge-transfer fluorescent dyes have been developed for use as molecular probes. These dyes are based on benzofuran nuclei with attached phenyl groups substituted with, variously, electron donors, electron acceptors, or combinations of donors and acceptors. Optionally, these dyes could be incorporated as parts of polymer backbones or as pendant groups or attached to certain surfaces via self-assembly-based methods. These dyes exhibit high fluorescence quantum yields -- ranging from 0.2 to 0.98, depending upon solvents and chemical structures. The wavelengths, quantum yields, intensities, and lifetimes of the fluorescence emitted by these dyes vary with (and, hence, can be used as indicators of) the polarities of solvents in which they are dissolved: In solvents of increasing polarity, fluorescence spectra shift to longer wavelengths, fluorescence quantum yields decrease, and fluorescence lifetimes increase. The wavelengths, quantum yields, intensities, and lifetimes are also expected to be sensitive to viscosities and/or glass-transition temperatures. Some chemical species -- especially amines, amino acids, and metal ions -- quench the fluorescence of these dyes, with consequent reductions in intensities, quantum yields, and lifetimes. As a result, the dyes can be used to detect these species. Another useful characteristic of these dyes is a capability for both two-photon and one-photon absorption. Typically, these dyes absorb single photons in the ultraviolet region of the spectrum (wavelengths < 400 nm) and emit photons in the long-wavelength ultraviolet, visible, and, when dissolved in some solvents, near-infrared regions. In addition, these dyes can be excited by two-photon absorption at near-infrared wavelengths (600 to 800 nm) to produce fluorescence spectra identical to those obtained in response to excitation by single photons at half the corresponding wavelengths (300 to 400 nm). While many prior fluorescent dyes exhibit high quantum yields, solvent-polarity- dependent fluorescence behavior, susceptibility to quenching by certain chemical species, and/or two-photon fluorescence, none of them has the combination of all of these attributes. Because the present dyes do have all of these attributes, they have potential utility as molecular probes in a variety of applications. Examples include (1) monitoring curing and deterioration of polymers; (2) monitoring protein expression; (3) high-throughput screening of drugs; (4) monitoring such chemical species as glucose, amines, amino acids, and metal ions; and (5) photodynamic therapy of cancers and other diseases.

Meador, Michael↗

NASA NTRS1996

Effect of Altered Gravity Environment on Tobacco Hornworm (Manduca Sexta) Development

Metamorphosis provides a unique condition for studying the role of gravity in development. Formation of new organs in a previously existing organism requires a highly active period of turnover of amino acids and proteins, and of changes in the endocrine profile. Furthermore, metamorphosis offers the advantage of studying a self-contained biological system. The tobacco hornworm provides a suitable species to study the effect of altered gravitational environment on invertebrate development. This species has been one of the most thoroughly investigated organisms in a variety of aspects of insect biology. M. sexta pharate adults can provide significant amounts of material with which to work, thus facilitating the study of metabolic aspects of adult development. During wandering, the period immediately following cessation of larval feeding, the larva burrows into the soil to form a pupation chamber. Despite burrowing down 25 to 30 cm, the insects reorient themselves to a slightly head-up (10 +/- 1 degree) position. Since light and temperature are not factors in this process, the larvae must sense the gravity vector. In our ground-based studies we had assessed whether developing adults might be sensitive to their gravitational environment by orienting pupae in a vertical head-up position within 24 to 48 h after pupal ecdysis. Our ground-based findings formed the foundation for determining which parameters would be evaluated in developing Manduca following spaceflight. Measurements were to include: (1) extent of development by all of the insects, (2) analysis of hemolymph obtained from half of the insects postflight for ecdysteroid, amino acid, urea, ammonia and trehalose concentrations, (3) further development of the other half of the insects to adult (moths), (4) analysis of the flight muscle protein content of the adults. Based on the first flight attempt in July, 1995, we modified the BRIC hardware to accommodate the insects. Our studies after BRIC-04 showed that sealing the top and bottom lids of the canisters with an 0-ring and with vacuum grease caused the insects to stop developing because of accumulation of carbon dioxide. Even though removing both the O-rings and vacuum grease permitted normal development, there was still some increase in carbon dioxide levels. Consequently, the canisters were vented and the vents covered with gas permeable membrane.

