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At least 1,081 records · Page 60

Two novel fish paralogs provide insights into the Rid family of imine deaminases active in pre-empting enamine/imine metabolic damage

Reactive Intermediate Deaminase (Rid) protein superfamily includes eight families among which the RidA is conserved in all domains of life. RidA proteins accelerate the deamination of the reactive 2-aminoacrylate (2AA), an enamine produced by some pyridoxal phosphate (PLP)-dependent enzymes. 2AA accumulation inhibits target enzymes with a detrimental impact on fitness. As a consequence of whole genome duplication, teleost fish have two ridA paralogs, while other extant vertebrates contain a single-copy gene. We investigated the biochemical properties of the products of two paralogs, identified in Salmo salar. Ss RidA-1 and Ss RidA-2 complemented the growth defect of a Salmonella enterica ridA mutant, an in vivo model of 2AA stress. In vitro, both proteins hydrolyzed 2-imino acids (IA) to keto-acids and ammonia. Ss RidA-1 was active on IA derived from nonpolar amino acids and poorly active or inactive on IA derived from other amino acids tested. In contrast, SsRidA-2 had a generally low catalytic efficiency, but showed a relatively higher activity with IA derived from L-Glu and aromatic amino acids. The crystal structures of Ss RidA-1 and Ss RidA-2 provided hints of the remarkably different conformational stability and substrate specificity. Overall, Ss RidA-1 is similar to the mammalian orthologs whereas SsRidA-2 displays unique properties likely generated by functional specialization of a duplicated ancestral gene.

59 BASIC BIOLOGICAL SCIENCES↗

Proteolytic dissection of Zab, the Z-DNA-binding domain of human ADAR1

Zalpha is a peptide motif that binds to Z-DNA with high affinity. This motif binds to alternating dC-dG sequences stabilized in the Z-conformation by means of bromination or supercoiling, but not to B-DNA. Zalpha is part of the N-terminal region of double-stranded RNA adenosine deaminase (ADAR1), a candidate enzyme for nuclear pre-mRNA editing in mammals. Zalpha is conserved in ADAR1 from many species; in each case, there is a second similar motif, Zbeta, separated from Zalpha by a more divergent linker. To investigate the structure-function relationship of Zalpha, its domain structure was studied by limited proteolysis. Proteolytic profiles indicated that Zalpha is part of a domain, Zab, of 229 amino acids (residues 133-361 in human ADAR1). This domain contains both Zalpha and Zbeta as well as a tandem repeat of a 49-amino acid linker module. Prolonged proteolysis revealed a minimal core domain of 77 amino acids (positions 133-209), containing only Zalpha, which is sufficient to bind left-handed Z-DNA; however, the substrate binding is strikingly different from that of Zab. The second motif, Zbeta, retains its structural integrity only in the context of Zab and does not bind Z-DNA as a separate entity. These results suggest that Zalpha and Zbeta act as a single bipartite domain. In the presence of substrate DNA, Zab becomes more resistant to proteases, suggesting that it adopts a more rigid structure when bound to its substrate, possibly with conformational changes in parts of the protein.

Non-NASA Center↗

Effects of hemorrhagic hypotension on tyrosine concentrations in rat spinal cord and plasma

Tyrosine is the precursor for catecholamine neurotransmitters. When catecholamine-containing neurons are physiologically active (as sympathoadrenal cells are in hypotension), tyrosine administration increases catecholamine synthesis and release. Since hypotension can alter plasma amino acid composition, the effects of an acute hypotensive insult on tyrosine concentrations in plasma and spinal cord were examined. Rats were cannulated and bled until the systolic blood pressure was 50 mmHg, or were kept normotensive for 1 h. Tyrosine and other large neutral amino acids (LNAA) known to compete with tyrosine for brain uptake were assayed in plasma and spinal cord. The rate at which intra-arterial (H-3)tyrosine disappeared from the plasma was also estimated in hemorrhaged and control rats. In plasma of hemorrhaged animals, both the tyrosine concentration and the tyrosine/LNAA ratio was elevated; moreover, the disappearance of (H-3)tyrosine was slowed. Tyrosine concentrations also increased in spinal cords of hemorrhaged-hypotensive rats when compared to normotensive controls. Changes in plasma amino acid patterns may thus influence spinal cord concentrations of amino acid precursors for neurotransmitters during the stress of hemorrhagic shock.

