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Search indexed NASA NTRS and DOE OSTI research on propulsion, heat transfer, battery materials and energy systems. Follow report and document links to the original sources.

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At least 19 records
All sourcesNASA NTRSDOE OSTI
DOE OSTI2025

A substrate-multiplexed platform for profiling enzymatic potential of plant family 1 glycosyltransferases

Plants have expanded various biosynthetic enzyme families to produce a wide diversity of natural products; however, most enzymes encoded in plant genomes remain uncharacterized, highlighting the need for new functional genomic approaches. Here, we report a platform enabling the rapid functional characterization of plant family 1 glycosyltransferases, which serve important roles in plant development, defense, and communication. Using substrate-multiplexed reactions, mass spectrometry, and automated analysis, we screen 85 enzymes against a diverse library of 453 natural products, for a total of nearly 40,000 possible reactions. The resulting dataset reveals a widespread promiscuity and a strong preference for planar, hydroxylated aromatic substrates among family 1 glycosyltransferases. We also characterize glycosyltransferases with an unusually wide substrate scope and with a non-canonical Cys-Asp catalytic dyad. This work establishes a widely-applicable enzymatic screening pipeline, reflects the immense glycosylation capability of plants, and has implications in biocatalysis, metabolic engineering, and gene discovery.

Sirirungruang, Sasilada↗
DOE OSTI2026

Functional characterization of glycosyltransferases in duckweed to enable predictive biology

Glycosyltransferases (GTs) catalyze the formation of glycosidic linkages to produce almost all complex carbohydrates. This project used a multi-disciplinary, high-throughput (HTP) biochemical and computational biology approach focused on duckweed as a model energy crop, to study carbohydrate metabolic processes. To achieve this, developed and carried out out high-throughput (HTP) functional characterization of plant glycosyltransferases (GTs) role of enzymatic microenvironments be assessed through a combined proteomic and computational biology approach, and the combined data was used to populate deep-learning frameworks to predict plant GT function. Functional validation achieved through this research is being used to assign gene function and study plant processes at the systems level to efficiently link the genome sequence with gene function. Together, the combined approaches used within this study provide a foundation for how computational prediction, in combination with high-throughput functional validation, can be used to study plant processes at the systems level and translate knowledge gained to efficiently link genome sequence with gene function in a species agnostic manner.

09 BIOMASS FUELS↗
DOE OSTI2024

Structural and biochemical basis for regiospecificity of the flavonoid glycosyltransferase UGT95A1

Glycosylation is a predominant strategy plants use to fine-tune the properties of small molecule metabolites to affect their bioactivity, transport, and storage. It is also important in biotechnology and medicine as many glycosides are utilized in human health. Small molecule glycosylation is largely carried out by family 1 glycosyltransferases. Here, we report a structural and biochemical investigation of UGT95A1, a family 1 GT enzyme from Pilosella officinarum that exhibits a strong, unusual regiospecificity for the 3'-O position of flavonoid acceptor substrate luteolin. We obtained an apo crystal structure to help drive the analyses of a series of binding site mutants, revealing that while most residues are tolerant to mutations, key residues M145 and D464 are important for overall glycosylation activity. Interestingly, E347 is crucial for maintaining the strong preference for 3'-O glycosylation, while R462 can be mutated to increase regioselectivity. The structural determinants of regioselectivity were further confirmed in homologous enzymes. Our study also suggests that the enzyme contains large, highly dynamic, disordered regions. We showed that while most disordered regions of the protein have little to no implication in catalysis, the disordered regions conserved among investigated homologs are important to both the overall efficiency and regiospecificity of the enzyme. This report represents a comprehensive in-depth analysis of a family 1 GT enzyme with a unique substrate regiospecificity and may provide a basis for enzyme functional prediction and engineering.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗
DOE OSTI2025

Decoding substrate specificity determining factors in glycosyltransferase-B enzymes – insights from machine learning models

