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At least 631 records · Page 35
All sourcesNASA NTRSDOE OSTI
DOE OSTI2025

Multi‐Omics Analyses Reveal Divergent Molecular Mechanisms Underlying Plant Biomass Conversion by Five Fungi

Fungal plant biomass conversion (FPBC) is of great importance to the global carbon cycle and has been increasingly applied for the production of biofuel and biochemicals from lignocellulose. However, the comprehensive understanding of relevant molecular mechanisms in different fungi remains challenging. Here, we comparatively analyzed the transcriptome, proteome and metabolome profile of four ascomycetes and one basidiomycete fungi during their growth on two common agricultural feedstocks (soybean hulls and corn stover). We revealed strong time‐, substrate‐ and species‐specific responses at multi‐omics levels for the tested fungi, highlighting species‐specific carbon utilization approaches and evolutionary adaptation to environmental niches. Notably, a remarkable expressional diversity of lignocellulose degrading enzymes, sugar transporter and metabolic genes, as well as industrially relevant metabolites were identified across different fungi and cultivation conditions. The findings improves our understanding of complex molecular networks underlying FPBC and fungal ecological roles, offering novel insights that can guide future genetic engineering of fungi for valorization of agriculture waste into value‐added bioproducts.

CAZy↗
DOE OSTI2024

Exploring biofiber properties and their influence on biocomposite tensile properties

Biofibers serve as effective reinforcements for neat polylactic acid (PLA) in biocomposites, offering an attractive opportunity to decarbonize the manufacturing sector of the United States by displacing fossil-based reinforcement fibers such as carbon fibers. Also, biofiber production can stimulate economic growth in rural economies, fueling sustainable development. PLA resins are commonly compounded with biofibers to create biocomposites suitable for additive manufacturing. PLA-biofiber composites often exhibit better overall material properties than neat (pure) PLA, but the associations between biofiber properties and the material properties of their biocomposites remain largely unexplored. Hence, this research delves into a comprehensive exploration of diverse biofibers, scrutinizing their physical and chemical attributes, including size, shape, ash content and biochemical composition. The study meticulously analyzes the flow properties of each biofiber and elucidates the ultimate tensile strengths and Young's modulus of corresponding biocomposite samples. Noteworthy correlations between biofiber and biocomposite tensile properties are uncovered, shedding light on critical interrelationships. The study introduces an approach employing regression models to predict the ultimate tensile strength and Young's modulus of biocomposites. These models, validated with a cross-validation technique, exhibit remarkable predictive accuracy, particularly in estimating ultimate tensile strength. © 2024 Oak Ridge National Laboratory managed by UT-Battelle, LLC and The Author(s). Polymer International published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.

36 MATERIALS SCIENCE↗
DOE OSTI2020

BdGT43B2 functions in xylan biosynthesis and is essential for seedling survival in Brachypodium distachyon

Abstract Xylan is the predominant hemicellulose in the primary cell walls of grasses, but its synthesis and interactions with other wall polysaccharides are complex and incompletely understood. To probe xylan biosynthesis, we generated CRISPR/Cas9 knockout and amiRNA knockdown lines of BdGT43B2 , an ortholog of the wheat TaGT43‐4 xylan synthase scaffolding protein in the IRX14 clade, in Brachypodium distachyon . Knockout of BdGT43B2 caused stunting and premature death in Brachypodium seedlings. Immunofluorescence labeling of xylans was greatly reduced in homozygous knockout BdGT43B2 mutants, whereas cellulose labeling was unchanged or slightly increased. Biochemical analysis showed reductions in digestible xylan in knockout mutant walls, and cell size was smaller in knockout leaves. BdGT43B2 knockdown plants appeared morphologically normal as adults, but showed slight reductions in seedling growth and small decreases in xylose content in isolated cell walls. Immunofluorescence labeling of xylan and cellulose staining was both reduced in BdGT43B2 knockdown plants. Together, these data indicate that BdGT43B2 functions in the synthesis of a form of xylan that is required for seedling growth and survival in Brachypodium distachyon .