Conlay, L. A.↗

Amino acidis derived from Titan tholins

The production of amino acids by acid treatment of Titan tholin is experimentally investigated. The synthesis of Titan tholin and the derivatization of amino acids to N-trifluoroacetyl isopropyl esters are described. The gas chromatography/mass spectroscopy analysis of the Titan tholins reveals the presence of glycine, alpha and beta alainine, and aspartic acid, and the total yield of amino acids is about 0.01.

Khare, Bishun N.↗

Formation, Survival and Evolution of Organics After Impact Processing of Asteroidal and Cometary Simulants

This work examines the formation and evolution of amino acids during impact events on the prebiotic Earth Laboratory experiments, which simulate impact processes, provide direct tests of questions regarding the conditions (concentration of amino acid, shock stress or impact velocity, matrix material) under which amino acid synthesis and polymerization occur.

exobiology↗

Developing a pipeline to expand the genetic code of diverse bacteria for microbial engineering

Microbial biotechnologies are key to addressing grand challenges to promote human health, reverse carbon emissions, recycle mixed plastic waste, remediate contaminated soils, and achieve sustainable economies. Synthetic biology has enabled design of diverse microbes and their proteins for useful purposes, but the narrowness of the natural genetic code limits functional diversity (e.g., biosynthesis) of engineered microbes. The natural genetic code defines the fundamental rules of translating genetic information into proteins comprised of 22 ‘canonical’ amino acids. However, using a technique called genetic code expansion (GCE), the chemical properties and therefore functions of proteins can be transformed by incorporation of one or more of ~200 chemically diverse ‘non-canonical’ amino acids. The effective application of genetic code expansion in diverse microbes has the potential to revolutionize biotechnology. However, despite over 50 years of research and its transformative potential, the application of genetic code expansion has been limited to a handful of bacterial species. In this project, we will perform three tasks to both overcome the barriers that prevent wide spread adoption of GCE as molecular tool and demonstrate its potential for biotechnological applications. Specifically, we will (1) develop a genetic engineering methodology that will enable use of GCE in a broad range of bacterial hosts, (2) use high-throughput functional genomics methods to identify physiological responses to both genetic code expansion and exposure to non-canonical amino acids in three different bacteria, and (3) demonstrate an application of GCE by selectively incorporate non-canonical amino acids into surface displayed peptides such as those used for biomining.

59 BASIC BIOLOGICAL SCIENCES↗

Thermal adaptation analyzed by comparison of protein sequences from mesophilic and extremely thermophilic Methanococcus species

The genome sequence of the extremely thermophilic archaeon Methanococcus jannaschii provides a wealth of data on proteins from a thermophile. In this paper, sequences of 115 proteins from M. jannaschii are compared with their homologs from mesophilic Methanococcus species. Although the growth temperatures of the mesophiles are about 50 degrees C below that of M. jannaschii, their genomic G+C contents are nearly identical. The properties most correlated with the proteins of the thermophile include higher residue volume, higher residue hydrophobicity, more charged amino acids (especially Glu, Arg, and Lys), and fewer uncharged polar residues (Ser, Thr, Asn, and Gln). These are recurring themes, with all trends applying to 83-92% of the proteins for which complete sequences were available. Nearly all of the amino acid replacements most significantly correlated with the temperature change are the same relatively conservative changes observed in all proteins, but in the case of the mesophile/thermophile comparison there is a directional bias. We identify 26 specific pairs of amino acids with a statistically significant (P < 0.01) preferred direction of replacement.

