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At least 793 records · Page 44
All sourcesNASA NTRSDOE OSTI
DOE OSTI2024

Characterization of lignin-degrading enzyme PmdC, which catalyzes a key step in the synthesis of polymer precursor 2-pyrone-4,6-dicarboxylic acid

Pyrone-2,4-dicarboxylic acid (PDC) is a valuable polymer precursor that can be derived from the microbial degradation of lignin. The key enzyme in the microbial production of PDC is 4-carboxy-2-hydroxymuconate-6-semialdehyde (CHMS) dehydrogenase, which acts on the substrate CHMS. We present the crystal structure of CHMS dehydrogenase (PmdC from Comamonas testosteroni) bound to the cofactor NADP, shedding light on its three-dimensional architecture, and revealing residues responsible for binding NADP. Using a combination of structural homology, molecular docking, and quantum chemistry calculations, we have predicted the binding site of CHMS. Key histidine residues in a conserved sequence are identified as crucial for binding the hydroxyl group of CHMS and facilitating dehydrogenation with NADP. Mutating these histidine residues results in a loss of enzyme activity, leading to a proposed model for the enzyme's mechanism. These findings are expected to help guide efforts in protein and metabolic engineering to enhance PDC yields in biological routes to polymer feedstock synthesis.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗
DOE OSTI2022

Amyloid-like amelogenin nanoribbons template mineralization via a low-energy interface of ion binding sites

Protein scaffolds direct the organization of amorphous precursors that transform into mineralized tissues, but the templating mechanism remains elusive. Motivated by models for the biomineralization of tooth enamel, wherein amyloid-like amelogenin nanoribbons guide the mineralization of apatite filaments, we investigated the impact of nanoribbon structure, sequence, and chemistry on amorphous calcium phosphate (ACP) nucleation. Using full-length human amelogenin and peptide analogs with an amyloid-like domain, films of β-sheet nanoribbons were self-assembled on graphite and characterized by in situ atomic force microscopy and molecular dynamics simulations. All sequences substantially reduce nucleation barriers for ACP by creating low-energy interfaces, while phosphoserines along the length of the nanoribbons dramatically enhance kinetic factors associated with ion binding. Furthermore, the distribution of negatively charged residues along the nanoribbons presents a potential match to the Ca–Ca distances of the multi-ion complexes that constitute ACP. These findings show that amyloid-like amelogenin nanoribbons provide potent scaffolds for ACP mineralization by presenting energetically and stereochemically favorable templates of calcium phosphate ion binding and suggest enhanced surface wetting toward calcium phosphates in general.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2024

Complete genomes of Asgard archaea reveal diverse integrated and mobile genetic elements

Asgard archaea are of great interest as the progenitors of Eukaryotes, but little is known about the mobile genetic elements (MGEs) that may shape their ongoing evolution. Here, we describe MGEs that replicate in Atabeyarchaeia, a wetland Asgard archaea lineage represented by two complete genomes. We used soil depth–resolved population metagenomic data sets to track 18 MGEs for which genome structures were defined and precise chromosome integration sites could be identified for confident host linkage. Additionally, we identified a complete 20.67 kbp circular plasmid and two family-level groups of viruses linked to Atabeyarchaeia, via CRISPR spacer targeting. Closely related 40 kbp viruses possess a hypervariable genomic region encoding combinations of specific genes for small cysteine-rich proteins structurally similar to restriction-homing endonucleases. One 10.9 kbp integrative conjugative element (ICE) integrates genomically into theAtabeyarchaeum deiterrae-1chromosome and has a 2.5 kbp circularizable element integrated within it. The 10.9 kbp ICE encodes an expressed Type IIG restriction-modification system with a sequence specificity matching an active methylation motif identified by Pacific Biosciences (PacBio) high-accuracy long-read (HiFi) metagenomic sequencing. Restriction-modification of Atabeyarchaeia differs from that of another coexisting Asgard archaea, Freyarchaeia, which has few identified MGEs but possesses diverse defense mechanisms, including DISARM and Hachiman, not found in Atabeyarchaeia. Overall, defense systems and methylation mechanisms of Asgard archaea likely modulate their interactions with MGEs, and integration/excision and copy number variation of MGEs in turn enable host genetic versatility.