36 MATERIALS SCIENCE↗
DOE OSTI2021

Catalase protects against nonenzymatic decarboxylations during photorespiration in Arabidopsis thaliana

Abstract Photorespiration recovers carbon that would be otherwise lost following the oxygenation reaction of rubisco and production of glycolate. Photorespiration is essential in plants and recycles glycolate into usable metabolic products through reactions spanning the chloroplast, mitochondrion, and peroxisome. Catalase in peroxisomes plays an important role in this process by disproportionating H 2 O 2 resulting from glycolate oxidation into O 2 and water. We hypothesize that catalase in the peroxisome also protects against nonenzymatic decarboxylations between hydrogen peroxide and photorespiratory intermediates (glyoxylate and/or hydroxypyruvate). We test this hypothesis by detailed gas exchange and biochemical analysis of Arabidopsis thaliana mutants lacking peroxisomal catalase. Our results strongly support this hypothesis, with catalase mutants showing gas exchange evidence for an increased stoichiometry of CO 2 release from photorespiration, specifically an increase in the CO 2 compensation point, a photorespiratory‐dependent decrease in the quantum efficiency of CO 2 assimilation, increase in the 12 CO 2 released in a 13 CO 2 background, and an increase in the postillumination CO 2 burst. Further metabolic evidence suggests this excess CO 2 release occurred via the nonenzymatic decarboxylation of hydroxypyruvate. Specifically, the catalase mutant showed an accumulation of photorespiratory intermediates during a transient increase in rubisco oxygenation consistent with this hypothesis. Additionally, end products of alternative hypotheses explaining this excess release were similar between wild type and catalase mutants. Furthermore, the calculated rate of hydroxypyruvate decarboxylation in catalase mutant is much higher than that of glyoxylate decarboxylation. This work provides evidence that these nonenzymatic decarboxylation reactions, predominately hydroxypyruvate decarboxylation, can occur in vivo when photorespiratory metabolism is genetically disrupted.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2024

A phosphorylation-deficient ribosomal protein eS6 is largely functional in Arabidopsis thaliana , rescuing mutant defects from global translation and gene expression to photosynthesis and growth

The eukaryote-specific ribosomal protein of the small subunit eS6 is phosphorylated through the target of rapamycin (TOR) kinase pathway. Although this phosphorylation event responds dynamically to environmental conditions and has been studied for over 50 years, its biochemical and physiological significance remains controversial and poorly understood. Here, we report data from Arabidopsis thaliana, which indicate that plants expressing only a phospho-deficient isoform of eS6 grow essentially normally under laboratory conditions. The eS6z (RPS6A) paralog of eS6 functionally rescued a double mutant in both rps6a and rps6b genes when expressed at approximately twice the wild-type dosage. A mutant isoform of eS6z lacking the major six phosphorylatable serine and threonine residues in its carboxyl-terminal tail also rescued the lethality, rosette growth, and polyribosome loading of the double mutant. This isoform also complemented many mutant phenotypes of rps6 that were newly characterized here, including photosynthetic efficiency, and most of the gene expression defects that were measured by transcriptomics and proteomics. However, compared with plants rescued with a phospho-enabled version of eS6z, the phospho-deficient seedlings retained a mild pointed-leaf phenotype, root growth was reduced, and certain cell cycle-related mRNAs and ribosome biogenesis proteins were misexpressed. The residual defects of the phospho-deficient seedlings could be understood as an incomplete rescue of the rps6 mutant defects. There was little or no evidence for gain-of-function defects. As previously published, the phospho-deficient eS6z also rescued the rps6a and rps6b single mutants; however, phosphorylation of the eS6y (RPS6B) paralog remained lower than predicted, further underscoring that plants can tolerate phospho-deficiency of eS6 well. Our data also yield new insights into how plants cope with mutations in essential, duplicated ribosomal protein isoforms.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2023

The yeast ALA synthase C‐terminus positively controls enzyme structure and function

Abstract 5‐Aminolevulinic acid synthase (ALAS) is a pyridoxal 5′‐phosphate (PLP)‐dependent enzyme that catalyzes the first and rate‐limiting step of heme biosynthesis in α‐proteobacteria and several non‐plant eukaryotes. All ALAS homologs contain a highly conserved catalytic core, but eukaryotes also have a unique C‐terminal extension that plays a role in enzyme regulation. Several mutations in this region are implicated in multiple blood disorders in humans. In Saccharomyces cerevisiae ALAS (Hem1), the C‐terminal extension wraps around the homodimer core to contact conserved ALAS motifs proximal to the opposite active site. To determine the importance of these Hem1 C‐terminal interactions, we determined the crystal structure of S. cerevisiae Hem1 lacking the terminal 14 amino acids (Hem1 ΔCT). With truncation of the C‐terminal extension, we show structurally and biochemically that multiple catalytic motifs become flexible, including an antiparallel β‐sheet important to Fold‐Type I PLP‐dependent enzymes. The changes in protein conformation result in an altered cofactor microenvironment, decreased enzyme activity and catalytic efficiency, and ablation of subunit cooperativity. These findings suggest that the eukaryotic ALAS C‐terminus has a homolog‐specific role in mediating heme biosynthesis, indicating a mechanism for autoregulation that can be exploited to allosterically modulate heme biosynthesis in different organisms.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2023