NASA Discipline Exobiology↗

Identification of the Tyrosine- and Phenylalanine-Derived Soluble Metabolomes of Sorghum

The synthesis of small organic molecules, known as specialized or secondary metabolites, is one mechanism by which plants resist and tolerate biotic and abiotic stress. Many specialized metabolites are derived from the aromatic amino acids phenylalanine (Phe) and tyrosine (Tyr). In addition, the improved characterization of compounds derived from these amino acids could inform strategies for developing crops with greater resilience and improved traits for the biorefinery. Sorghum and other grasses possess phenylalanine ammonia-lyase (PAL) enzymes that generate cinnamic acid from Phe and bifunctional phenylalanine/tyrosine ammonia-lyase (PTAL) enzymes that generate cinnamic acid and p -coumaric acid from Phe and Tyr, respectively. Cinnamic acid can, in turn, be converted into p -coumaric acid by cinnamate 4-hydroxylase. Thus, Phe and Tyr are both precursors of common downstream products. Not all derivatives of Phe and Tyr are shared, however, and each can act as a precursor for unique metabolites. In this study, 13 C isotopic-labeled precursors and the recently developed Precursor of Origin Determination in Untargeted Metabolomics (PODIUM) mass spectrometry (MS) analytical pipeline were used to identify over 600 MS features derived from Phe and Tyr in sorghum. These features comprised 20% of the MS signal collected by reverse-phase chromatography and detected through negative-ionization. Ninety percent of the labeled mass features were derived from both Phe and Tyr, although the proportional contribution of each precursor varied. In addition, the relative incorporation of Phe and Tyr varied between metabolites and tissues, suggesting the existence of multiple pools of p -coumaric acid that are fed by the two amino acids. Furthermore, Phe incorporation was greater for many known hydroxycinnamate esters and flavonoid glycosides. In contrast, mass features derived exclusively from Tyr were the most abundant in every tissue. The Phe- and Tyr-derived metabolite library was also utilized to retrospectively annotate soluble MS features in two brown midrib mutants ( bmr6 and bmr12 ) identifying several MS features that change significantly in each mutant.

09 BIOMASS FUELS↗

Molecular cloning and characterization of a novel isoform of the non-canonical poly(A) polymerase PAPD7

Highlights: ► So far, only an enzymatically inactive isoform of PAPD7 was reported. ► The novel isoform: PAPD7 l shows robust nucleotidyl transferase activity. ► The newly identified amino terminal region is required for the activity. ► PAPD7 l localizes to the nucleoplasm. ► The N terminal region identified is also required for the nuclear localization. - Abstract: Non-canonical poly(A) polymerases (ncPAPs) catalyze the addition of poly(A) tail to the 3′ end of RNA to play pivotal roles in the regulation of gene expression and also in quality control. Here we identified a novel isoform of the 7th member of ncPAPs: PAPD7 (PAPD7 l), which contains 230 extra amino acids at the amino terminus of the previously identified PAPD7 (PAPD7 s). In sharp contrast to the inactive PAPD7 s, PAPD7 l showed robust nucleotidyl transferase activity when tethered to an RNA. A region required for the activity was localized to 187–219 aa, and this region was also required for the nuclear retention of PAPD7 l. Western blot analysis revealed that 94 kDa band (corresponding to PAPD7 l) but not 62 kDa band (corresponding to PAPD7 s) detected by PAPD7 antibody was specifically depleted by treatment with PAPD7 siRNA in both HeLa and U2OS cells. These results suggest that PAPD7 l is the major and active isoform of PAPD7 expressed in cells.

60 APPLIED LIFE SCIENCES↗

Aliphatic amines in the Murchison meteorite

The paper reports on the determination of aliphatic amines in water extracts of the Murchison meteorite. The amines were analyzed by gas chromatography both as the free amines and as 2,4-dinitrophenyl (DNP) derivatives. The results give evidence for the presence of all of the possible primary aliphatic monoamines (eight) with fewer than five carbon atoms. Two of the seven possible secondary or tertiary aliphatic monoamines were identified. The identified primary amines total 80 nmol per g meteorite, and seem to be chemically or physically trapped in the meteorite. Similarities between the water-extractable amines and amino acids suggest that (1) a simple carbon compound, methane, for example, is the precursor of meteorite amines and amino acids, and (2) both amines and amino acids are extracted from the meteorite both as such and in the form of acid-hydrolyzable derivative or precursor species.