Biochemistry & Molecular Biology↗
DOE OSTI2024

Psychosocial experiences are associated with human brain mitochondrial biology

Psychosocial experiences affect brain health and aging trajectories, but the molecular pathways underlying these associations remain unclear. Normal brain function relies on energy transformation by mitochondria oxidative phosphorylation (OxPhos). Two main lines of evidence position mitochondria both as targets and drivers of psychosocial experiences. On the one hand, chronic stress exposure and mood states may alter multiple aspects of mitochondrial biology; on the other hand, functional variations in mitochondrial OxPhos capacity may alter social behavior, stress reactivity, and mood. But are psychosocial exposures and subjective experiences linked to mitochondrial biology in the human brain? By combining longitudinal antemortem assessments of psychosocial factors with postmortem brain (dorsolateral prefrontal cortex) proteomics in older adults, we find that higher well-being is linked to greater abundance of the mitochondrial OxPhos machinery, whereas higher negative mood is linked to lower OxPhos protein content. Combined, positive and negative psychosocial factors explained 18 to 25% of the variance in the abundance of OxPhos complex I, the primary biochemical entry point that energizes brain mitochondria. Moreover, interrogating mitochondrial psychobiological associations in specific neuronal and nonneuronal brain cells with single-nucleus RNA sequencing (RNA-seq) revealed strong cell-type-specific associations for positive psychosocial experiences and mitochondria in glia but opposite associations in neurons. As a result, these “mind-mitochondria” associations were masked in bulk RNA-seq, highlighting the likely underestimation of true psychobiological effect sizes in bulk brain tissues. Thus, self-reported psychosocial experiences are linked to human brain mitochondrial phenotypes.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2021

Epigenetic Control of Drought Response in Sorghum (EPICON)

Genetic manipulation of crops to increase the presence of desirable traits has been critical to increasing agricultural productivity. These changes have primarily involved modification of the plant’s DNA sequence. However, there is increasing evidence that environmental responses are also mediated by epigenetics, which involves heritable changes without changes in DNA sequence. Epigenetic changes have been shown to play a major role in regulating plant responses to drought, an increasing problem worldwide due to climate change. In general, exposure of plants to water limitation triggers epigenetic changes, which include remodeling of chromatin, the network of DNA, RNA and various proteins making up chromosomes, and related changes in regulatory mechanisms. EPICON’s efforts focus on unraveling the role epigenetic signals play in acclimation to and recovery from drought through effects on individual transcription factors or transcriptional networks that direct entire metabolic pathways. To achieve this goal we will follow responses to water deprivation in sorghum, a widely cultivated cereal with recognized drought tolerance. In EPICON’s field trials, sorghum will be grown under controlled irrigation conditions. Leaf and root samples will be taken to perform molecular phenotyping to track changes in epigenetic, transcriptomic, metabolomic and proteomic footprints. Analysis of this data will provide a better understanding of the epigenetic processes related to drought tolerance, leading to our ultimate goal of identifying transcriptional regulators and pathways controlling drought resistance. The identified genetic targets and their regulatory pathways will be used in future efforts to improve growth of sorghum and other crops in the field and in marginal lands under water-limiting conditions.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2022

Effects of Microbial-Mineral Interactions on Organic Carbon Stabilization in a Ponderosa Pine Root Zone: A Micro-Scale Approach

Soil microbial communities affect the formation of micro-scale mineral-associated organic matter (MAOM) where complex processes, including adhesion, aggregate formation, microbial mineral weathering and soil organic matter stabilization occur in a narrow zone of large biogeochemical gradients. Here we designed a field study to examine carbon stabilization mechanisms by using in-growth mesh bags containing biotite that were placed in a ponderosa pine root zone for 6 months and compared to the surrounding bulk soil. We sought to determine the composition of the microbial community in the mesh bags compared to the surrounding soils, analyze the direct interactions between microbes and biotite, and finally identify the nature of the newly formed MAOM within the mesh-bags. Our results revealed that minerals in the mesh bags were colonized by a microbial community that produced organic matter in situ. The 16S rRNA gene sequencing and ITS2 region characterization showed phylogenetic similarity between the mesh bag and bulk soil archaea/bacteria and fungi microbiomes, with significant differences in alpha- and beta-diversity and species abundances. Organic matter pools in the mesh bags, analyzed by Fourier transform ion cyclotron resonance mass spectrometry, contained protein- (peptides) and lipid-like compounds while the bulk soil OM was comprised of lignin-like and carboxyl-rich alicyclic molecules. These results support that the newly formed biotite associated organic compounds have a microbial signature in the mesh bags. High-resolution electron microscopy documented strongly adhered organic compounds to biotite surfaces, formation of microaggregates, elemental uptake at the microbe (organic matter)-mineral interface, and distortion of biotite layers. Overall, this study shows the direct and indirect involvement of soil microbial communities from the root zone of ponderosa pine in the formation of MAOM, soil organic carbon stabilization, microaggregation, and mineral weathering at micro- and nano-scales.