Engineering highly stable variants of Corynactis californica green fluorescent proteins

Fluorescent proteins (FPs) are versatile biomarkers that facilitate effective detection and tracking of macromolecules of interest in real time. Engineered FPs such as superfolder green fluorescent protein (sfGFP) and superfolder Cherry (sfCherry) have exceptional refolding capability capable of delivering fluorescent readout in harsh environments where most proteins lose their native functions. Our recent work on the development of a split FP from a species of strawberry anemone, Corynactis californica, delivered pairs of fragments with up to threefold faster complementation than split GFP. We present the biophysical, biochemical, and structural characteristics of five full-length variants derived from these split C. californica GFP (ccGFP). These ccGFP variants are more tolerant under chemical denaturation with up to 8 kcal/mol lower unfolding free energy than that of the sfGFP. It is likely that some of these ccGFP variants could be suitable as biomarkers under more adverse environments where sfGFP fails to survive. A structural analysis suggests explanations of the variations in stabilities among the ccGFP variants.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2024

RrA, an enzyme from Rhodospirillum rubrum , is a prototype of a new family of short‐chain L‐asparaginases

Abstract L‐Asparaginases (ASNases) catalyze the hydrolysis of L‐Asn to L‐Asp and ammonia. Members of the ASNase family are used as drugs in the treatment of leukemia, as well as in the food industry. The protomers of bacterial ASNases typically contain 300–400 amino acids (typical class 1 ASNases). In contrast, the chain of ASNase from Rhodospirillum rubrum , reported here and referred to as RrA, consists of only 172 amino acid residues. RrA is homologous to the N‐terminal domain of typical bacterial class 1 ASNases and exhibits millimolar affinity for L‐Asn. In this study, we demonstrate that RrA belongs to a unique family of cytoplasmic, short‐chain ASNases (scASNases). These proteins occupy a distinct region in the sequence space, separate from the regions typically assigned to class 1 ASNases. The scASNases are present in approximately 7% of eubacterial species, spanning diverse bacterial lineages. They seem to be significantly enriched in species that encode for more than one class 1 ASNase. Here, we report biochemical, biophysical, and structural properties of RrA, a member of scASNases family. Crystal structures of the wild‐type RrA, both with and without bound L‐Asp, as well as structures of several RrA mutants, reveal topologically unique tetramers. Moreover, the active site of one protomer is complemented by two residues (Tyr21 and Asn26) from another protomer. Upon closer inspection, these findings clearly outline scASNases as a stand‐alone subfamily of ASNases that can catalyze the hydrolysis of L‐Asn to L‐Asp despite the lack of the C‐terminal domain that is present in all ASNases described structurally to date.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2024

Design of a symmetry‐broken tetrahedral protein cage by a method of internal steric occlusion

Abstract Methods in protein design have made it possible to create large and complex, self‐assembling protein cages with diverse applications. These have largely been based on highly symmetric forms exemplified by the Platonic solids. Prospective applications of protein cages would be expanded by strategies for breaking the designed symmetry, for example, so that only one or a few (instead of many) copies of an exterior domain or motif might be displayed on their surfaces. Here we demonstrate a straightforward design approach for creating symmetry‐broken protein cages able to display singular copies of outward‐facing domains. We modify the subunit of an otherwise symmetric protein cage through fusion to a small inward‐facing domain, only one copy of which can be accommodated in the cage interior. Using biochemical methods and native mass spectrometry, we show that co‐expression of the original subunit and the modified subunit, which is further fused to an outward‐facing anti‐GFP DARPin domain, leads to self‐assembly of a protein cage presenting just one copy of the DARPin protein on its exterior. This strategy of designed occlusion provides a facile route for creating new types of protein cages with unique properties.

Gladkov, Nika↗
DOE OSTI2024

Understanding the stability of a plastic‐degrading Rieske iron oxidoreductase system