Jungclaus, G.↗

Adsorption of modified Arg, Lys, Asp, and GIn to dry and hydrated ZnO surface: A density functional theory study

The interface of biological molecules with inorganic surfaces has been the subject of several recent studies. Experimentally some amino acids are evidenced to play a critical role in the adhesion and selectivity on oxide surfaces; however, detailed information on how the water molecules on the hydrated surface are able to mediate the adsorption is still missing. Accurate total energy ab initio calculations based on dispersion-corrected density functional theory have been performed to investigate the adsorption of selected amino acids on the hydrated ZnO(101¯0) surface, and the results are presented and discussed in this paper. We have also investigated the role played by water in the determination of the most energetically favorable adsorption configurations of the selected amino acids. As a result, we have found that for some amino acids the most energetically favorable configurations involve the deprotonation of the molecule if the water screening is not effective.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗

The Origin and Evolution of Organic Matter in Carbonaceous Chondrites and Links to Their Parent Bodies

The nature of organic matter in meteorites reveals information about early solar system chemistry and the histories of parent bodies as recorded in the effects of physical and chemical processes that occurred over the past 4.5 billion years. Asteroids and their fragments impact the Earth with approximately 40 million kg of material each year and contributed to the inventory of organics available for the origin of life. Analyses of primitive carbonaceous chondrites over the last five decades have revealed a major insoluble organic component, as well as a complex and highly diverse suite of soluble organic molecules that includes aliphatic and aromatic hydrocarbons, carboxylic acids, hydroxy acids, N-heterocycles, polyols, amino acids, amines, and many other molecules that have not yet been identified. Thermal and aqueous alteration in primitive asteroids played an important role in the formation and destruction organics, including amplification of L-amino acid and D-sugar acid enantiomeric excesses that may have contributed to the origin of homochirality in life on Earth.

Chondrites↗

Deletion of Type I glutamine synthetase deregulates nitrogen metabolism and increases ethanol production in Clostridium thermocellum

Clostridium thermocellum rapidly deconstructs cellulose and ferments resulting hydrolysis products into ethanol and other products, and is thus a promising platform organism for the development of cellulosic biofuel production via consolidated bioprocessing. And while recent metabolic engineering strategies have targeted eliminating canonical fermentation products (acetate, lactate, formate, and H 2 ), C. thermocellum also secretes amino acids, which has limited ethanol yields in engineered strains to approximately 70% of the theoretical maximum. To decrease amino acid secretion, we attempted to reduce ammonium assimilation by deleting the Type I glutamine synthetase (glnA) in C. thermocellum. Deletion of glnA reduced levels of secreted valine and total amino acids by 53% and 44% respectively, and increased ethanol yields by 53%. RNA-seq analysis revealed that genes encoding the RNF-complex were more highly expressed in ΔglnA and may have a role in improving NADH-availability for ethanol production. While a significant up-regulation of genes involved in nitrogen assimilation and urea uptake suggested that deletion of glnA induces a nitrogen starvation response, metabolomic analysis showed an increase in intracellular glutamine and α-ketoglutarate levels indicative of nitrogen-rich conditions. Here, we propose that deletion of glnA causes deregulation of nitrogen metabolism, leading to overexpression of nitrogen metabolism genes and, in turn, elevated glutamine/α-ketoglutarate levels. Here we demonstrate that perturbation of nitrogen assimilation is a promising strategy to redirect flux from the production of nitrogenous compounds toward biofuels in C. thermocellum.

59 BASIC BIOLOGICAL SCIENCES↗

Genomics, Exometabolomics, and Metabolic Probing Reveal Conserved Proteolytic Metabolism of Thermoflexus hugenholtzii and Three Candidate Species From China and Japan