Substrate specificity is an essential characteristic of any enzyme's function and an understanding of the factors that determine this specificity is crucial for enzyme engineering. Unlike the structure of an enzyme which is directly impacted by its sequence, substrate specificity as an enzyme attribute involves a rather indirect relationship with sequence as it also depends on structural aspects that dictate substrate accessibility and active site dynamics. In this study, we explore the performance of classifier-based machine learning models trained on curated sequence and structural data for a class of glycosyltransferases (GTs), namely GT-Bs, to understand their substrate specificity determining factors. GTs enable the transfer of sugar moieties to other biomolecules such as oligosaccharides or proteins and are found in all kingdoms of life. In plants, GTs participate in the biosynthesis of plant cell wall biopolymers (e.g.: hemicelluloses and pectins) and are an integral part of the enzymatic machinery that enables the storage of carbon and energy as plant biomass. To elucidate the substrate specificity of uncharacterized GT-Bs, we constructed multi-label machine learning models (Support Vector Classifier, K-Nearest Neighbors, Gaussian Naïve-Bayes, Random Forest) that incorporate both sequence and structural features. These models achieve good predictive accuracies on test datasets. However, despite our use of structural information, we highlight that there is further scope for improvement in training these models to draw interpretable relationships between sequence, structure and substrate specificity determining motifs in GT-Bs.

97 MATHEMATICS AND COMPUTING↗
DOE OSTI2024

Putative rhamnogalacturonan-II glycosyltransferase identified through callus gene editing bypasses embryo lethality

Rhamnogalacturonan II (RG-II) is a structurally complex and conserved domain of the pectin present in the primary cell walls of vascular plants. Borate crosslinking of RG-II is required for plants to grow and develop normally. Mutations that alter RG-II structure also affect crosslinking and are lethal or severely impair growth. Thus, few genes involved in RG-II synthesis have been identified. Here we developed a method to generate viable loss-of-function Arabidopsis (Arabidopsis thaliana) mutants in callus tissue via CRISPR/Cas9-mediated gene editing. We combined this with a candidate gene approach to characterize the male gametophyte defective 2 (MPG2) gene that encodes a putative family GT29 glycosyltransferase. Plants homozygous for this mutation do not survive. We showed that in the callus mutant cell walls, RG-II does not crosslink normally because it lacks 3-deoxy-D-manno-octulosonic acid (Kdo) and thus cannot form the α-L-Rhap-(1→5)-α-D-kdop-(1→ sidechain. We suggest that MGP2 encodes an inverting RG-II CMP-β-Kdo transferase (RCKT1). Our discovery provides further insight into the role of sidechains in RG-II dimerization. Our method also provides a viable strategy for further identifying proteins involved in the biosynthesis of RG-II.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2024

Identification of glycosyltransferases mediating 2‐ O ‐arabinopyranosyl and 2‐ O ‐galactosyl substitutions of glucuronosyl side chains of xylan

SUMMARY Xylan is one of the major hemicelluloses in plant cell walls and its xylosyl backbone is often decorated at O ‐2 with glucuronic acid (GlcA) and/or methylglucuronic acid (MeGlcA) residues. The GlcA/MeGlcA side chains may be further substituted with 2‐ O ‐arabinopyranose (Ara p ) or 2‐ O ‐galactopyranose (Gal) residues in some plant species, but the enzymes responsible for these substitutions remain unknown. During our endeavor to investigate the enzymatic activities of Arabidopsis MUR3‐clade members of the GT47 glycosyltransferase family, we found that one of them was able to transfer Ara p from UDP‐Ara p onto O ‐2 of GlcA side chains of xylan, and thus it was named xylan 2‐ O ‐arabinopyranosyltransferase 1 (AtXAPT1). The function of AtXAPT1 was verified in planta by its T‐DNA knockout mutation showing a loss of the Ara p substitution on xylan GlcA side chains. Further biochemical characterization of XAPT close homologs from other plant species demonstrated that while the poplar ones had the same catalytic activity as AtXAPT1, those from Eucalyptus , lemon‐scented gum, sea apple, 'Ohi'a lehua, duckweed and purple yam were capable of catalyzing both 2‐ O ‐Ara p and 2‐ O ‐Gal substitutions of xylan GlcA side chains albeit with differential activities. Sequential reactions with XAPTs and glucuronoxylan methyltransferase 3 (GXM3) showed that XAPTs acted poorly on MeGlcA side chains, whereas GXM3 could efficiently methylate arabinosylated or galactosylated GlcA side chains of xylan. Furthermore, molecular docking and site‐directed mutagenesis analyses of Eucalyptus XAPT1 revealed critical roles of several amino acid residues at the putative active site in its activity. Together, these findings establish that XAPTs residing in the MUR3 clade of family GT47 are responsible for 2‐ O ‐arabinopyranosylation and 2‐ O ‐galactosylation of GlcA side chains of xylan.