58 GEOSCIENCES↗
DOE OSTI2023

Biochemical basis for the formation of organ-specific volatile blends in mint

Above-ground material of members of the mint family is commercially distilled to extract essential oils, which are then formulated into a myriad of consumer products. Most of the research aimed at characterizing the processes involved in the formation of terpenoid oil constituents has focused on leaves. We now demonstrate, by investigating three mint species, peppermint ( Mentha ˣ piperita L.), spearmint ( Mentha spicata L.) and horsemint ( Mentha longifolia (L.) Huds.; accessions CMEN 585 and CMEN 584), that other organs – namely stems, rhizomes and roots – also emit volatiles and that the terpenoid volatile composition of these organs can vary substantially from that of leaves, supporting the notion that substantial, currently underappreciated, chemical diversity exists. Differences in volatile quantities released by plants whose roots had been dipped in a Verticillium dahliae -spore suspension (experimental) or dipped in water (controls) were evident: increases of some volatiles in the root headspace of mint species that are susceptible to Verticillium wilt disease (peppermint and M. longifolia CMEN 584) were detected, while the quantities of certain volatiles decreased in rhizomes of species that show resistance to the disease (spearmint and M. longifolia CMEN 585). To address the genetic and biochemical basis underlying chemical diversity, we took advantage of the newly sequenced M. longifolia CMEN 585 genome to identify candidate genes putatively coding for monoterpene synthases (MTSs), the enzymes that catalyze the first committed step in the biosynthesis of monoterpenoid volatiles. The functions of these genes were established by heterologous expression in Escherichia coli , purification of the corresponding recombinant proteins, and enzyme assays, thereby establishing the existence of MTSs with activities to convert a common substrate, geranyl diphosphate, to (+)-α-terpineol, 1,8-cineole, γ-terpinene, and (–)-bornyl diphosphate, but were not active with other potential substrates. In conjunction with previously described MTSs that catalyze the formation of (–)-β-pinene and (–)-limonene, the product profiles of the MTSs identified here can explain the generation of all major monoterpene skeletons represented in the volatiles released by different mint organs.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2022

Structure of the divergent human astrovirus MLB capsid spike

Despite their worldwide prevalence and association with human disease, the molecular bases of human astrovirus (HAstV) infection and evolution remain poorly characterized. Here, we report the structure of the capsid protein spike of the divergent HAstV MLB clade (HAstV MLB). While the structure shares a similar folding topology with that of classical-clade HAstV spikes, it is otherwise strikingly different. We find no evidence of a conserved receptor-binding site between the MLB and classical HAstV spikes, suggesting that MLB and classical HAstVs utilize different receptors for host-cell attachment. We provide evidence for this hypothesis using a novel HAstV infection competition assay. Comparisons of the HAstV MLB spike structure with structures predicted from its sequence reveal poor matches, but template-based predictions were surprisingly accurate relative to machine-learning-based predictions. Our data provide a foundation for understanding the mechanisms of infection by diverse HAstVs and can support structure determination in similarly unstudied systems.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2020

Major Revisions in Arthropod Phylogeny Through Improved Supermatrix, With Support for Two Possible Waves of Land Invasion by Chelicerates