Thermoflexus hugenholtzii JAD2 T , the only cultured representative of the Chloroflexota order Thermoflexales, is abundant in Great Boiling Spring (GBS), NV, United States, and close relatives inhabit geothermal systems globally. However, no defined medium exists for T. hugenholtzii JAD2 T and no single carbon source is known to support its growth, leaving key knowledge gaps in its metabolism and nutritional needs. Here, we report comparative genomic analysis of the draft genome of T. hugenholtzii JAD2 T and eight closely related metagenome-assembled genomes (MAGs) from geothermal sites in China, Japan, and the United States, representing “Candidatus Thermoflexus japonica,” “Candidatus Thermoflexus tengchongensis,” and “Candidatus Thermoflexus sinensis.” Genomics was integrated with targeted exometabolomics and 13 C metabolic probing of T. hugenholtzii. The Thermoflexus genomes each code for complete central carbon metabolic pathways and an unusually high abundance and diversity of peptidases, particularly Metallo- and Serine peptidase families, along with ABC transporters for peptides and some amino acids. The T. hugenholtzii JAD2 T exometabolome provided evidence of extracellular proteolytic activity based on the accumulation of free amino acids. However, several neutral and polar amino acids appear not to be utilized, based on their accumulation in the medium and the lack of annotated transporters. Adenine and adenosine were scavenged, and thymine and nicotinic acid were released, suggesting interdependency with other organisms in situ. Metabolic probing of T. hugenholtzii JAD2 T using 13 C-labeled compounds provided evidence of oxidation of glucose, pyruvate, cysteine, and citrate, and functioning glycolytic, tricarboxylic acid (TCA), and oxidative pentose-phosphate pathways (PPPs). However, differential use of position-specific 13 C-labeled compounds showed that glycolysis and the TCA cycle were uncoupled. Thus, despite the high abundance of Thermoflexus in sediments of some geothermal systems, they appear to be highly focused on chemoorganotrophy, particularly protein degradation, and may interact extensively with other microorganisms in situ.

59 BASIC BIOLOGICAL SCIENCES↗

A Peptide-Based Method for 13C Metabolic Flux Analysis in Microbial Communities

The study of intracellular metabolic fluxes and inter-species metabolite exchange for microbial communities is of crucial importance to understand and predict their behaviour. The most authoritative method of measuring intracellular fluxes, 13C Metabolic Flux Analysis (13C MFA), uses the labeling pattern obtained from metabolites (typically amino acids) during 13C labeling experiments to derive intracellular fluxes. However, these metabolite labeling patterns cannot easily be obtained for each of the members of the community. Here we propose a new type of 13C MFA that infers fluxes based on peptide labeling, instead of amino acid labeling. The advantage of this method resides in the fact that the peptide sequence can be used to identify the microbial species it originates from and, simultaneously, the peptide labeling can be used to infer intracellular metabolic fluxes. Peptide identity and labeling patterns can be obtained in a high-throughput manner from modern proteomics techniques. We show that, using this method, it is theoretically possible to recover intracellular metabolic fluxes in the same way as through the standard amino acid based 13C MFA, and quantify the amount of information lost as a consequence of using peptides instead of amino acids. We show that by using a relatively small number of peptides we can counter this information loss. We computationally tested this method with a well-characterized simple microbial community consisting of two species.

59 BASIC BIOLOGICAL SCIENCES↗

Unusual Metabolism and Hypervariation in the Genome of a Gracilibacterium (BD1-5) from an Oil-Degrading Community

The candidate phyla radiation (CPR) comprises a large monophyletic group of bacterial lineages known almost exclusively based on genomes obtained using cultivation-independent methods. Within the CPR, Gracilibacteria (BD1-5) are particularly poorly understood due to undersampling and the inherent fragmented nature of available genomes. Here, we report the first closed, curated genome of a gracilibacterium from an enrichment experiment inoculated from the Gulf of Mexico and designed to investigate hydrocarbon degradation. The gracilibacterium rose in abundance after the community switched to dominance by Colwellia . Notably, we predict that this gracilibacterium completely lacks glycolysis, the pentose phosphate and Entner-Doudoroff pathways. It appears to acquire pyruvate, acetyl coenzyme A (acetyl-CoA), and oxaloacetate via degradation of externally derived citrate, malate, and amino acids and may use compound interconversion and oxidoreductases to generate and recycle reductive power. The initial genome assembly was fragmented in an unusual gene that is hypervariable within a repeat region. Such extreme local variation is rare but characteristic of genes that confer traits under pressure to diversify within a population. Notably, the four major repeated 9-mer nucleotide sequences all generate a proline-threonine-aspartic acid (PTD) repeat. The genome of an abundant Colwellia psychrerythraea population has a large extracellular protein that also contains the repeated PTD motif. Although we do not know the host for the BD1-5 cell, the high relative abundance of the C. psychrerythraea population and the shared surface protein repeat may indicate an association between these bacteria. CPR bacteria are generally predicted to be symbionts due to their extensive biosynthetic deficits. Although monophyletic, they are not monolithic in terms of their lifestyles. The organism described here appears to have evolved an unusual metabolic platform not reliant on glucose or pentose sugars. Its biology appears to be centered around bacterial host-derived compounds and/or cell detritus. Amino acids likely provide building blocks for nucleic acids, peptidoglycan, and protein synthesis. We resolved an unusual repeat region that would be invisible without genome curation. The nucleotide sequence is apparently under strong diversifying selection, but the amino acid sequence is under stabilizing selection. Finally, the amino acid repeat also occurs in a surface protein of a coexisting bacterium, suggesting colocation and possibly interdependence.