Tischler, Marc E.↗

DOE OSTI2018

PlantSEED enables automated annotation and reconstruction of plant primary metabolism with improved compartmentalization and comparative consistency

Genome-scale metabolic reconstructions help us to understand and engineer metabolism. Next-generation sequencing technologies are delivering genomes and transcriptomes for an ever-widening range of plants. While such omic data can, in principle, be used to compare metabolic reconstructions in different species, organs and environmental conditions, these comparisons require a standardized framework for the reconstruction of metabolic networks from transcript data. We previously introduced PlantSEED as a framework covering primary metabolism for 10 species. We have now expanded PlantSEED to include 39 species and provide tools that enable automated annotation and metabolic reconstruction from transcriptome data. The algorithm for automated annotation in PlantSEED propagates annotations using a set of signature k-mers (short amino acid sequences characteristic of particular proteins) that identify metabolic enzymes with an accuracy of about 97%. PlantSEED reconstructions are built from a curated template that includes consistent compartmentalization for more than 100 primary metabolic subsystems. Together, the annotation and reconstruction algorithms produce reconstructions without gaps and with more accurate compartmentalization than existing resources. These tools are available via the PlantSEED web interface at , which enables users to upload, annotate and reconstruct from private transcript data and simulate metabolic activity under various conditions using flux balance analysis. We demonstrate the ability to compare these metabolic reconstructions with a case study involving growth on several nitrogen sources in roots of four species.

Flux Balance Analysis↗

DOE OSTI2016

Exploring the Mechanism of β-Lactam Ring Protonation in the Class A β-lactamase Acylation Mechanism Using Neutron and X-ray Crystallography

The catalytic mechanism of class A beta-lactamases is often debated due in part to the large number of amino acids that interact with bound beta-lactam substrates. The role and function of the conserved residue Lys 73 in the catalytic mechanism of class A type beta-lactamase enzymes is still not well understood after decades of scientific research. To better elucidate the functions of this vital residue, we used both neutron and high-resolution X-ray diffraction to examine both the structures of the ligand free protein and the acyl-enzyme complex of perdeuterated E166A Toho-1 beta-lactamase with the antibiotic cefotaxime. The E166A mutant lacks a critical glutamate residue that has a key role in the deacylation step of the catalytic mechanism, allowing the acyl-enzyme adduct to be captured for study. In our ligand free structures, Lys 73 is present in a single conformation, however in all of our acyl-enzyme structures, Lys 73 is present in two different conformations, in which one conformer is closer to Ser 70 while the other conformer is positioned closer to Ser 130, which supports the existence of a possible pathway by which proton transfer from Lys 73 to Ser 130 can occur. This and further clarifications of the role of Lys 73 in the acylation mechanism may facilitate the design of inhibitors that capitalize on the enzymes native machinery.

38 RADIATION CHEMISTRY, RADIOCHEMISTRY, AND NUCLEA↗

DOE OSTI2017

Functional Characterization of Four Putative δ1-Pyrroline-5-Carboxylate Reductases from Bacillus subtilis

In most living organisms, the amino acid proline is synthesized starting from both glutamate and ornithine. In prokaryotes, in the absence of an ornithine cyclodeaminase that has been identified to date only in a small number of soil and plant bacteria, these pathways share the last step, the reduction of δ1-pyrroline-5-carboxylate (P5C) catalyzed by P5C reductase (EC 1.5.1.2). In several species, multiple forms of P5C reductase have been reported, possibly reflecting the dual function of proline. Aside from its common role as a building block of proteins, proline is indeed also involved in the cellular response to osmotic and oxidative stress conditions. Genome analysis of Bacillus subtilis identifies the presence of four genes (ProH, ProI, ProG, and ComER) that, based on bioinformatic and phylogenic studies, were defined as respectively coding a putative P5C reductase. Here we describe the cloning, heterologous expression, functional analysis and small-angle X-ray scattering studies of the four affinity-purified proteins. Results showed that two of them, namely ProI and ComER, lost their catalytic efficiency or underwent subfunctionalization. In the case of ComER, this could be likely explained by the loss of the ability to form a dimer, which has been previously shown to be an essential structural feature of the catalytically active P5C reductase. The properties of the two active enzymes are consistent with a constitutive role for ProG, and suggest that ProH expression may be beneficial to satisfy an increased need for proline.

59 BASIC BIOLOGICAL SCIENCES↗

DOE OSTI2018

Structures of the Mycobacterium tuberculosis GlpX protein (class II fructose-1,6-bisphosphatase): implications for the active oligomeric state, catalytic mechanism and citrate inhibition

The crystal structures of native class II fructose-1,6-bisphosphatase (FBPaseII) from Mycobacterium tuberculosis at 2.6 Å resolution and two active-site protein variants are presented. The variants were complexed with the reaction product fructose 6-phosphate (F6P). The Thr84Ala mutant is inactive, while the Thr84Ser mutant has a lower catalytic activity. The structures reveal the presence of a 222 tetramer, similar to those described for fructose-1,6/sedoheptulose-1,7-bisphosphatase from Synechocystis (strain 6803) as well as the equivalent enzyme from Thermosynechococcus elongatus . This homotetramer corresponds to a homologous oligomer that is present but not described in the crystal structure of FBPaseII from Escherichia coli and is probably conserved in all FBPaseIIs. The constellation of amino-acid residues in the active site of FBPaseII from M. tuberculosis ( Mt FBPaseII) is conserved and is analogous to that described previously for the E. coli enzyme. Moreover, the structure of the active site of the partially active (Thr84Ser) variant and the analysis of the kinetics are consistent with the previously proposed catalytic mechanism. The presence of metabolites in the crystallization medium (for example citrate and malonate) and in the corresponding crystal structures of Mt FBPaseII, combined with their observed inhibitory effect, could suggest the existence of an uncharacterized inhibition of this class of enzymes besides the allosteric inhibition by adenosine monophosphate observed for the Synechocystis enzyme. The structural and functional insights derived from the structure of Mt FBPaseII will provide critical information for the design of lead inhibitors, which will be used to validate this target for future chemical intervention.