Zhong, Ruiqin↗
DOE OSTI2026

A Maize GT14 Family Glycosyltransferase Affects Cell Wall Composition and Carbohydrate Export from Source Leaves

Sucrose translocation from photosynthetic leaves to distant parts of a plant, such as seeds and roots, is a critical aspect of plant growth and development and a major determinant of crop yield. To identify genes contributing to this process in maize (Zea mays), we isolated four allelic mutants, carbohydrate partitioning defective7, 48, 49 (cpd7, cpd48, cpd49) and a UniformMu insertion (mu1049954), all of which exhibited reduced growth and fertility and hyperaccumulation of starch and soluble sugars in mature leaves. Consistent with carbohydrate accumulation, cpd7 mutants exhibited reduced sucrose export from mature leaves. Cpd7 encodes a Golgi-resident glucuronosyltransferase belonging to the Glycosyltransferase14 (GT14) family, which is involved in decoration of type II arabinogalactan proteins. No previously described GT14 mutants exhibit reduced sucrose transport or carbohydrate partitioning defects. Additionally, we show that mature leaves of cpd7 mutants have reduced cellulose content and an altered cell wall composition. Further, cpd7 mutants exhibit ectopic phloem lignification likely as a compensatory mechanism for reduced cell wall integrity. Collectively, our data suggest that Cpd7 functions to facilitate cell wall development in the phloem, which is required for efficient sucrose export from mature maize leaves.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2026

A characterization of recombinant Arabidopsis FRIABLE1 (FRB1) reveals robust rhamnogalacturonan-I rhamnosyltransferase activity and critical catalytic residues

Plant cell walls are glycan-rich extracellular matrices that fundamentally impact essential cellular processes, such as growth, adhesion, and cell shape acquisition. Understanding plant cell wall glycans requires the identification and characterization of the biosynthetic enzymes that produce these polymers. Most successful in vitro protein expression studies of plant cell wall glycosyltransferases have relied on insect, fungal/yeast, or human cell expression systems, whereas prokaryotic expression systems have been generally unsuccessful. Here, we show that Arabidopsis FRIABLE1 (FRB1)/rhamnogalacturonan-I rhamnosyltransferase 8 (RRT8) can be produced in Escherichia coli RosettaGami2 cells as N-terminal maltose-binding protein fusion proteins containing C-terminal 6X-His-tags. We also report the catalytic constants of FRB1/RRT8 with apparent K M and K cat values of 226 μM and 33 min -1 for UDP-Rhamnose and 117 μM and 28.7 min -1 for rhamnogalacturonan-I (RG-I), respectively. We examine the catalytic activities of mutated FRB1/RRT8 proteins based on an AlphaFold 3-generated FRB1/RRT8 protein structural model with a virtually docked UDP-Rha donor. Enzymatic characterization of the mutated and wildtype FRB1/RRT8 protein confirmed that mutation of predicted catalytic site amino acid residues resulted in a 20-fold reduction in RRT activity. FRB1 also robustly polymerizes RG-I in combination with RG-I galacturonosyltransferase 1. These results show how a robust E. coli expression system combined with artificial intelligence tools can be used to increase understanding of plant cell wall glycosyltransferase structure and function.