59 BASIC BIOLOGICAL SCIENCES↗

Persistent fibrosis in the liver of choline-deficient and iron-supplemented L-amino acid-defined diet-induced nonalcoholic steatohepatitis rat due to continuing oxidative stress after choline supplementation

Nonalcoholic steatohepatitis (NASH) is characterized by combined pathology of steatosis, lobular inflammation, fibrosis, and hepatocellular degeneration, with systemic symptoms of diabetes or hyperlipidemia, all in the absence of alcohol abuse. Given the therapeutic importance and conflicting findings regarding the potential for healing the histopathologic features of NASH in humans, particularly fibrosis, we investigated the reversibility of NASH-related findings in Wistar rats fed a choline-deficient and iron-supplemented L-amino acid-defined (CDAA) diet for 12 weeks, with a recovery period of 7 weeks, during which the diets were switched to a choline-sufficient and iron-supplemented L-amino acid-defined (CSAA) one. Analysis showed that steatosis and inflammation were significantly resolved by the end of the recovery period, along with decreases in AST and ALT activities within 4 weeks. In contrast, fibrosis remained even after the recovery period, to an extent similar to that in continuously CDAA-fed animals. Real-time reverse transcriptase-polymerase chain reaction, Western blot, and immunohistochemical investigations revealed that expression of some factors indicating oxidative stress (CYP2E1, 4-HNE, and iNOS) were elevated, whereas catalase and SOD1 were decreased, and a hypoxic state and CD34-positive neovascularization were evident even after the recovery period, although the fibrogenesis pathway by activated α-SMA-positive hepatic stellate cells via TGF-β and TIMPs decreased to the CSAA group level. In conclusion, persistent fibrosis was noted after the recovery period of 7 weeks, possibly due to sustained hypoxia and oxidative stress supposedly caused by capillarization. Otherwise, histopathological features of steatosis and inflammation, as well as serum AST and ALT activities, were recovered. - Highlights: ► NASH-like liver lesions are induced in rats by feeding a CDAA diet. ► Steatosis and lobular inflammation are resolved after switching to a CSAA diet. ► Fibrosis is sustained, possibly due to continuing hypoxia and oxidative stress.

60 APPLIED LIFE SCIENCES↗

AdenPredictor: accurate prediction of the adenylation domain specificity of nonribosomal peptide biosynthetic gene clusters in microbial genomes

Microbial natural products represent a major source of bioactive compounds for drug discovery. Among these molecules, nonribosomal peptides (NRPs) represent a diverse class that include antibiotics, immunosuppressants, anticancer agents, toxins, siderophores, pigments, and cytostatics. The discovery of novel NRPs remains a laborious process because many NRPs consist of nonstandard amino acids that are assembled by nonribosomal peptide synthetases (NRPSs). Adenylation domains (A-domains) in NRPSs are responsible for selection and activation of monomers appearing in NRPs. During the past decade, several support vector machine-based algorithms have been developed for predicting the specificity of the monomers present in NRPs. These algorithms utilize physiochemical features of the amino acids present in the A-domains of NRPSs. In this article, we benchmarked the performance of various machine learning algorithms and features for predicting specificities of NRPSs and we showed that the extra trees model paired with one-hot encoding features outperforms the existing approaches. Moreover, we show that unsupervised clustering of 453560 A-domains reveals many clusters that correspond to potentially novel amino acids. While it is challenging to predict the chemical structure of these amino acids, we developed novel techniques to predict their various properties, including polarity, hydrophobicity, charge, and presence of aromatic rings, carboxyl, and hydroxyl groups.

59 BASIC BIOLOGICAL SCIENCES↗