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1. Xyloglucan Xylosyltransferase 1 Displays Promiscuity Toward Donor Substrates During In vitro Reactions

   https://doi.org/10.1093/pcp/pcab114  

   Ehrlich, Jacqueline J; Weerts, Richard M; Shome, Sayane; Culbertson, Alan T; Honzatko, Richard B; Jernigan, Robert L; Zabotina, Olga A (July 2021, Plant and Cell Physiology)

   null (Ed.)

   Abstract Glycosyltransferases (GTs) are a large family of enzymes that add sugars to a broad range of acceptor substrates, including polysaccharides, proteins, and lipids, by utilizing a wide variety of donor substrates in the form of activated sugars. Individual GTs have generally been considered to exhibit a high level of substrate specificity, but this has not been thoroughly investigated across the extremely large set of GTs. Here we investigate Xyloglucan Xylosyltransferase 1 (XXT1), a GT involved in synthesis of the plant cell wall polysaccharide, xyloglucan. Xyloglucan has a glucan backbone, with initial side chain substitutions exclusively composed of xylose from UDP-Xylose. While this conserved substitution pattern suggests a high substrate specificity for XXT1, our in vitro kinetic studies elucidate a more complex set of behavior. Kinetic studies demonstrate comparable kcat values for reactions with UDP-Xylose and UDP-Glucose, while reactions with UDP-Arabinose and UDP-Galactose are over 10-fold slower. Using kcat/Km as a measure of efficiency, UDP-Xylose is 8-fold more efficient as a substrate than the next best alternative, UDP-Glucose. To the best of our knowledge, we are the first to demonstrate that not all plant XXTs are highly substrate specific, and some do show significant promiscuity in their in vitro reactions. Kinetic parameters alone likely do not explain the high substrate selectivity in planta, suggesting there are additional control mechanisms operating during polysaccharide biosynthesis. Improved understanding of substrate specificity of the GTs will aid in protein engineering, development of diagnostic tools, and understanding of biological systems. 

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2. Biochemical and Genetic Analysis Identify CSLD3 as a beta-1,4-glucan Synthase that Functions during Plant Cell Wall Synthesis.

   https://doi.org/doi: 10.1105/tpc.19.00637  

   *Yang J., *Bak G. (May 2020, The plant cell)

   In plants, changes in cell size and shape during development fundamentally depend on the ability to synthesize and modify cell wall polysaccharides. The main classes of cell wall polysaccharides produced by terrestrial plants are cellulose, hemicelluloses, and pectins. Members of the cellulose synthase (CESA) and cellulose synthase-like (CSL) families encode glycosyltransferases that synthesize the β-1,4-linked glycan backbones of cellulose and most hemicellulosic polysaccharides that comprise plant cell walls. Cellulose microfibrils are the major load-bearing component in plant cell walls and are assembled from individual β-1,4-glucan polymers synthesized by CESA proteins that are organized into multimeric complexes called CESA complexes, in the plant plasma membrane. During distinct modes of polarized cell wall deposition, such as in the tip growth that occurs during the formation of root hairs and pollen tubes or de novo formation of cell plates during plant cytokinesis, newly synthesized cell wall polysaccharides are deposited in a restricted region of the cell. These processes require the activity of members of the CESA-like D subfamily. However, while these CSLD polysaccharide synthases are essential, the nature of the polysaccharides they synthesize has remained elusive. Here, we use a combination of genetic rescue experiments with CSLD-CESA chimeric proteins, in vitro biochemical reconstitution, and supporting computational modeling and simulation, to demonstrate that Arabidopsis (Arabidopsis thaliana) CSLD3 is a UDP-glucose-dependent β-1,4-glucan synthase that forms protein complexes displaying similar ultrastructural features to those formed by CESA6. 

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3. Towards Computationally Guided Design and Engineering of a Neisseria meningitidis Serogroup W Capsule Polymerase with Altered Substrate Specificity

   https://doi.org/10.3390/pr9122192  

   Paudel, Subhadra; Wachira, James; McCarthy, Pumtiwitt C. (December 2021, Processes)