glycosyltransferase↗
DOE OSTI2025

Structure and mechanism of biosynthesis of Streptococcus mutans cell wall polysaccharide

Streptococcus mutans, the causative agent of human dental caries, expresses a cell wall attached Serotype c-specific Carbohydrate (SCC) that is critical for cell viability. SCC consists of a polyrhamnose backbone of →3)α-Rha(1 → 2)α-Rha(1→ repeats with glucose (Glc) side-chains and glycerol phosphate (GroP) decorations. This study reveals that SCC has one predominant and two more minor Glc modifications. The predominant Glc modification, α-Glc, attached to position 2 of 3-rhamnose, is installed by SccN and SccM glycosyltransferases and is the site of the GroP addition. The minor Glc modifications are β-Glc linked to position 4 of 3-rhamnose installed by SccP and SccQ glycosyltransferases, and α-Glc attached to position 4 of 2-rhamnose installed by SccN working in tandem with an unknown enzyme. Both the major and the minor β-Glc modifications control bacterial morphology, but only the GroP and major Glc modifications are critical for biofilm formation.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2025

Structural basis of undecaprenyl phosphate glycosylation leading to polymyxin resistance in Gram-negative bacteria

In Gram-negative bacteria, the enzymatic modification of Lipid A with aminoarabinose (L-Ara4N) leads to resistance against polymyxin antibiotics and cationic antimicrobial peptides. ArnC, an integral membrane glycosyltransferase, attaches a formylated form of aminoarabinose to the lipid undecaprenyl phosphate, enabling its association with the bacterial inner membrane. Here, we present cryo-electron microscopy structures of ArnC from S. enterica in apo and nucleotide-bound conformations. These structures reveal a conformational transition that takes place upon binding of the partial donor substrate. Using coarse-grained and atomistic simulations, we provide insights into substrate coordination before and during catalysis, and we propose a catalytic mechanism that may operate on all similar metal-dependent polyprenyl phosphate glycosyltransferases. The reported structures provide a new target for drug design aiming to combat polymyxin resistance.

Ashraf, Khuram U. [Rutgers University, Newark, NJ ↗
DOE OSTI2026

A conserved glycan motif induces broadly reactive functional antibodies against the zoonotic pathogen Streptococcus suis

Streptococcus suis is a largely neglected but emerging bacterial zoonotic pathogen of global concern for animal welfare, antibiotic resistance development, and human health. No effective vaccines are now available. Here, we identified and characterized the function and structure of two cell wall polysaccharide variants in pathogenic S. suis strains using genetic deletion and (heterologous) complementation, lectin staining, glycan composition analysis, and specialized NMR spectroscopy. Both glycan variants were anionic polymers that differed in the presence of glucose in the side chain as a result of allelic variation in a glycosyltransferase gene. Deletion of this variable glycosyltransferase revealed an identical glycan “core” and affected S. suis morphology and lysozyme resistance. Immunization of pigs with this core domain elicited antibodies that recognized antigenically diverse pathogenic S. suis strains and induced complement deposition on encapsulated pathogenic S. suis strains. This study provides valuable insights for developing next-generation glycoconjugate vaccines, whereby a single-glycan target could protect against the emerging zoonotic pathogen S. suis.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2025

Empirical evidence that glucan-interacting amino acid side chains within the transmembrane channel collectively facilitate cellulose synthase function