Deep phylogeny involving arthropod lineages is difficult to recover because the erosion of phylogenetic signals over time leads to unreliable multiple sequence alignment (MSA) and subsequent phylogenetic reconstruction. One way to alleviate the problem is to assemble a large number of gene sequences to compensate for the weakness in each individual gene. Such an approach has led to many robustly supported but contradictory phylogenies. A close examination shows that the supermatrix approach often suffers from two shortcomings. The first is that MSA is rarely checked for reliability and, as will be illustrated, can be poor. The second is that, to alleviate the problem of homoplasy at the third codon position of protein-coding genes due to convergent evolution of nucleotide frequencies, phylogeneticists may remove or degenerate the third codon position but may do it improperly and introduce new biases. We performed extensive reanalysis of one of such “big data” sets to highlight these two problems, and demonstrated the power and benefits of correcting or alleviating these problems. Our results support a new group with Xiphosura and Arachnopulmonata (Tetrapulmonata + Scorpiones) as sister taxa. This favors a new hypothesis in which the ancestor of Xiphosura and the extinct Eurypterida (sea scorpions, of which many later forms lived in brackish or freshwater) returned to the sea after the initial chelicerate invasion of land. Our phylogeny is supported even with the original data but processed with a new “principled” codon degeneration. We also show that removing the 1673 codon sites with both AGN and UCN codons (encoding serine) in our alignment can partially reconcile discrepancies between nucleotide-based and AA-based tree, partly because two sequences, one with AGN and the other with UCN, would be identical at the amino acid level but quite different at the nucleotide level.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2022

Protein cost minimization promotes the emergence of coenzyme redundancy

Metabolism relies on a small class of molecules (coenzymes) that serve as universal donors and acceptors of key chemical groups and electrons. Although metabolic networks crucially depend on structurally redundant coenzymes [e.g., NAD(H) and NADP(H)] associated with different enzymes, the criteria that led to the emergence of this redundancy remain poorly understood. Our combination of modeling and structural and sequence analysis indicates that coenzyme redundancy may not be essential for metabolism but could rather constitute an evolved strategy promoting efficient usage of enzymes when biochemical reactions are near equilibrium. Our work suggests that early metabolism may have operated with fewer coenzymes and that adaptation for metabolic efficiency may have driven the rise of coenzyme diversity in living systems.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2021

Data for Cloning and Characterization of a Panel of Mitochondrial Targeting Sequences for Compartmentalization Engineering in Saccharomyces cerevisiae

Mitochondrion is generally considered as the most promising subcellular organelle for compartmentalization engineering. Much progress has been made in reconstituting whole metabolic pathways in the mitochondria of yeast to harness the precursor pools (i.e., pyruvate and acetyl-CoA), bypass competing pathways, and minimize transportation limitations. However, only a few mitochondrial targeting sequences (MTSs) have been characterized (i.e., MTS of COX4), limiting the application of compartmentalization engineering for multigene biosynthetic pathways in the mitochondria of yeast. In the present study, based on the mitochondrial proteome, a total of 20 MTSs were cloned and the efficiency of these MTSs in targeting heterologous proteins, including the Escherichia coli FabI and enhanced green fluorescence protein (EGFP) into the mitochondria was evaluated by growth complementation and confocal microscopy. After systematic characterization, six of the well-performed MTSs were chosen for the colocalization of complete biosynthetic pathways into the mitochondria. As proof of concept, the full α-santalene biosynthetic pathway consisting of 10 expression cassettes capable of converting acetyl-coA to α-santalene was compartmentalized into the mitochondria, leading to a 3.7-fold improvement in the production of α-santalene. The newly characterized MTSs should contribute to the expanded metabolic engineering and synthetic biology toolbox for yeast mitochondrial compartmentalization engineering.

Conversion↗
DOE OSTI2022

Multiple Mutations Associated with Emergent Variants Can Be Detected as Low-Frequency Mutations in Early SARS-CoV-2 Pandemic Clinical Samples