   Heavy metal contamination of drinking water is a public health concern that requires the development of more efficient bioremediation techniques. Absorption technologies, including biosorption, provide opportunities for improvements to increase the diversity of target metal ions and overall binding capacity. Microorganisms are a key component in wastewater treatment plants, and they naturally bind metal ions through surface macromolecules but with limited capacity. The long-term goal of this work is to engineer capsule polymerases to synthesize molecules with novel functionalities. In previously published work, we showed that the Neisseria meningitidis serogroup W (NmW) galactose–sialic acid (Gal–NeuNAc) heteropolysaccharide binds lead ions effectively, thereby demonstrating the potential for its use in environmental decontamination applications. In this study, computational analysis of the NmW capsule polymerase galactosyltransferase (GT) domain was used to gain insight into how the enzyme could be modified to enable the synthesis of N-acetylgalactosamine–sialic acid (GalNAc–NeuNAc) heteropolysaccharide. Various computational approaches, including molecular modeling with I-TASSER and molecular dynamics (MD) simulations with NAMD, were utilized to identify key amino acid residues in the substrate binding pocket of the GT domain that may be key to conferring UDP-GalNAc specificity. Through these combined strategies and using BshA, a UDP-GlcNAc transferase, as a structural template, several NmW active site residues were identified as mutational targets to accommodate the proposed N-acetyl group in UDP-GalNAc. Thus, a rational approach for potentially conferring new properties to bacterial capsular polysaccharides is demonstrated. 

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4. Dysregulation of a nucleotidyltransferase induces division and surface glycan defects in Escherichia coli by altering related metabolite levels

   https://doi.org/10.1073/pnas.2510285122  

   Zheng, Maggie C; Bryant, Joseph C; Koid, Andrea; Sheshova, Mia; Kim, Hanee; Jorgenson, Matthew A; Lupoli, Tania J (January 2026, Proceedings of the National Academy of Sciences)

   The cell surface of gram-negativeEscherichia coliis rich in glycoconjugates, including O-antigen (O-Ag) and enterobacterial common antigen polysaccharides, which mediate interactions with the environment. Enzymes called nucleotidyltransferases produce nucleoside diphosphate sugars (NDP-sugars), the precursors to these and many other cellular glycans. Across bacteria, glucose-1-phosphate thymidylyltransferases (G1P-Ts) couple deoxythymidine triphosphate (dTTP) and glucose-1-phosphate to produce a key secondary metabolite, deoxythymidine diphosphate-glucose (dTDP-Glc). Flux through many glycan biosynthetic pathways is regulated through feedback inhibition at an allosteric site of the conserved bacterial G1P-T RmlA. Here, we sought to address the cellular consequences of G1P-T dysregulation on metabolic flux andE. coliphysiology. Expression of a hyperactive RmlA variant inE. colicells lacking native G1P-Ts abrogated growth, induced morphological defects, and increased sensitivity toward cell division inhibitors. These defects were suppressed through nucleotide supplementation, leading to the hypothesis that growth and division defects result from observed depletion of nucleotide pools required for DNA synthesis. Inspection of nucleotide metabolite compositions inE. colilysates carrying mutant G1P-T, however, also indicated increased cellular concentrations of the primary metabolite uridine diphosphate glucose (UDP-Glc), which is known to block cell division. We found that high intracellular levels of UDP-Glc also unexpectedly halt the expression of O-Ag, which reveals that this metabolite is key for coordinating surface glycan biosynthesis and DNA precursor production prior to cell division. Overall, this work highlights the importance of G1P-T regulation and uncovers additional roles of UDP-Glc as a molecular sensor that mediates diverse bacterial biosynthetic processes. 

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5. Characterization of the need for galactofuranose during the Neurospora crassa life cycle

   https://doi.org/10.1016/j.fgb.2023.103826  

   Schaff, Hayden; Dey, Protyusha; Heiss, Christian; Keiser, Griffin; Moro, Tatiana Rojo; Azadi, Parastoo; Patel, Pavan; Free, Stephen J. (October 2023, Fungal Genetics and Biology)

   Galactofuranose is a constituent of the cell walls of filamentous fungi. The galactofuranose can be found as a component of N-linked oligosaccharides, in O-linked oligosaccharides, in GPI-anchored galactomannan, and in free galactomannan. The Neurospora genome contains a single UDP-galactose mutase gene (ugm-1/NCU01824) and two UDP-galactofuranose translocases used to import UDP-galactofuranose into the lumen of the Golgi apparatus (ugt-1/NCU01826 and ugt-2/NCU01456). Our results demonstrate that loss of galactofuranose synthesis or its translocation into the lumen of the secretory pathway affects the morphology and growth rate of the vegetative hyphae, the production of conidia (asexual spores), and dramatically affects the sexual stages of the life cycle. In mutants that are unable to make galactofuranose or transport it into the lumen of the Golgi apparatus, ascospore development is aborted soon after fertilization and perithecium maturation is aborted prior to the formation of the neck and ostiole. The Neurospora genome contains three genes encoding possible galactofuranosyltransferases from the GT31 family of glycosyltransferases (gfs-1/NCU05878, gfs-2/NCU07762, and gfs-3/NCU02213) which might be involved in generating galactofuranose-containing oligosaccharide structures. Analysis of triple KO mutants in GT31 glycosyltransferases shows that these mutants have normal morphology, suggesting that these genes do not encode vital galactofuranosyltransferases. 

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