The fundamental mechanism of cellulose synthesis is widely conserved across Kingdoms and depends on cellulose synthases, which are processive, dual-function, family 2 glycosyltransferases (GT-2). These enzymes polymerize glucose on the cytoplasmic side of the plasma membrane and export the glucan chain to the cell surface through an integral transmembrane (TM) channel. Structural studies of active plant cellulose synthases (CESAs) have revealed interactions between the nascent glucan chain and the side chains of polar, charged, and aromatic amino acid residues that line the TM channel. However, the functional consequences of modifying these side chains have not been tested in vivo in CESAs or other processive GT-2s. To test this, we used an established in vivo assay based on genetic complementation of CESA5 in the moss, Physcomitrium patens. For accurate prediction of glucan-interacting amino acid residues, we generated a complete homotrimeric molecular model of PpCESA5 using a combination of homology and de novo modeling. All-atom molecular dynamics-based analyses of contact metrics and interaction energy identified 23 amino acid residues with high propensity to interact with the nascent glucan chain within the TM channel or on the apoplastic surface of PpCESA5. Mutating any one of 18 of these amino acid residues to alanine, thereby removing their side chains, abolished or impaired CESA function, with the strongest effects observed upon the loss of charged amino acid side chains. This provides direct evidence to support the hypothesis that multiple amino acid residues collectively maintain a smooth energy landscape within the TM channel to facilitate glucan translocation.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2024

Enzymatic Routes to Designer Hemicelluloses for Use in Biobased Materials

Various enzymes can be used to modify the structure of hemicelluloses directly in vivo or following extraction from biomass sources, such as wood and agricultural residues. Generally, these enzymes can contribute to designer hemicelluloses through four main strategies: (1) enzymatic hydrolysis such as selective removal of side groups by glycoside hydrolases (GH) and carbohydrate esterases (CE), (2) enzymatic cross-linking, for instance, the selective addition of side groups by glycosyltransferases (GT) with activated sugars, (3) enzymatic polymerization by glycosynthases (GS) with activated glycosyl donors or transglycosylation, and (4) enzymatic functionalization, particularly via oxidation by carbohydrate oxidoreductases and via amination by amine transaminases. Thus, this Perspective will first highlight enzymes that play a role in regulating the degree of polymerization and side group composition of hemicelluloses, and subsequently, it will explore enzymes that enhance cross-linking capabilities and incorporate novel chemical functionalities into saccharide structures. These enzymatic routes offer a precise way to tailor the properties of hemicelluloses for specific applications in biobased materials, contributing to the development of renewable alternatives to conventional materials derived from fossil fuels.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗
DOE OSTI2024

Complete biosynthesis of QS-21 in engineered yeast

QS-21 is a potent vaccine adjuvant and remains the only saponin-based adjuvant that has been clinically approved for use in humans. However, owing to the complex structure of QS-21, its availability is limited. Today, the supply depends on laborious extraction from the Chilean soapbark tree or on low-yielding total chemical synthesis. Here we demonstrate the complete biosynthesis of QS-21 and its precursors, as well as structural derivatives, in engineered yeast strains. The successful biosynthesis in yeast requires fine-tuning of the host’s native pathway fluxes, as well as the functional and balanced expression of 38 heterologous enzymes. The required biosynthetic pathway spans seven enzyme families—a terpene synthase, P450s, nucleotide sugar synthases, glycosyltransferases, a coenzyme A ligase, acyl transferases and polyketide synthases—from six organisms, and mimics in yeast the subcellular compartmentalization of plants from the endoplasmic reticulum membrane to the cytosol. Finally, by taking advantage of the promiscuity of certain pathway enzymes, we produced structural analogues of QS-21 using this biosynthetic platform. This microbial production scheme will allow for the future establishment of a structure–activity relationship, and will thus enable the rational design of potent vaccine adjuvants.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2025

Genomic and transcriptomic characterization of carbohydrate-active enzymes in the anaerobic fungus Neocallimastix cameroonii var. constans