Genetic analysis of intra-host viral populations provides unique insight into pre-emergent mutations that may contribute to the genotype of future variants. Clinical samples positive for SARS-CoV-2 collected in California during the first months of the pandemic were sequenced to define the dynamics of mutation emergence as the virus became established in the state. Deep sequencing of 90 nasopharyngeal samples showed that many mutations associated with the establishment of SARS-CoV-2 globally were present at varying frequencies in a majority of the samples, even those collected as the virus was first detected in the US. A subset of mutations that emerged months later in consensus sequences were detected as subconsensus members of intra-host populations. Spike mutations P681H, H655Y, and V1104L were detected prior to emergence in variant genotypes, mutations were detected at multiple positions within the furin cleavage site, and pre-emergent mutations were identified in the nucleocapsid and the envelope genes. Because many of the samples had a very high depth of coverage, a bioinformatics pipeline, “Mappgene”, was established that uses both iVar and LoFreq variant calling to enable identification of very low-frequency variants. This enabled detection of a spike protein deletion present in many samples at low frequency and associated with a variant of concern.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2021

Large-scale survey of prion protein genetic variability in scrapie disease-free goats from the United States

Scrapie is a slowly progressive neurodegenerative disease of small ruminants caused by an accumulation of an abnormal isoform of prion protein in the central nervous system. Polymorphisms of the prion protein gene (PRNP) strongly modulate scrapie resistance and incubation period in goats. The aim of this study was to identify PRNP genetic variability in goats across the United States. Blood from a total of 6,029 apparent scrapie disease-free goats from 654 operations and 19 breeds were analyzed. Sequencing of PRNP revealed 26 genotypes with different rates based on eight codons. The GG127, RR154, and QQ222 genotypes were predominant and showed a remarkably high rate across all goats. The QK222 and NS146 genotypes, known to be protective against scrapie, were found in 0.6% [with 95% CI = (0.3, 1.2)] and 22.0% [95% CI = (19.1, 25.2)] of goats, respectively. The QK222 genotype was found in 23.1% of Oberhasli goats tested, with 95%CI = (3.9, 68.7)] and 22.0% of Toggenburg goats tested with 95%CI = (9.7, 42.5)], while NS146 was found in 65.5% of Savannah goats tested, with 95%CI = (30.8, 89.9), 36.7% of Boer goats tested, with 95%CI = (33.1, 40.4), 36.3% of Nubian goats tested, with 95%CI = (27.0, 46.7)], and 35.6% of LaMancha goats tested, with 95%CI = (22.8, 50.8%). The MM142 and IM142 genotypes were found more frequently in goats on dairy operations, while the HR143, NS146, and ND146 genotypes were found more frequently in goats on meat operations. Goats in the east region had a higher percentage of goats with RH154, RQ211, and QK222 genotypes than goats in the west region. The results of this study showed high genetic variability of PRNP among the U.S. goat population, with differences by location and breed, and may serve as a rationale for development of goat breeding programs at the national level to mitigate the risk of scrapie.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2022

Low Pathogenicity H7N3 Avian Influenza Viruses Have Higher Within-Host Genetic Diversity Than a Closely Related High Pathogenicity H7N3 Virus in Infected Turkeys and Chickens

Within-host viral diversity offers a view into the early stages of viral evolution occurring after a virus infects a host. In recent years, advances in deep sequencing have allowed for routine identification of low-frequency variants, which are important sources of viral genetic diversity and can potentially emerge as a major virus population under certain conditions. We examined within-host viral diversity in turkeys and chickens experimentally infected with closely related H7N3 avian influenza viruses (AIVs), specifically one high pathogenicity AIV (HPAIV) and two low pathogenicity AIV (LPAIVs) with different neuraminidase protein stalk lengths. Consistent with the high mutation rates of AIVs, an abundance of intra-host single nucleotide variants (iSNVs) at low frequencies of 2–10% was observed in all samples collected. Furthermore, a small number of common iSNVs were observed between turkeys and chickens, and between directly inoculated and contact-exposed birds. Notably, the LPAIVs have significantly higher iSNV diversities and frequencies of nonsynonymous changes than the HPAIV in both turkeys and chickens. These findings highlight the dynamics of AIV populations within hosts and the potential impact of genetic changes, including mutations in the hemagglutinin gene that confers the high pathogenicity pathotype, on AIV virus populations and evolution.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2022

Chapter 20: Advances and Application of CRISPR-Cas Systems

A new gene-editing technique called clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated (Cas) proteins have revolutionized genome engineering because of its high efficiency, relatively low cost, and ease of use compared with other techniques such as zinc-finger nucleases and transcription activator-like effector nucleases. CRISPR-Cas systems have transformed biological research, quickly becoming the preferred method for engineering specific genome sequences in industrially relevant microbes, important food crops, and human cells. This powerful new tool has the potential for limitless applications ranging from the treatment of human diseases to improving food security to the generation of renewable and sustainable bioproducts and biofuels. In this chapter, we will discuss advances and applications for these CRISPR-Cas systems in biological engineering.