Abstract Rieske oxygenases (ROs) are a diverse metalloenzyme class with growing potential in bioconversion and synthetic applications. We postulated that ROs are nonetheless underutilized because they are unstable. Terephthalate dioxygenase (TPA DO PDB ID 7Q05 ) is a structurally characterized heterohexameric α 3 β 3 RO that, with its cognate reductase (TPA RED ), catalyzes the first intracellular step of bacterial polyethylene terephthalate plastic bioconversion. Here, we showed that the heterologously expressed TPA DO /TPA RED system exhibits only ~300 total turnovers at its optimal pH and temperature. We investigated the thermal stability of the system and the unfolding pathway of TPA DO through a combination of biochemical and biophysical approaches. The system's activity is thermally limited by a melting temperature ( T m ) of 39.9°C for the monomeric TPA RED , while the independent T m of TPA DO is 50.8°C. Differential scanning calorimetry revealed a two‐step thermal decomposition pathway for TPA DO with T m values of 47.6 and 58.0°C (Δ H = 210 and 509 kcal mol −1 , respectively) for each step. Temperature‐dependent small‐angle x‐ray scattering and dynamic light scattering both detected heat‐induced dissociation of TPA DO subunits at 53.8°C, followed by higher‐temperature loss of tertiary structure that coincided with protein aggregation. The computed enthalpies of dissociation for the monomer interfaces were most congruent with a decomposition pathway initiated by β‐β interface dissociation, a pattern predicted to be widespread in ROs. As a strategy for enhancing TPA DO stability, we propose prioritizing the re‐engineering of the β subunit interfaces, with subsequent targeted improvements of the subunits.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2025

Streptococcus pneumoniae HtrA is a dynamic and monomeric virulence factor capable of forming larger oligomeric complexes

Abstract High‐temperature requirement A (HtrA) proteases are a conserved family of serine proteases central to protein quality control and bacterial virulence. While Gram‐negative and human HtrAs are structurally well studied, Gram‐positive homologs remain essentially uncharacterized. Here, we present the first integrated structural and mechanistic analysis of a Gram‐positive HtrA, from Streptococcus pneumoniae , a virulence factor essential for adhesion and infection in vivo. Proteomic profiling of an htrA knockout and cleavage assays demonstrate that S. pneumoniae HtrA is required for protein quality control, with the PDZ domain mediating substrate recognition. Biochemically, S. pneumoniae HtrA exists exclusively as a monomer in solution, a striking divergence from canonical trimeric HtrAs that we show is shared with other Gram‐positive homologs. NMR analyses reveal that the monomer dynamically samples open and closed conformations, while cryo‐EM of a catalytic mutant identifies a hexamer stabilized by a unique LoopA–PDZ interaction. Together, these findings define S. pneumoniae HtrA as a dynamic monomer with interdomain coupling between its protease and PDZ domains, establishing Gram‐positive HtrAs as a mechanistically divergent subgroup within the HtrA family.

Lee, Eunjeong [Department of Biochemistry and Mole↗
DOE OSTI2022

Structural and functional analysis of the human cone‐rod homeobox transcription factor

Abstract The cone‐rod homeobox (CRX) protein is a critical K50 homeodomain transcription factor responsible for the differentiation and maintenance of photoreceptor neurons in the vertebrate retina. Mutant alleles in the human gene encoding CRX result in a variety of distinct blinding retinopathies, including retinitis pigmentosa, cone‐rod dystrophy, and Leber congenital amaurosis. Despite the success of using in vitro biochemistry, animal models, and genomics approaches to study this clinically relevant transcription factor over the past 25 years since its initial characterization, there are no high‐resolution structures in the published literature for the CRX protein. In this study, we use bioinformatic approaches and small‐angle X‐ray scattering (SAXS) structural analysis to further understand the biochemical complexity of the human CRX homeodomain (CRX‐HD). We find that the CRX‐HD is a compact, globular monomer in solution that can specifically bind functional cis‐regulatory elements encoded upstream of retina‐specific genes. This study presents the first structural analysis of CRX, paving the way for a new approach to studying the biochemistry of this protein and its disease‐causing mutant protein variants.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2024

Molecular Evolution of the H5 and H7 Highly Pathogenic Avian Influenza Virus Haemagglutinin Cleavage Site Motif

ABSTRACT Avian influenza viruses are ubiquitous in the Anatinae subfamily of aquatic birds and occasionally spill over to poultry. Infection with low pathogenicity avian influenza viruses generally leads to subclinical or mild clinical disease. In contrast, highly pathogenic avian influenza viruses emerge from low pathogenic forms and can cause severe disease associated with extraordinarily high mortality rates. Here, we describe the natural history of avian influenza virus, with a focus on H5Nx and H7Nx subtypes, and the emergence of highly pathogenic forms; we review the biology of AIV; we examine cleavage of haemagglutinin by host cell enzymes with a particular emphasis on the biochemical properties of the proprotein convertases, and trypsin and trypsin‐like proteases; we describe mechanisms implicated in the functional evolution of the haemagglutinin cleavage site motif that leads to emergence of HPAIVs; and finally, we discuss the diversity of H5 and H7 haemagglutinin cleavage site sequence motifs. It is crucial to understand the molecular attributes that drive the emergence and evolution of HPAIVs with pandemic potential to inform risk assessments and mitigate the threat of HPAIVs to poultry and human populations.

Luczo, Jasmina M. [Australian Animal Health Labora↗