Anaerobic gut fungi effectively degrade lignocellulose in the guts of large herbivores, but there remain a limited number of isolated, publicly available, and sequenced strains that impede our understanding of the role of anaerobic fungi within microbial communities. We isolated and characterized a new fungal isolate, Neocallimastix cameroonii var. constans, providing a transcriptomic and genomic understanding of its ability to degrade diverse carbohydrates. This anaerobic fungal strain was stably cultivated for multiple years in vitro among members of an initial enrichment microbial community derived from goat feces, and it demonstrated the ability to pair with other microbial members, namely, archaeal methanogens to produce methane from lignocellulose. Genomic analysis revealed a higher number of predicted carbohydrate-active enzymes encoded in the N. cameroonii var. constans genome compared to most other sequenced anaerobic fungi. The carbohydrate-active enzyme profile for this isolate contained 660 glycoside hydrolases, 160 carbohydrate esterases, 194 glycosyltransferases, and 85 polysaccharide lyases. Differential gene expression analysis showed the upregulation of thousands of genes (including predicted carbohydrate-active enzymes) when N. cameroonii var. constans was grown on lignocellulose (reed canary grass) compared to less complex substrates, such as cellulose (filter paper), cellobiose, and glucose. AlphaFold was used to predict functions of transcriptionally active yet poorly annotated genes, revealing feruloyl esterases that likely play an important role in lignocellulose degradation by anaerobic fungi. The combination of this strain's genomic and transcriptomic characterization, omics-informed structural prediction, and robustness in microbial co-culture make it a well-suited platform to conduct future investigations into bioprocessing and enzyme discovery.

CAZymes↗
DOE OSTI2024

Glycosyl transferase GT2 genes mediate the biosynthesis of an unusual (1,3;1,4)‐β‐glucan exopolysaccharide in the bacterium Sarcina ventriculi

Abstract Linear, unbranched (1,3;1,4)‐β‐glucans (mixed‐linkage glucans or MLGs) are commonly found in the cell walls of grasses, but have also been detected in basal land plants, algae, fungi and bacteria. Here we show that two family GT2 glycosyltransferases from the Gram‐positive bacteriumSarcina ventriculiare capable of synthesizing MLGs. Immunotransmission electron microscopy demonstrates that MLG is secreted as an exopolysaccharide, where it may play a role in organizing individual cells into packets that are characteristic ofSarcinaspecies. Heterologous expression of these two genes shows that they are capable of producing MLGsin planta, including an MLG that is chemically identical to the MLG secreted fromS. ventriculicells but which has regularly spaced (1,3)‐β‐linkages in a structure not reported previously for MLGs. The tandemly arranged, paralogous pair of genes are designatedSvBmlgs1andSvBmlgs2. The data indicate that MLG synthases have evolved different enzymic mechanisms for the incorporation of (1,3)‐β‐ and (1,4)‐β‐glucosyl residues into a single polysaccharide chain. Amino acid variants associated with the evolutionary switch from (1,4)‐β‐glucan (cellulose) to MLG synthesis have been identified in the active site regions of the enzymes. The presence of MLG synthesis in bacteria could prove valuable for large‐scale production of MLG for medical, food and beverage applications.

Biochemistry & Molecular Biology↗
DOE OSTI2026

Biochemical characterization of xyloglucan galactosyltransferases MUR3 and XLT2 from Spirodela polyrhiza

Glycosyltransferases (GTs) are the primary enzymes responsible for the biosynthesis of the complex polysaccharides in plant cell walls. Given the important role of GTs in plants, it is necessary to undertake their functional characterization to better understand plant cell wall synthesis pathways to develop improved feedstocks for efficient conversion into fuels and products to support the emerging bioeconomy. The GT47 family in plants represents a unique target for characterization due to the substantial diversity of donor and acceptor substrates observed within a single family. Here, we have carried out the biochemical characterization of MUR3 and XLT2 orthologs from the aquatic monocot Spirodela polyrhiza. Our findings support existing genetic and phylogenetic data classifying these enzymes as regio-specific galactosyltransferases involved in xyloglucan (XyG) sidechain biosynthesis. In addition, we have identified novel characteristics for both enzymes, such as in vitro arabinopyranosyltransferase activity and distinctiveness in xyloglucan reducing end specificity.

54 ENVIRONMENTAL SCIENCES↗