Wolf, Nina M.↗

DOE OSTI2018

Identification of the I38T PA Substitution as a Resistance Marker for Next-Generation Influenza Virus Endonuclease Inhibitors

ABSTRACT The clinical severity and annual occurrence of influenza virus epidemics, combined with the availability of just a single class of antivirals to treat infections, underscores the urgent need to develop new anti-influenza drugs. The endonuclease activity within the viral acidic polymerase (PA) protein is an attractive target for drug discovery due to the critical role it plays in viral gene transcription. RO-7 is a next-generation PA endonuclease inhibitor of influenza A and B viruses, but its drug resistance potential is unknown. Through serial passage of influenza A(H1N1) viruses in MDCK cells under selective pressure of RO-7, we identified an I38T substitution within the PA endonuclease domain that conferred in vitro resistance to RO-7 (up to a 287-fold change in 50% effective concentration [EC 50 ]). I38T emerged between 5 and 10 passages, and when introduced into recombinant influenza A(H1N1) viruses, alone conferred RO-7 resistance (up to an 81-fold change in EC 50 ). Cocrystal structures of mutant and wild-type endonuclease domains with RO-7 provided the structural basis of resistance, where a key hydrophobic interaction between RO-7 and the Ile38 side chain is compromised when mutated to the polar threonine. While Ile38 does not have a crucial role in coordinating the endonuclease active site, the switch to threonine does affect the polymerase activity of some viruses and influences RO-7 affinity for the PA N target (i.e., the ≈200-residue N-terminal domain of PA). However, the change does not lead to a complete loss of replication activity in vitro . Our results predict that RO-7-resistant influenza viruses carrying the I38T substitution may emerge under treatment. This should be taken into consideration for clinical surveillance and in refinement of these drugs. IMPORTANCE The effectiveness of antiviral drugs can be severely compromised by the emergence of resistant viruses. Therefore, determination of the mechanisms by which viruses become resistant is critical for drug development and clinical use. RO-7 is a compound that potently inhibits influenza virus replication and belongs to a new class of drugs in late-stage clinical trials for treatment of influenza virus infection. Here we demonstrate that a single amino acid change acquired under prolonged virus exposure to RO-7 renders influenza viruses significantly less susceptible to its inhibitory effects. We have discovered how the mutation can simultaneously interfere with drug activity and still maintain efficient virus replication. These findings have important implications for the development of more effective derivatives of RO-7-like drugs and provide guidance for how to monitor the emergence of resistance.

Jones, Jeremy C.↗

DOE OSTI2015

The reduced genomes of Parcubacteria (OD1) contain signatures of a symbiotic lifestyle

Candidate phylum OD1 bacteria (also referred to as Parcubacteria) have been identified in broad range of anoxic environments through community survey analysis. Although none of these species have been isolated in the laboratory, several genome sequences have been reconstructed from metagenomic sequence data and single-cell sequencing. The organisms have small (generally <1 Mb) genomes with severely reduced metabolic capabilities. We have reconstructed 8 partial to near-complete OD1 genomes from oxic groundwater samples, and compared them against existing genomic data. The conserved core gene set comprises 202 genes, or ~28% of the genomic complement. ‘Housekeeping’ genes and genes for biosynthesis of peptidoglycan and Type IV pilus production are conserved. Gene sets for biosynthesis of cofactors, amino acids, nucleotides and fatty acids are absent entirely or greatly reduced. The only aspects of energy metabolism conserved are the non-oxidative branch of the pentose-phosphate shunt and central glycolysis. These organisms also lack some activities conserved in almost all other known bacterial genomes, including signal recognition particle, pseudouridine synthase A, and FAD synthase. Pan-genome analysis indicates a broad genotypic diversity and perhaps a highly fluid gene complement, indicating historical adaptation to a wide range of growth environments and a high degree of specialization. The genomes were examined for signatures suggesting either a free-living, streamlined lifestyle or a symbiotic lifestyle. The lack of biosynthetic capabilities and DNA repair, along with the presence of potential attachment and adhesion proteins suggest the Parcubacteria are ectosymbionts or parasites of other organisms. The wide diversity of genes that potentially mediate cell-cell contact suggests a broad range of partner/prey organisms across the phylum.

candidate phyla↗