bacterial↗
DOE OSTI2025

The genome of the polyextremophilic yeast, Naganishia friedmannii, reveals adaptations involved in stress response pathways, carbohydrate metabolism expansion, and a limited DNA repair repertoire

Here we report the draft genome sequence of Naganishia friedmannii (formerly Cryptococcus friedmannii) isolate, a Basidiomycota yeast commonly found in some of the most extreme environments of the Earth's cryosphere. We isolated N. friedmannii strain Llullensis from soils at 6000 m above sea level on Volcán Llullaillaco, Argentina. The genome was 22.2 Mb with 6251 identified protein coding genes. Proteins known to be associated with thermal, osmotic, and radiation stress were identified in the genome. Comparative analysis with seven other Naganishia genomes revealed unique features underlying its polyextremophilic lifestyle. Naganishia friedmannii showed an expansion of genes involved in breaking down plant-derived carbohydrates, supporting the hypothesis that it survives at high elevations by metabolizing wind-deposited organic matter. Surprisingly, many genes involved in cell-cycle checkpoints and DNA repair were missing, as in several other Naganishia species. This extensive loss may be adaptive in extreme environments prone to abiotic stress, where a high mutation rate could generate advantageous traits, and reduced cell-cycle control may allow for faster reproduction that would be advantageous for rapid growth during brief periods of soil wetting following rare snow events.

Vimercati, Lara↗
DOE OSTI2022

Expansion of the global RNA virome reveals diverse clades of bacteriophages

High-throughput RNA sequencing offers broad opportunities to explore the Earth RNA virome. Mining 5,150 diverse metatranscriptomes uncovered >2.5 million RNA virus contigs. Analysis of >330,000 RNA-dependent RNA polymerases (RdRPs) shows that this expansion corresponds to a 5-fold increase of the known RNA virus diversity. Gene content analysis revealed multiple protein domains previously not found in RNA viruses and implicated in virus-host interactions. Extended RdRP phylogeny supports the monophyly of the five established phyla and reveals two putative additional bacteriophage phyla and numerous putative additional classes and orders. The dramatically expanded phylum Lenarviricota, consisting of bacterial and related eukaryotic viruses, now accounts for a third of the RNA virome. Identification of CRISPR spacer matches and bacteriolytic proteins suggests that subsets of picobirnaviruses and partitiviruses, previously associated with eukaryotes, infect prokaryotic hosts.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2025

Identification and characterization of mono- and bifunctional galactan synthases in the pediatric pathogen Kingella kingae

The emerging pediatric pathogen Kingella kingae elaborates a lipopolysaccharide (LPS) that is extended with a galactofuranose homopolymer called galactan, which is a key virulence determinant that contributes to resistance to complement-mediated and neutrophil-mediated killing. Previous work has demonstrated that the pamABCDE locus is required for galactan synthesis. In this study, mutational studies suggested that the pamC gene product is a UDP-galactofuranose (Galf) transferase and is the galactan synthase. Analysis of genome sequence data revealed two distinct pamC alleles designated pamC1 and pamC2, which correlate with the two galactan structures in K. kingae. Examination of isogenic mutants expressing either pamC1 or pamC2 demonstrated that the pamC alleles are the determinants of galactan structure. Experiments with recombinant PamC1 and PamC2 in vitro established that these proteins are galactan synthases capable of extending synthetic Galf disaccharide acceptors in the presence of UDP-Galf. Homology analysis identified critical amino acids that are essential for PamC1 and PamC2 enzymatic activity both in vitro and in K. kingae. Structural analysis of the in vitro-modified synthetic acceptors implicated PamC1 as a monofunctional enzyme capable of generating a β-(1 → 5) Galf linkage and PamC2 as a bifunctional enzyme capable of generating β-(1 → 3) and β-(1 → 6) Galf linkages. This study advances our understanding of the GT2 family of UDP-galactofuranosyltransferases.

60 APPLIED LIFE SCIENCES↗